技术领域technical field
本发明涉及基因工程和分子生物学领域，具体涉及假病毒包装载体及包装系统，其用于制备假病毒的用途，以及假病毒的制备方法。The invention relates to the fields of genetic engineering and molecular biology, in particular to a pseudovirus packaging carrier and a packaging system, its use for preparing pseudoviruses, and a preparation method for pseudoviruses.
背景技术Background technique
假病毒(pseudovirus)是病毒衣壳蛋白或者包膜蛋白包裹携带有报告基因的异源核酸形成的类似于真病毒的具有感染性的病毒颗粒，由于被包裹的核酸不具有复制形成病毒的全部核酸序列的能力，所以假病毒只有一轮的感染力，操作比较安全。Pseudovirus (pseudovirus) is an infectious virus particle similar to a true virus formed by viral capsid protein or envelope protein wrapping heterologous nucleic acid carrying a reporter gene, because the wrapped nucleic acid does not have all the nucleic acids that replicate to form a virus The ability to sequence, so the fake virus only has one round of infectivity, and the operation is relatively safe.
假病毒主要通过多质粒共转染HEK293T或293FT细胞，在细胞内自行复制、表达组装形成，通过收集培养基上清或者裂解细胞等方法获得。Pseudoviruses are mainly co-transfected with multiple plasmids into HEK293T or 293FT cells, self-replicated, expressed and assembled in the cells, and obtained by collecting the supernatant of the culture medium or lysing the cells.
假病毒具备和真病毒一样的感染性，进入细胞的过程和真病毒一样，但其进入细胞后不具有复制产生病毒的能力，因而没有危害，可以更安全地用于中和抗体和抗病毒药物检测。Pseudoviruses have the same infectivity as real viruses, and the process of entering cells is the same as that of real viruses, but they do not have the ability to replicate and produce viruses after entering cells, so they are harmless and can be used more safely for neutralizing antibodies and antiviral drugs detection.
对传染性强、致病性强的病毒，如SARS-CoV，Ebola病毒，狂犬病毒，H7N9等的研究具有极大的危险性，而且数量有限，而假病毒安全性好，并可以通过转染技术大量制备，为这些危险性高的病毒研究提供了方便。Research on highly infectious and pathogenic viruses, such as SARS-CoV, Ebola virus, rabies virus, H7N9, etc., is extremely dangerous, and the number is limited, while pseudoviruses are safe and can be transfected The mass preparation of technology provides convenience for the research of these highly dangerous viruses.
假病毒体系有多种，主要包括以慢病毒载体构建的假病毒，以病毒蛋白自组装形成的假病毒，以及以病毒基因改造、插入报告基因所形成的假病毒。慢病毒载体是以HIV-1为基础发展起来的，包含了包装、转染、稳定整合所需要的遗传信息，可提供所有转录并包装RNA到重组假病毒所需要的全部辅助蛋白，利用表达载体和包装质粒同时共转染细胞，在细胞中进行病毒包装，包装好的病毒颗粒分泌到细胞外培养基中。There are many kinds of pseudovirus systems, mainly including pseudoviruses constructed with lentiviral vectors, pseudoviruses formed by self-assembly of viral proteins, and pseudoviruses formed by genetic modification of viruses and insertion of reporter genes. The lentiviral vector is developed on the basis of HIV-1, contains the genetic information required for packaging, transfection, and stable integration, and can provide all the auxiliary proteins required for transcription and packaging of RNA to recombinant pseudoviruses, using expression vectors The cells are co-transfected with the packaging plasmid at the same time, the virus is packaged in the cell, and the packaged virus particles are secreted into the extracellular medium.
假病毒体系的特点是克服了传统方法的缺陷，使检测技术更安全、简便、快速、高通量，而且使一些本身很难培养的病毒也能采用培养的方法进行检测。The pseudovirus system is characterized by overcoming the shortcomings of traditional methods, making the detection technology safer, simpler, faster, and high-throughput, and enabling some viruses that are difficult to culture themselves to be detected by culture methods.
美国国立卫生研究院(NIH)提供了两个HIV-1为基础的质粒，分别是不携带Fluc基因的pSG3.Δenv和携带Fluc基因的NL4-3.Fluc.R-.E-。种辉辉、聂建辉等利用pSG3.Δenv建立了HIV假病毒检测中和抗体的方法，另外国内外很多研究人员利用NL4-3.Fluc.R-.E-构建了许多假病毒，例如SARS-CoV，Ebola病毒，狂犬病毒，流感病毒等。由于pSG3.Δenv不含报告基因，因此只能用于感染表达荧光素酶的TZM-bl等细胞，限制了假病毒的感染细胞范围；而NL4-3.Fluc.R-.E-可以表达荧光素酶，从而提供检测的信号，但是该质粒用到的启动子为HIV长末端重复序列(LTR)，表达效率较低。The National Institutes of Health (NIH) of the United States provides two HIV-1-based plasmids, pSG3.Δenv without the Fluc gene and NL4-3.Fluc.R-.E- with the Fluc gene. Zhong Huihui, Nie Jianhui, etc. used pSG3.Δenv to establish a method for HIV pseudovirus detection of neutralizing antibodies. In addition, many researchers at home and abroad used NL4-3.Fluc.R-.E- to construct many pseudoviruses, such as SARS-CoV , Ebola virus, rabies virus, influenza virus, etc. Since pSG3.Δenv does not contain a reporter gene, it can only be used to infect cells such as TZM-bl expressing luciferase, which limits the range of infected cells of pseudoviruses; while NL4-3.Fluc.R-.E- can express fluorescence primease, thereby providing a detection signal, but the promoter used in this plasmid is the HIV long terminal repeat (LTR), and the expression efficiency is low.
发明内容Contents of the invention
本发明提供了一种能够高效表达荧光素酶的假病毒包装载体及包装系统，具体包括以下几个方面。The invention provides a pseudovirus packaging vector and packaging system capable of highly expressing luciferase, specifically including the following aspects.
本发明第一方面涉及一种假病毒包装载体，其是在载体pSG3.Δenv中可操作地连接有荧光素酶基因和调节该基因转录的启动子。The first aspect of the present invention relates to a pseudovirus packaging vector, which is operably connected with a luciferase gene and a promoter that regulates the transcription of the gene in the vector pSG3.Δenv.
在本发明的一个实施方案中，其是在载体pSG3.Δenv中nef基因的上游可操作地连接有荧光素酶基因和调节该基因转录的启动子。In one embodiment of the present invention, a luciferase gene and a promoter regulating the transcription of the gene are operably linked upstream of the nef gene in the vector pSG3.Δenv.
在本发明的一个实施方案中，其是在载体pSG3.Δenv第11946位至14103之间可操作地连接有荧光素酶基因和调节该基因转录的启动子。In one embodiment of the present invention, a luciferase gene and a promoter regulating the transcription of the gene are operably linked between positions 11946 to 14103 of the vector pSG3.Δenv.
在本发明的一个具体实施方案中，其是在载体pSG3.Δenv第13967处可操作地连接有荧光素酶基因和调节该基因转录的启动子。In a specific embodiment of the present invention, a luciferase gene and a promoter regulating the transcription of the gene are operably linked at position 13967 of the vector pSG3.Δenv.
在本发明的一个实施方案中，其中所述的荧光素酶选自萤火虫荧光素酶、海肾荧光素酶。In one embodiment of the present invention, wherein said luciferase is selected from firefly luciferase and Renilla luciferase.
