CN103103197A - CD138-resistant monoclonal antibody variable region sequence as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a CD138-resistant monoclonal antibody variable region sequence. The CD138-resistant monoclonal antibody variable region sequence comprises six nucleotide sequences, wherein the nucleotide sequences are 58208(1)-VH, 58208(1)-VL, 58208(2)-VH, 58208(2)-VL, 59166-VH and 59166-VL. Polypeptide synthesized by CD138 proteins and U266 cells are used as antigens; after mice are immunized, the mice of which the corresponding antibody expression is positive are detected; after splenocyte is separated; the splenocyte and myeloma cell are fused; after culture in HAT culture medium, an antibody expression positive clone is acquired by detection, and a positive hybridoma total ribonucleic acid (RNA) is extracted; because the mRNA in the total RNA is used as a template, cDNA of the corresponding antibody gene is obtained by inverse transcription; and the heavy chain variable region and light chain variable region of the corresponding antibody are acquired by using a specific primer polymerase chain reaction (PCR) so as to clone and sequence.

Description

Translated fromChinese

抗CD138单克隆抗体可变区序列及其制备方法和应用Anti-CD138 monoclonal antibody variable region sequence and its preparation method and application

技术领域technical field

本发明涉及生物医药技术领域，更具体地，涉及抗CD138单克隆抗体可变区序列及其制备方法和应用。The present invention relates to the technical field of biomedicine, and more specifically, to the variable region sequence of an anti-CD138 monoclonal antibody and its preparation method and application.

背景技术Background technique

Syndecans家族有4个成员，分别为Syndecan-1、Syndecan-2、Syndecan-3、Syndecan-4，其中对Syndecan-1(以下简称为CD138)的研究最为广泛。CD138是一种蛋白聚糖，通过与肝素结合生长因子、可溶性基质成分等多种效应器相互作用来调节细胞行为。CD138分子是跨膜粘结蛋白聚糖家族成员,由胞浆段、跨膜段和胞外段组成，CD138是一类非常复杂的大分子复合物，分子量约为85～92kD，由棱核心蛋白和未分枝的糖胺聚糖链共价结合形成的一类糖结合物，