在本发明的一个具体实施方案中，所述萤火虫荧光素酶基因的序列如SEQ ID NO：3所示。In a specific embodiment of the present invention, the sequence of the firefly luciferase gene is shown in SEQ ID NO:3.
在本发明的一个实施方案中，其中所述的启动子为CMV启动子，优选地，所述CMV启动子的序列如SEQ ID NO：18所示。In one embodiment of the present invention, wherein the promoter is a CMV promoter, preferably, the sequence of the CMV promoter is shown in SEQ ID NO:18.
在本发明的一个实施方案中，其中所述荧光素酶基因和调节该基因转录的启动子的序列如SEQ ID NO：6所示。In one embodiment of the present invention, the sequence of the luciferase gene and the promoter regulating the transcription of the gene is shown in SEQ ID NO:6.
在本发明的一个具体实施方案中，所述假病毒包装载体具有如SEQ ID NO：7所示的序列。In a specific embodiment of the present invention, the pseudovirus packaging vector has the sequence shown in SEQ ID NO:7.
本发明另一方面涉及一种假病毒包装系统，其包含本发明第一方面任一项的假病毒包装载体和至少一种真核表达载体。Another aspect of the present invention relates to a pseudovirus packaging system, which comprises the pseudovirus packaging vector of any one of the first aspect of the present invention and at least one eukaryotic expression vector.
在本发明的实施方案中其中所述本发明第一方面任一项的假病毒包装载体作为假病毒包装的骨架载体，所述真核表达载体作为假病毒包装的外源基因表达载体。In an embodiment of the present invention, the pseudovirus packaging vector of any one of the first aspect of the present invention is used as a backbone vector for pseudovirus packaging, and the eukaryotic expression vector is used as an exogenous gene expression vector for pseudovirus packaging.
在本发明的一个实施方案中，其中所述的真核表达载体是将载体pcDNA3.1(+)中CMV启动子至T7启动子的序列替换为CAG启动子、LTR启动子或包含有人巨细胞病毒主要即刻早期蛋白基因(Human cytomegalovirus major immediate-early protein gene)增强子/启动子、人巨细胞病毒主要即刻早期蛋白基因外显子1和人巨细胞病毒主要即刻早期蛋白基因内含子1的序列。In one embodiment of the present invention, wherein the eukaryotic expression vector is to replace the sequence from the CMV promoter to the T7 promoter in the vector pcDNA3.1 (+) with a CAG promoter, an LTR promoter or a human giant cell Human cytomegalovirus major immediate-early protein gene (Human cytomegalovirus major immediate-early protein gene) enhancer/promoter, human cytomegalovirus major immediate-early protein gene exon 1 and human cytomegalovirus major immediate-early protein gene intron 1 sequence.
在本发明的一个实施方案中，所述pcDNA3.1(+)中CMV启动子至T7启动子的序列为第229-896位的序列。In one embodiment of the present invention, the sequence from the CMV promoter to the T7 promoter in the pcDNA3.1(+) is the sequence at positions 229-896.
在本发明的一个实施方案中，所述CAG启动子的序列如SEQ ID NO：10所示。In one embodiment of the present invention, the sequence of the CAG promoter is shown in SEQ ID NO:10.
在本发明的一个实施方案中，所述LTR启动子的序列如SEQ ID NO：13所示。In one embodiment of the present invention, the sequence of the LTR promoter is shown in SEQ ID NO:13.
在本发明的一个实施方案中，其中所述的包含有人巨细胞病毒主要即刻早期蛋白基因(Human cytomegalovirus major immediate-early protein gene)增强子/启动子、人巨细胞病毒主要即刻早期蛋白基因外显子1和人巨细胞病毒主要即刻早期蛋白基因内含子1的序列如SEQ ID NO：16所示。In one embodiment of the present invention, wherein said human cytomegalovirus major immediate-early protein gene (Human cytomegalovirus major immediate-early protein gene) enhancer/promoter, human cytomegalovirus major immediate-early protein gene external expression The sequences of intron 1 and human cytomegalovirus major immediate early protein gene intron 1 are shown in SEQ ID NO:16.
在本发明的一个实施方案中，所述真核表达载体具有如SEQ ID NO：17所示的序列。In one embodiment of the present invention, the eukaryotic expression vector has the sequence shown in SEQ ID NO:17.
在本发明的一个实施方案中，其中所述的真核表达载体含有外源基因，优选地，所述外源基因为病毒的膜蛋白基因。In one embodiment of the present invention, wherein the eukaryotic expression vector contains a foreign gene, preferably, the foreign gene is a membrane protein gene of a virus.
在本发明的一个实施方案中，所述病毒为有包膜病毒，优选地，所述有包膜病毒包括DNA病毒、RNA病毒和逆转录病毒，例如选自冠状病毒(例如SARS-CoV)，疱疹病毒，痘病毒，嗜肝病毒(如丙肝病毒)，丝状病毒(如Ebola病毒)，弹状病毒(例如狂犬病毒)，流感病毒，副粘病毒(如麻疹病毒、呼吸道合胞病毒)，黄病毒(例如登革病毒)，囊膜病毒，布尼亚病毒，逆转录病毒。In one embodiment of the present invention, the virus is an enveloped virus, preferably, the enveloped virus includes DNA viruses, RNA viruses and retroviruses, for example selected from coronaviruses (such as SARS-CoV), Herpesviruses, poxviruses, hepatotropic viruses (eg, hepatitis C virus), filoviruses (eg, Ebola virus), rhabdoviruses (eg, rabies virus), influenza viruses, paramyxoviruses (eg, measles virus, respiratory syncytial virus), Flaviviruses (eg dengue virus), enveloped viruses, bunyaviruses, retroviruses.
本发明还涉及一种真核表达载体，其是将载体pcDNA3.1(+)中CMV启动子至T7启动子的序列替换为CAG启动子、LTR启动子或包含有人巨细胞病毒主要即刻早期蛋白基因(Human cytomegalovirus major immediate-early protein gene)增强子/启动子、人巨细胞病毒主要即刻早期蛋白基因外显子1和人巨细胞病毒主要即刻早期蛋白基因内含子1的序列。The present invention also relates to a eukaryotic expression vector, which replaces the sequence from the CMV promoter to the T7 promoter in the carrier pcDNA3.1(+) with a CAG promoter, an LTR promoter or contains the main immediate early protein of human cytomegalovirus Gene (Human cytomegalovirus major immediate-early protein gene) enhancer/promoter, human cytomegalovirus major immediate-early protein gene exon 1 and human cytomegalovirus major immediate-early protein gene intron 1 sequence.
在本发明的一个实施方案中，所述pcDNA3.1(+)中CMV启动子至T7启动子的序列为第229-896位的序列。In one embodiment of the present invention, the sequence from the CMV promoter to the T7 promoter in the pcDNA3.1(+) is the sequence at positions 229-896.
在本发明的一个实施方案中，所述CAG启动子的序列如SEQ ID NO：10所示。In one embodiment of the present invention, the sequence of the CAG promoter is shown in SEQ ID NO:10.
在本发明的一个实施方案中，所述LTR启动子的序列如SEQ ID NO：13所示。In one embodiment of the present invention, the sequence of the LTR promoter is shown in SEQ ID NO:13.
在本发明的一个实施方案中，其中所述的包含有人巨细胞病毒主要即刻早期蛋白基因(Human cytomegalovirus major immediate-early protein gene)增强子/启动子、人巨细胞病毒主要即刻早期蛋白基因外显子1和人巨细胞病毒主要即刻早期蛋白基因内含子1的序列如SEQ ID NO：16所示。In one embodiment of the present invention, wherein said human cytomegalovirus major immediate-early protein gene (Human cytomegalovirus major immediate-early protein gene) enhancer/promoter, human cytomegalovirus major immediate-early protein gene external expression The sequences of intron 1 and human cytomegalovirus major immediate early protein gene intron 1 are shown in SEQ ID NO:16.
在本发明的一个具体实施方案中，所述真核表达载体具有如SEQ ID NO：17所示的序列。In a specific embodiment of the present invention, the eukaryotic expression vector has the sequence shown in SEQ ID NO:17.
在本发明的一个实施方案中，其还含有外源基因，优选地，所述外源基因为病毒的膜蛋白基因。In one embodiment of the present invention, it also contains a foreign gene, preferably, the foreign gene is a membrane protein gene of a virus.
在本发明的一个实施方案中，所述病毒为有包膜病毒，优选地，所述有包膜病毒包括DNA病毒、RNA病毒和逆转录病毒，例如选自冠状病毒(例如SARS-CoV)，疱疹病毒，痘病毒，嗜肝病毒(如丙肝病毒)，丝状病毒(如Ebola病毒)，弹状病毒(例如狂犬病毒)，流感病毒，副粘病毒(如麻疹病毒、呼吸道合胞病毒)，黄病毒(例如登革病毒)，囊膜病毒，布尼亚病毒，逆转录病毒。In one embodiment of the present invention, the virus is an enveloped virus, preferably, the enveloped virus includes DNA viruses, RNA viruses and retroviruses, for example selected from coronaviruses (such as SARS-CoV), Herpesviruses, poxviruses, hepatotropic viruses (eg, hepatitis C virus), filoviruses (eg, Ebola virus), rhabdoviruses (eg, rabies virus), influenza viruses, paramyxoviruses (eg, measles virus, respiratory syncytial virus), Flaviviruses (eg dengue virus), enveloped viruses, bunyaviruses, retroviruses.
本发明还涉及宿主细胞，其含有本发明第一方面任一项的假病毒包装载体、本发明任一项的假病毒包装系统或本发明任一项的真核表达载体。The present invention also relates to a host cell containing the pseudovirus packaging vector of any one of the first aspect of the present invention, the pseudovirus packaging system of any one of the present invention or the eukaryotic expression vector of any one of the present invention.
在本发明的一个实施方案中，所述细胞为真核细胞，例如为HEK293、HEK293T、HEK293FT等细胞。In one embodiment of the present invention, the cells are eukaryotic cells, such as HEK293, HEK293T, HEK293FT and other cells.
本发明还涉及假病毒，其由本发明第一方面任一项的假病毒包装载体、本发明任一项的假病毒包装系统、本发明任一项的真核表达载体或本发明任一项的宿主细胞制备得到。The present invention also relates to a pseudovirus, which is composed of the pseudovirus packaging vector of any one of the first aspect of the present invention, the pseudovirus packaging system of any one of the present invention, the eukaryotic expression vector of any one of the present invention or any one of the present invention The host cells are prepared.
本发明还涉及本发明任一项的假病毒包装载体、本发明任一项的假病毒包装系统、本发明任一项的真核表达载体或本发明任一项的宿主细胞在制备假病毒中的用途。The present invention also relates to any pseudovirus packaging vector of the present invention, any pseudovirus packaging system of the present invention, any eukaryotic expression vector of the present invention or any host cell of the present invention in preparing pseudoviruses the use of.
在本发明的一个实施方案中，其中所述的假病毒为有包膜病毒的假病毒，所述有包膜病毒包括DNA病毒、RNA病毒和逆转录病毒，例如选自冠状病毒(例如SARS-CoV)，疱疹病毒，痘病毒，嗜肝病毒(如丙肝病毒)，丝状病毒(如Ebola病毒)，弹状病毒(例如狂犬病毒)，流感病毒，副粘病毒(如麻疹病毒、呼吸道合胞病毒)，黄病毒(例如登革病毒)，囊膜病毒，布尼亚病毒，逆转录病毒。In one embodiment of the present invention, wherein said pseudovirus is a pseudovirus of enveloped virus, said enveloped virus includes DNA virus, RNA virus and retrovirus, for example selected from coronavirus (such as SARS- CoV), herpes virus, pox virus, hepatotropic virus (eg, hepatitis C virus), filovirus (eg, Ebola virus), rhabdovirus (eg, rabies virus), influenza virus, paramyxovirus (eg, measles virus, respiratory syncytial virus) viruses), flaviviruses (e.g. dengue virus), enveloped viruses, bunyaviruses, retroviruses.
本发明还涉及一种假病毒的制备方法，其包括以下步骤：The present invention also relates to a kind of preparation method of pseudovirus, it comprises the following steps:
将本发明第一方面任一项的假病毒包装载体和本发明任一项的真核表达载体共转染真核细胞，收集细胞培养上清，即得到假病毒。Co-transfect eukaryotic cells with the pseudovirus packaging vector of any one of the first aspect of the present invention and the eukaryotic expression vector of any one of the present invention, and collect the cell culture supernatant to obtain the pseudovirus.
在本发明的实施方案中，所述真核细胞例如选自HEK293、HEK293T、HEK293FT等细胞。In an embodiment of the present invention, the eukaryotic cells are, for example, selected from HEK293, HEK293T, HEK293FT and other cells.
在本发明的一个实施方案中，提供了一种携带萤火虫荧光素酶报告基因的新的慢病毒载体pSG3.Δenv.Fluc，它是双链环状DNA，长度为16.4kb左右。利用pSG3.Δenv的HpaI酶切位点，设计In-Fusion连接引物，PCR扩增荧光素酶基因，用In-Fusion HD EnzymePremix将PCR产物和酶切载体，连接得到pSG3.Δenv.Fluc质粒。In one embodiment of the present invention, a new lentiviral vector pSG3.Δenv.Fluc carrying a firefly luciferase reporter gene is provided, which is a double-stranded circular DNA with a length of about 16.4kb. Using the HpaI restriction site of pSG3.Δenv, design In-Fusion ligation primers, PCR amplify the luciferase gene, use In-Fusion HD EnzymePremix to digest the PCR product and the vector, and connect to obtain the pSG3.Δenv.Fluc plasmid.
在本发明的一个实施方案中，提供了一种新的慢病毒载体pSG3.Δenv.cmvFluc，它是双链环状DNA，长度为17.1kb左右，其是在所得载体pSG3.Δenv.Fluc的基础上添加CMV启动子到荧光素酶基因上游。具体地，利用pSG3.Δenv的HpaI酶切位点，设计In-Fusion连接引物，从带有荧光素酶基因的pcDNA3.1质粒中PCR扩增带有CMV启动子的荧光素酶基因，用In-Fusion HD Enzyme Premix将PCR产物和酶切载体，连接得到pSG3.Δenv.cmvFluc质粒。In one embodiment of the present invention, a new lentiviral vector pSG3.Δenv.cmvFluc is provided, which is a double-stranded circular DNA with a length of about 17.1kb, which is the basis of the obtained vector pSG3.Δenv.Fluc Add the CMV promoter upstream of the luciferase gene. Specifically, use the HpaI restriction site of pSG3.Δenv to design In-Fusion connection primers, PCR amplify the luciferase gene with the CMV promoter from the pcDNA3.1 plasmid with the luciferase gene, and use In -Fusion HD Enzyme Premix The PCR product and the vector were ligated to obtain the pSG3.Δenv.cmvFluc plasmid.
在本发明的一个实施方案中，提供了一种新的真核表达质粒pCAG3.1，它是双链环状DNA，长度为6.5kb左右。其是在pcDNA3.1+表达载体基础上，用CAG(CMV early enhancerchickenβactin)启动子替换掉CMV启动子。根据pcDNA3.1+的序列，利用MluI和NheI酶切位点，设计In-Fusion连接引物，PCR扩增得到CAG启动子目的片段，用In-Fusion HD EnzymePremix将PCR产物和酶切载体，连接得到pCAG3.1质粒。In one embodiment of the present invention, a new eukaryotic expression plasmid pCAG3.1 is provided, which is a double-stranded circular DNA with a length of about 6.5 kb. It replaces the CMV promoter with the CAG (CMV early enhancer chicken βactin) promoter on the basis of the pcDNA3.1+ expression vector. According to the sequence of pcDNA3.1+, use MluI and NheI restriction sites to design In-Fusion ligation primers, PCR amplify to obtain the target fragment of CAG promoter, and use In-Fusion HD EnzymePremix to connect the PCR product and restriction vector to obtain pCAG3.1 plasmid.
在本发明的一个实施方案中，提供了一种新的真核表达质粒pLTR3.1，它是双链环状DNA，长度为5.6kb左右。考虑假病毒制备需将真核表达质粒和HIV骨架质粒同时转染293T细胞，于是将HIV LTR启动子替换到pcDNA3.1+质粒，骨架质粒在细胞内表达调控蛋白可以作用于LTR，从而提高LTR下游基因的表达量。根据pcDNA3.1+的序列，利用MluI和NheI酶切位点，设计In-Fusion连接引物，PCR扩增得到HIV LTR启动子目的片段，用In-Fusion HDEnzyme Premix将PCR产物和酶切载体，连接得到pLTR3.1质粒。In one embodiment of the present invention, a new eukaryotic expression plasmid pLTR3.1 is provided, which is a double-stranded circular DNA with a length of about 5.6 kb. Considering that pseudovirus preparation needs to transfect 293T cells with eukaryotic expression plasmid and HIV backbone plasmid at the same time, the HIV LTR promoter is replaced with pcDNA3.1+ plasmid, and the regulatory protein expressed by the backbone plasmid in the cell can act on LTR, thereby increasing LTR The expression level of downstream genes. According to the sequence of pcDNA3.1+, use MluI and NheI restriction sites to design In-Fusion ligation primers, PCR amplify the target fragment of HIV LTR promoter, and use In-Fusion HDEnzyme Premix to connect the PCR product and restriction vector to ligate The pLTR3.1 plasmid was obtained.
在本发明的一个实施方案中，提供了一种新的真核表达质粒pCMV3.1，它是双链环状DNA，长度为6.3kb左右。在pcDNA3.1+表达载体基础上，将CMV增强子/启动子、人巨细胞病毒主要即刻早期蛋白基因外显子1和人巨细胞病毒主要即刻早期蛋白基因内含子1替换原来的CMV启动子。根据pcDNA3.1+的序列，利用MluI和NheI酶切位点，设计In-Fusion连接引物，PCR扩增得到包括CMV增强子/启动子、人巨细胞病毒主要即刻早期蛋白基因外显子1和人巨细胞病毒主要即刻早期蛋白基因内含子1的目的片段，用In-Fusion HD EnzymePremix将PCR产物和酶切载体，连接得到pCMV3.1质粒。In one embodiment of the present invention, a new eukaryotic expression plasmid pCMV3.1 is provided, which is a double-stranded circular DNA with a length of about 6.3kb. On the basis of pcDNA3.1+ expression vector, replace the original CMV promoter with CMV enhancer/promoter, human cytomegalovirus major immediate early protein gene exon 1 and human cytomegalovirus major immediate early protein gene intron 1 son. According to the sequence of pcDNA3.1+, MluI and NheI restriction sites were used to design In-Fusion connection primers, and PCR amplification included CMV enhancer/promoter, human cytomegalovirus major immediate early protein gene exon 1 and The target fragment of intron 1 of the major immediate early protein gene of human cytomegalovirus was ligated with the PCR product and the vector by In-Fusion HD EnzymePremix to obtain the pCMV3.1 plasmid.
发明的有益效果Beneficial Effects of the Invention
本发明的假病毒包装系统能够高效、便利地制备假病毒，为病毒的研究和疫苗的制备等提供了有力的工具。The pseudovirus packaging system of the invention can efficiently and conveniently prepare pseudoviruses, and provides a powerful tool for virus research and vaccine preparation.
附图说明Description of drawings
图1骨架质粒pSG3.Δenv.Fluc的构建示意图。Figure 1 Schematic diagram of the construction of the backbone plasmid pSG3.Δenv.Fluc.
图2骨架质粒pSG3.Δenv.cmvFluc的构建示意图。Fig. 2 Schematic diagram of the construction of backbone plasmid pSG3.Δenv.cmvFluc.
图3真核表达质粒pCAG3.1的构建示意图。Fig. 3 Schematic diagram of the construction of eukaryotic expression plasmid pCAG3.1.
图4真核表达质粒pLTR3.1的构建示意图。Fig. 4 Schematic diagram of the construction of eukaryotic expression plasmid pLTR3.1.
图5真核表达质粒pCMV3.1的构建示意图。Fig. 5 Schematic diagram of the construction of eukaryotic expression plasmid pCMV3.1.
图6骨架质粒表达荧光素酶效率的比较。Figure 6 Comparison of luciferase expression efficiency of backbone plasmids.
图7真核质粒表达荧光素酶效率的比较。Fig. 7 Comparison of eukaryotic plasmid expression luciferase efficiency.
图8双质粒共转染比例的确定。Fig. 8 Determination of double plasmid co-transfection ratio.
图9不同真核表达质粒和骨架质粒间包装假病毒的效果，其中每组图从左至右依次为pCAG-CVS、pLTR-CVS、pCMV-CVS。Figure 9 The effect of packaging pseudoviruses between different eukaryotic expression plasmids and backbone plasmids, wherein each group of pictures is pCAG-CVS, pLTR-CVS, pCMV-CVS from left to right.
图10不同真核表达质粒和骨架质粒间包装假病毒的效果，其中每组图从左至右依次为pCAG-CTN、pLTR-CTN、pCMV-CTN。Figure 10 Effects of packaging pseudoviruses between different eukaryotic expression plasmids and backbone plasmids, wherein each group of pictures is pCAG-CTN, pLTR-CTN, pCMV-CTN from left to right.
图11狂犬病人免疫球蛋白对狂犬街毒株假病毒的抑制效果。Fig. 11 Inhibitory effect of rabies patient immunoglobulin on rabies street strain pseudovirus.
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。实施例中涉及的PCR、酶切、连接，或感受态细胞制备、转化等分子生物学方法均参考国内外相关文献。Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products. For the molecular biology methods such as PCR, enzyme digestion, ligation, or competent cell preparation and transformation involved in the examples, refer to relevant domestic and foreign literatures.
实施例1骨架质粒pSG3.Δenv.Fluc的构建Example 1 Construction of Backbone Plasmid pSG3.Δenv.Fluc
步骤1.用限制性内切酶HpaI对质粒pSG3.Δenv(NIH惠赠)进行完全酶切，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收酶切后产生的14.7kb的片段。Step 1. Plasmid pSG3.Δenv (gifted by NIH) was completely digested with restriction endonuclease HpaI, electrophoresed on agarose gel, and the 14.7 kb fragment generated after digestion was recovered with the gel recovery kit of Qiagen company.
步骤2.用Takara公司的Primestar DNA聚合酶PCR扩增pcDNA3.1-Fluc中的(本实验室构建，将Fluc基因插入pcDNA3.1的BamH1和XhoI之间得到)荧光素酶基因，扩增引物为：Step 2. use the Primestar DNA polymerase PCR amplification of Takara company in pcDNA3.1-Fluc (this laboratory constructs, Fluc gene is inserted between BamH1 and XhoI of pcDNA3.1 and obtains) luciferase gene, amplification primer for:
p1F：p1F:
AAGAATAGTGCTGTTGCCACCATGGAAGATGCCAAAAACAT(SEQ ID NO：1)AAGAATAGTGCTGTTGCCACCATGGAAGATGCCAAAAACAT (SEQ ID NO: 1)
p1R：p1R:
GACATTAAGCAAGTTTTATTACACGGCGATCTTGCCGCCCT(SEQ ID NO：2)GACATTAAGCAAGTTTTATTACACGGCGATCTTGCCGCCCT (SEQ ID NO: 2)
98℃1min预变性，98℃10s，68℃2min延伸，共35个循环，最后72℃延长10min。得到1.7kb的目的片段，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收。得到的荧光素酶目的片段的序列如SEQ ID NO：3所示。Pre-denaturation at 98°C for 1min, extension at 98°C for 10s, extension at 68°C for 2min, a total of 35 cycles, and a final extension at 72°C for 10min. The target fragment of 1.7 kb was obtained, electrophoresed on agarose gel, and recovered with a gel extraction kit from Qiagen Company. The sequence of the obtained luciferase target fragment is shown in SEQ ID NO:3.
gccaccatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaataa(SEQ ID NO：3)gccaccatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttcc acctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaataa(SEQ ID NO：3)
步骤3.将步骤1得到的线性化载体及步骤2得到的荧光素酶目的片段混匀，加入In-Fusion HD Enzyme Premix(购自Takara)，于PCR仪中50℃反应15min。Step 3. Mix the linearized vector obtained in step 1 and the target luciferase fragment obtained in step 2, add In-Fusion HD Enzyme Premix (purchased from Takara), and react in a PCR machine at 50°C for 15 minutes.
步骤4.将步骤3的连接混合液全部直接转化E.coil Stbl2感受态细胞，冰浴30min后，42℃热激1min，30℃复苏90min，涂布于含100μg/ml氨苄霉素的LB平板。挑取单菌落，提取DNA并酶切鉴定，确定1.7kb的荧光素酶基因插入成功，该质粒命名为pSG3.Δenv.Fluc。质粒构建的示意图如图1所示。Step 4. Transform all the ligation mixture in step 3 directly into E.coil Stbl2 competent cells. After 30 minutes in ice bath, heat shock at 42°C for 1 minute, recover at 30°C for 90 minutes, and spread on LB plates containing 100 μg/ml ampicillin . A single colony was picked, DNA was extracted and identified by enzyme digestion, and it was confirmed that the 1.7kb luciferase gene was successfully inserted, and the plasmid was named pSG3.Δenv.Fluc. The schematic diagram of plasmid construction is shown in Figure 1.
实施例2骨架质粒pSG3.Δenv.cmvFluc的构建Example 2 Construction of Backbone Plasmid pSG3.Δenv.cmvFluc
步骤1.用限制性内切酶HpaI对质粒pSG3.Δenv进行完全酶切，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收酶切后产生的14.7kb的片段。Step 1. The plasmid pSG3.Δenv was completely digested with the restriction endonuclease HpaI, electrophoresed on agarose gel, and the 14.7 kb fragment produced after the digest was recovered with the gel recovery kit of Qiagen company.
步骤2.用Takara公司的Primestar DNA聚合酶PCR扩增pcDNA3.1-Fluc中上游携带CMV启动子的荧光素酶基因，扩增引物为：Step 2. Use the Primestar DNA polymerase PCR of Takara Company to amplify the luciferase gene carrying the CMV promoter in the upstream of pcDNA3.1-Fluc, and the amplification primers are:
p3F:AAGAATAGTGCTGTTTGCTTCGCGATGTACGGGCCAGA(SEQ ID NO：4)p3F:AAGAATAGTGCTGTTTGCTTCGCGATGTACGGGCCAGA (SEQ ID NO: 4)
p3R:GACATTAAGCAAGTTTTATTACACGGCGATCTTGCCGCCCT(SEQ ID NO：5)p3R: GACATTAAGCAAGTTTTTATTACACGGCGATCTTGCCGCCCT (SEQ ID NO: 5)
98℃1min预变性，98℃10s，68℃2.5min延伸，共35个循环，最后72℃延长10min。得到2.3kb的目的片段，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收。得到的携带CMV启动子的荧光素酶目的片段的序列如SEQ ID NO：6所示。Pre-denaturation at 98°C for 1min, extension at 98°C for 10s, extension at 68°C for 2.5min, a total of 35 cycles, and a final extension at 72°C for 10min. The target fragment of 2.3 kb was obtained, electrophoresed on agarose gel, and recovered with a gel extraction kit from Qiagen Company. The sequence of the luciferase target fragment carrying the CMV promoter is shown in SEQ ID NO:6.
tgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggccaccatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaataa(SEQ ID NO：6)。tgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggccaccatggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttctt catgcccgtgttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaaggacggctggctgcacagcggcgac atcgcctactgggacgaggacgagcacttcttcatcgtggaccggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacaccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtgtaataa(SEQ ID NO：6)。
其中CMV启动子的序列如下所示：Wherein the sequence of the CMV promoter is as follows:
tgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggccacc(SEQ ID NO：18)所示。tgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccgagctcggccacc(SEQ ID NO：18)所示。
步骤3.将步骤1得到的线性化载体及步骤2得到的携带CMV启动子的荧光素酶目的片段混匀，加入In-Fusion HD Enzyme Premix，于PCR仪中50℃反应15min。Step 3. Mix the linearized vector obtained in step 1 and the target luciferase fragment carrying the CMV promoter obtained in step 2, add In-Fusion HD Enzyme Premix, and react in a PCR machine at 50°C for 15 minutes.
步骤4.将步骤3的连接混合液全部直接转化E.coil Stbl2感受态细胞，冰浴30min后，42℃热激1min，30℃复苏90min，涂布于含100μg/ml氨苄霉素的LB平板。挑取单菌落，提取DNA并酶切鉴定，确定2.3kb的目的基因插入成功，该质粒命名为pSG3.Δenv.cmvFluc。质粒构建的示意图如图2所示，其完整序列如SEQ ID NO：7所示。Step 4. Transform all the ligation mixture in step 3 directly into E.coil Stbl2 competent cells. After 30 minutes in ice bath, heat shock at 42°C for 1 minute, recover at 30°C for 90 minutes, and spread on LB plates containing 100 μg/ml ampicillin . A single colony was picked, DNA was extracted and identified by enzyme digestion, and it was confirmed that the 2.3kb target gene was successfully inserted, and the plasmid was named pSG3.Δenv.cmvFluc. The schematic diagram of plasmid construction is shown in Figure 2, and its complete sequence is shown in SEQ ID NO:7.
实施例3真核表达质粒pCAG3.1的构建Example 3 Construction of eukaryotic expression plasmid pCAG3.1
步骤1.用限制性内切酶MluI和NheI对质粒pcDNA3.1+(购自Invitrogen，货号V790-20，序列信息参见http://www.lifetechnologies.com/order/catalog/product/V79020？ICID＝search-product)进行完全酶切，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收酶切后产生的4.7kb的片段。Step 1. Plasmid pcDNA3.1+ (purchased from Invitrogen, Cat. No. V790-20, see http://www.lifetechnologies.com/order/catalog/product/V79020?ICID for sequence information) with restriction endonucleases MluI and NheI =search-product) for complete digestion, agarose gel electrophoresis, and the 4.7 kb fragment produced after digestion was recovered with the gel recovery kit of Qiagen Company.
步骤2.用Takara公司的Primestar DNA聚合酶PCR扩增CAG启动子序列(北京百奥赛图惠赠)，扩增引物为：Step 2. Use Takara's Primestar DNA polymerase to PCR amplify the CAG promoter sequence (gifted by Beijing Biocytogen), and the amplification primers are:
p5F:GGCCAGATATACGCGCTAGTTATTAATAGTAATCAATTACG(SEQ ID NO：8)p5F: GGCCAGATACGCGCGCTAGTTATTAATAGTAATCAATTACG (SEQ ID NO: 8)
p5R:AAGTTTAAACGCTAGAATTCTTTGCCAAAATGATGAGAC(SEQ ID NO：9)p5R: AAGTTTAAACGCTAGAATTCTTTGCCAAAATGATGAGAC (SEQ ID NO: 9)
98℃1min预变性，98℃10s，68℃2min延伸，共35个循环，最后72℃延长10min。得到1.7kb的目的片段，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收。得到的CAG启动子的序列如SEQ ID NO：10所示。Pre-denaturation at 98°C for 1min, extension at 98°C for 10s, extension at 68°C for 2min, a total of 35 cycles, and a final extension at 72°C for 10min. The target fragment of 1.7 kb was obtained, electrophoresed on agarose gel, and recovered with a gel extraction kit from Qiagen Company. The sequence of the obtained CAG promoter is shown in SEQ ID NO:10.
ctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcgtcggtcgggctgcaaccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaatt(SEQ ID NO：10)ctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcg gccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcgtcggtcgggctgcaaccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaatt(SEQ ID NO：10)
步骤3.将步骤1得到的线性化载体及步骤2得到的CAG启动子目的片段混匀，加入In-Fusion HD Enzyme Premix，于PCR仪中50℃反应15min。Step 3. Mix the linearized vector obtained in step 1 and the target fragment of the CAG promoter obtained in step 2, add In-Fusion HD Enzyme Premix, and react in a PCR machine at 50°C for 15 minutes.
步骤4.将步骤3的连接混合液全部直接转化E.coil Stbl2感受态细胞，冰浴30min后，42℃热激1min，30℃复苏90min，涂布于含100μg/ml氨苄霉素的LB平板。挑取单菌落，提取DNA并酶切鉴定，确定1.7kb的目的基因插入成功，该质粒命名为pCAG3.1。质粒构建的示意图如图3所示。Step 4. Transform all the ligation mixture in step 3 directly into E.coil Stbl2 competent cells. After 30 minutes in ice bath, heat shock at 42°C for 1 minute, recover at 30°C for 90 minutes, and spread on LB plates containing 100 μg/ml ampicillin . A single colony was picked, DNA was extracted and identified by enzyme digestion, and it was confirmed that the 1.7kb target gene was successfully inserted, and the plasmid was named pCAG3.1. The schematic diagram of plasmid construction is shown in Figure 3.
实施例4真核表达质粒pLTR3.1的构建Example 4 Construction of eukaryotic expression plasmid pLTR3.1
步骤1.用限制性内切酶MluI和NheI对质粒pcDNA3.1+进行完全酶切，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收酶切后产生的4.7kb的片段。Step 1. Completely digest the plasmid pcDNA3.1+ with restriction endonucleases MluI and NheI, perform agarose gel electrophoresis, and recover the 4.7 kb fragment generated after the digestion with a gel recovery kit from Qiagen.
步骤2.用Takara公司的Primestar DNA聚合酶PCR扩增NL4-3.Fluc.R-.E-质粒中的(NIH惠赠)HIV LTR序列，扩增引物为：Step 2. use the Primestar DNA polymerase PCR amplification NL4-3.Fluc.R-.E-plasmid (NIH gift) HIV LTR sequence of Takara company, amplification primer is:
p7F:GGCCAGATATACGCG GCAGTATCTCGAGACCTAGAA(SEQ ID NO：11)p7F:GGCCAGATACGCGGCAGTATCTCGAGACCTAGAA (SEQ ID NO: 11)
p7R:AAGTTTAAACGCTAG TACCTCCTGGGTGCTAGAGA(SEQ ID NO：12)p7R: AAGTTTAAACGCTAG TACCTCCTGGGTGCTAGAGA (SEQ ID NO: 12)
98℃1min预变性，98℃10s，68℃1min延伸，共35个循环，最后72℃延长10min。得到900bp的目的片段，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收。得到的LTR序列如SEQ ID NO：13所示。Pre-denaturation at 98°C for 1min, extension at 98°C for 10s, extension at 68°C for 1min, a total of 35 cycles, and a final extension at 72°C for 10min. The target fragment of 900bp was obtained, electrophoresed on agarose gel, and recovered with a gel recovery kit from Qiagen. The resulting LTR sequence is shown in SEQ ID NO:13.
Gcagtatctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaacaatgctgcttgtgcctggctagaagcacaagaggaggaagaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattggcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatggaatggatgaccctgagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgacatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatgctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcacccaggaggta(SEQID NO：13)Gcagtatctcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaacaatgctgcttgtgcctggctagaagcacaagaggaggaagaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattggcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatggaatggatgaccctgagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgacatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctgggcgggactggggagtggcgagccctcagatgctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcacccaggaggta(SEQID NO：13)
步骤3.将步骤1得到的线性化载体及步骤2得到的LTR序列目的片段混匀，加入In-Fusion HD Enzyme Premix，于PCR仪中50℃反应15min。Step 3. Mix the linearized vector obtained in step 1 and the target fragment of the LTR sequence obtained in step 2, add In-Fusion HD Enzyme Premix, and react in a PCR machine at 50°C for 15 minutes.
步骤4.将步骤3的连接混合液全部直接转化E.coil Stbl2感受态细胞，冰浴30min后，42℃热激1min，30℃复苏90min，涂布于含100μg/ml氨苄霉素的LB平板。挑取单菌落，提取DNA并酶切鉴定，确定900bp的目的基因插入成功，该质粒命名为pLTR3.1。质粒构建的示意图如图4所示。Step 4. Transform all the ligation mixture in step 3 directly into E.coil Stbl2 competent cells. After 30 minutes in ice bath, heat shock at 42°C for 1 minute, recover at 30°C for 90 minutes, and spread on LB plates containing 100 μg/ml ampicillin . A single colony was picked, DNA was extracted and identified by enzyme digestion, and it was confirmed that the 900bp target gene was successfully inserted, and the plasmid was named pLTR3.1. The schematic diagram of plasmid construction is shown in Figure 4.
实施例5真核表达质粒pCMV3.1的构建Example 5 Construction of eukaryotic expression plasmid pCMV3.1
步骤1.用限制性内切酶MluI和NheI对质粒pcDNA3.1+进行完全酶切，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收酶切后产生的4.7kb的片段。Step 1. Completely digest the plasmid pcDNA3.1+ with restriction endonucleases MluI and NheI, perform agarose gel electrophoresis, and recover the 4.7 kb fragment generated after the digestion with a gel recovery kit from Qiagen.
步骤2.用Takara公司的Primestar DNA聚合酶PCR扩增pDRVISV1.0载体(参见CN1560259A，中国疾病预防控制中心性病艾滋病预防控制中心邵一鸣教授惠赠)中的人巨细胞病毒主要即刻早期蛋白基因(Human cytomegalovirus major immediate-earlyprotein gene)增强子/启动子、人巨细胞病毒主要即刻早期蛋白基因外显子1、人巨细胞病毒主要即刻早期蛋白基因内含子1序列(CMV IE 5’UTR～CMV IE intron)，扩增引物为：Step 2. use the Primestar DNA polymerase PCR amplification of Takara company to amplify the human cytomegalovirus main immediate early protein gene (Human cytomegalovirus major immediate-early protein gene) enhancer/promoter, human cytomegalovirus major immediate early protein gene exon 1, human cytomegalovirus major immediate early protein gene intron 1 sequence (CMV IE 5'UTR～CMV IE intron ), the amplification primers are:
P6F:GGCCAGATATACGCGACCGCCATGTTGACATTGATT(SEQ ID NO：14)P6F: GGCCAGATACGCGACCGCCATGTTGACATTGATT (SEQ ID NO: 14)
P6R:AAGTTTAAACGCTAGCGTGTCGACGACGGTGACTGC(SEQ ID NO：15)P6R: AAGTTTAAACGCTAGCGTGTCGACGACGGTGACTGC (SEQ ID NO: 15)
98℃1min预变性，98℃10s，68℃2min延伸，共35个循环，最后72℃延长10min。得到1.7kb的目的片段，琼脂糖凝胶电泳，用Qiagen公司的胶回收试剂盒回收。得到的CMV启动子序列如SEQ ID NO：16所示。Pre-denaturation at 98°C for 1min, extension at 98°C for 10s, extension at 68°C for 2min, a total of 35 cycles, and a final extension at 72°C for 10min. The target fragment of 1.7 kb was obtained, electrophoresed on agarose gel, and recovered with a gel extraction kit from Qiagen Company. The obtained CMV promoter sequence is shown in SEQ ID NO:16.
accgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagactctataggcacacccctttggctcttatgcatgctatactgtttttggcttggggcctatacacccccgcttccttatgctataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactcccctattggtgacgatactttccattactaatccataacatggctctttgccacaactatctctattggctatatgccaatactctgtccttcagagactgacacggactctgtatttttacaggatggggtcccatttattatttacaaattcacatatacaacaacgccgtcccccgtgcccgcagtttttattaaacatagcgtgggatctccacgcgaatctcgggtacgtgttccggacatgggctcttctccggtagcggcggagcttccacatccgagccctggtcccatgcctccagcggctcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggcacagcacaatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtctgaaaatgagcgtggagattgggctcgcacggctgacgcagatggaagacttaaggcagcggcagaagaagatgcaggcagctgagttgttgtattctgataagagtcagaggtaactcccgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacg(SEQ ID NO：16)accgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccgcggccgggaacggtgcattggaacgcggattccccgtgccaagagtgacgtaagtaccgcctatagactctataggcacacccctttggctcttatgcatgctatactgtttttggcttggggcctatacacccccgcttccttatgctataggtgatggtatagcttagcctataggtgtgggttattgaccattattgaccactcccctattggtgacgatactttccattactaatccataacatggctctttgccacaactatctctattggctatatgccaatactctgtccttcagagactgacacggactct gtatttttacaggatggggtcccatttattatttacaaattcacatatacaacaacgccgtcccccgtgcccgcagtttttattaaacatagcgtgggatctccacgcgaatctcgggtacgtgttccggacatgggctcttctccggtagcggcggagcttccacatccgagccctggtcccatgcctccagcggctcatggtcgctcggcagctccttgctcctaacagtggaggccagacttaggcacagcacaatgcccaccaccaccagtgtgccgcacaaggccgtggcggtagggtatgtgtctgaaaatgagcgtggagattgggctcgcacggctgacgcagatggaagacttaaggcagcggcagaagaagatgcaggcagctgagttgttgtattctgataagagtcagaggtaactcccgttgcggtgctgttaacggtggagggcagtgtagtctgagcagtactcgttgctgccgcgcgcgccaccagacataatagctgacagactaacagactgttcctttccatgggtcttttctgcagtcaccgtcgtcgacacg(SEQ ID NO：16)
步骤3.将步骤1得到线性化载体及步骤2得到的CMV启动子目的片段混匀，加入In-Fusion HD Enzyme Premix，于PCR仪中50℃反应15min。Step 3. Mix the linearized vector obtained in step 1 and the target fragment of the CMV promoter obtained in step 2, add In-Fusion HD Enzyme Premix, and react in a PCR machine at 50°C for 15 minutes.
步骤4.将步骤3的连接混合液全部直接转化E.coil Stbl2感受态细胞，冰浴30min后，42℃热激1min，30℃复苏90min，涂布于含100μg/ml氨苄霉素的LB平板。挑取单菌落，提取DNA并酶切鉴定，确定1.7kb的目的基因插入成功，该质粒命名为pCMV3.1。质粒构建的示意图如图5所示，其完整序列如SEQ ID NO：17所示。Step 4. Transform all the ligation mixture in step 3 directly into E.coil Stbl2 competent cells. After 30 minutes in ice bath, heat shock at 42°C for 1 minute, recover at 30°C for 90 minutes, and spread on LB plates containing 100 μg/ml ampicillin . A single colony was picked, DNA was extracted and identified by enzyme digestion, and it was confirmed that the 1.7kb target gene was successfully inserted, and the plasmid was named pCMV3.1. The schematic diagram of plasmid construction is shown in Figure 5, and its complete sequence is shown in SEQ ID NO:17.
实施例6骨架质粒表达荧光素酶效率的比较The comparison of embodiment 6 backbone plasmid expression luciferase efficiency
根据lipofectamine2000转染试剂的说明书，将相同量的骨架质粒转染293T细胞，48h后，细胞吹打均匀，稀释后，使用Promega’s Bright-GloTM萤光素酶检测试剂盒，检测化学发光值，结果见图6。其中，NL4-3.Fluc.R-.E-、pSG3Δenv.Fluc发光结果无显著差异，而pSG3Δenv.cmvFluc的发光效果明显优于前两者。According to the instructions of lipofectamine2000 transfection reagent, the same amount of backbone plasmid was transfected into 293T cells. After 48 hours, the cells were blown evenly. After dilution, the chemiluminescence value was detected using Promega's Bright-GloTM luciferase detection kit. Image 6. Among them, there was no significant difference in the luminescence results of NL4-3.Fluc.R-.E- and pSG3Δenv.Fluc, but the luminescence effect of pSG3Δenv.cmvFluc was significantly better than the former two.
实施例7真核质粒表达荧光素酶效率的比较The comparison of embodiment 7 eukaryotic plasmid expression luciferase efficiency
为检测构建的pCMV3.1、pCAG3.1、pLTR3.1真核表达质粒的表达效率，将荧光素酶基因分别构建到pCMV3.1、pCAG3.1、pLTR3.1质粒中，得到pCMV-Fluc、pCAG-Fluc、pLTR-Fluc质粒。根据lipofectamine2000转染试剂的说明书，将相同量质粒转染293T细胞，48h后，细胞吹打均匀，稀释后，使用Promega’s Bright-GloTM萤光素酶检测试剂盒，检测化学发光值，结果见图7。其中，pCMV-Fluc质粒的荧光值最高，初步确定pCMV3.1质粒具有较高的表达效率。In order to detect the expression efficiency of the constructed pCMV3.1, pCAG3.1, pLTR3.1 eukaryotic expression plasmids, the luciferase gene was respectively constructed into pCMV3.1, pCAG3.1, pLTR3.1 plasmids to obtain pCMV-Fluc, pCAG-Fluc, pLTR-Fluc plasmids. According to the instructions of lipofectamine2000 transfection reagent, the same amount of plasmid was transfected into 293T cells. After 48 hours, the cells were pipetted evenly. After dilution, the chemiluminescence value was detected using Promega's Bright-GloTM luciferase detection kit. The results are shown in Figure 7 . Among them, the pCMV-Fluc plasmid has the highest fluorescence value, and it is preliminarily determined that the pCMV3.1 plasmid has a higher expression efficiency.
实施例8双质粒共转染比例的确定Determination of Example 8 Double-plasmid Co-transfection Ratio
将CVS-11膜蛋白(GenBank ID:GQ918139)构建到pcDNA3.1+质粒中，得到pcDNA3.1-cvs质粒。将包膜质粒pcDNA3.1-cvs和HIV骨架质粒(pSG3Δenv.Fluc)按照一定质量比(2：1、1.5：1、1：1、1：1.5、1：2、1：3)分别转染293T细胞，48h左右收集培养基上清，将收集的假病毒感染293T细胞，48h后检测RLU。结果如图8所示。The CVS-11 membrane protein (GenBank ID: GQ918139) was constructed into the pcDNA3.1+ plasmid to obtain the pcDNA3.1-cvs plasmid. The envelope plasmid pcDNA3.1-cvs and the HIV backbone plasmid (pSG3Δenv.Fluc) were transfected according to a certain mass ratio (2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3) For 293T cells, collect the supernatant of the medium at about 48 hours, infect the 293T cells with the collected pseudovirus, and detect RLU after 48 hours. The result is shown in Figure 8.
从图中可以看出，膜蛋白表达质粒和骨架质粒质量比为1：2时，可以得倒最佳的假病毒包装效果。It can be seen from the figure that when the mass ratio of the membrane protein expression plasmid and the backbone plasmid is 1:2, the best pseudovirus packaging effect can be obtained.
实施例9狂犬假病毒的构建The construction of embodiment 9 rabies pseudovirus
分别将CVS-11膜蛋白(GenBank ID:GQ918139)和CTN-1膜蛋白(GenBank ID:FJ959397)分别插入到pCAG3.1、pLTR3.1、pCMV3.1质粒中，得到pCAG-cvs、pLTR-cvs、pCMV-cvs和pCAG-CTN、pLTR-CTN、pCMV-CTN等狂犬膜蛋白真核表达质粒。Insert CVS-11 membrane protein (GenBank ID: GQ918139) and CTN-1 membrane protein (GenBank ID: FJ959397) into pCAG3.1, pLTR3.1, pCMV3.1 plasmids respectively to obtain pCAG-cvs, pLTR-cvs , pCMV-cvs and pCAG-CTN, pLTR-CTN, pCMV-CTN and other rabies membrane protein eukaryotic expression plasmids.
为检测不同真核表达质粒和骨架质粒间包装假病毒的效果的不同，分别将两组质粒进行交叉组合共转染已提前在六孔细胞培养板中培养好的293T细胞，48h后，收集假病毒。感染293T细胞，48h后检测发光值，得到的结果见表1、表2和图9、图10。In order to detect the difference in the effect of packaging pseudoviruses between different eukaryotic expression plasmids and backbone plasmids, the two groups of plasmids were cross-combined and co-transfected into 293T cells that had been cultured in six-well cell culture plates in advance. After 48 hours, the pseudoviruses were collected. Virus. After infecting 293T cells, the luminescence value was detected after 48 hours. The results obtained are shown in Table 1, Table 2 and Figures 9 and 10.
表1不同质粒组合共转染细胞的荧光值(RLU)Table 1 Fluorescence value (RLU) of co-transfected cells with different plasmid combinations
表2不同质粒组合共转染细胞的荧光值(RLU)Table 2 Fluorescence values (RLU) of co-transfected cells with different plasmid combinations
结果发现：pSG3.cmvFluc作为骨架质粒得到的假病毒的滴度比其他骨架质粒明显要高许多，pCMV3.1表达质粒和pSG3.cmvFluc骨架质粒具有最佳的包装效果，可以包装出较高滴度的狂犬假病毒。It was found that the titer of the pseudovirus obtained by using pSG3.cmvFluc as the backbone plasmid is significantly higher than that of other backbone plasmids, and the pCMV3.1 expression plasmid and the pSG3.cmvFluc backbone plasmid have the best packaging effect and can package higher titers rabies pseudovirus.
实施例10高滴度狂犬街毒株假病毒制备和中和实验Example 10 High titer rabies street strain pseudovirus preparation and neutralization experiment
将含有狂犬街毒株膜蛋白的pCMV-cvs质粒和pSG3.Δenv.cmvFluc骨架质粒按质量比1：2，利用lipofectamine2000转染试剂共转染293T细胞，6h左右换成新鲜的培养基，48h后收集培养基上清，分装冻存于-80℃备用。然后进行病毒滴定。取冻存的假病毒，3倍系列稀释加入96孔板中(见表3)，然后消化293T细胞，计数5×105/ml，每孔加入100μl，感染48h后，检测发光，利用Reed-Muench公式计算TCID50为5×104/ml。Co-transfect 293T cells with the pCMV-cvs plasmid containing the membrane protein of the rabies street strain and the pSG3.Δenv.cmvFluc backbone plasmid at a mass ratio of 1:2, using lipofectamine2000 transfection reagent, and replace it with fresh medium for about 6 hours, and after 48 hours The culture supernatant was collected, aliquoted and frozen at -80°C for later use. Virus titration was then performed. Take the frozen pseudovirus, add 3-fold serial dilutions to the 96-well plate (see Table 3), then digest the 293T cells, count 5×105 /ml, add 100 μl to each well, and detect the luminescence after 48 hours of infection, using Reed- Muench formula calculates TCID50 as 5×104 /ml.
表3table 3
注：VC为病毒对照，CC为细胞对照。Note: VC is virus control, CC is cell control.
狂犬街毒株假病毒中和实验：Rabies street strain pseudovirus neutralization experiment:
根据滴定结果，狂犬病人免疫球蛋白(中国食品药品检定研究院虫媒病毒疫苗室提供狂犬病人免疫球蛋白标准品和抗狂犬病人免疫球蛋白供试品)分别进行3倍系列稀释，再加入50TCID50的假病毒，37℃孵育1h，消化细胞，计数5×105/ml，每孔加入100μl，感染48h后，检测发光，计算抑制率，结果如图11所示。According to the titration results, the rabies patient immunoglobulin (the standard product of rabies patient immunoglobulin and the test product of anti-rabies patient immunoglobulin provided by the Arbovirus Vaccine Room of China Institute of Food and Drug Control) were serially diluted 3 times respectively, and then 50TCID50 The pseudovirus was incubated at 37°C for 1 hour, the cells were digested, counted at 5×105 /ml, and 100 μl was added to each well. After 48 hours of infection, the luminescence was detected and the inhibition rate was calculated. The results are shown in Figure 11.
利用graphpad计算得，免疫球蛋白的IC50为4×10-4IU/ml左右。Calculated by graphpad, the IC50 of the immunoglobulin is about 4×10-4 IU/ml.
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.
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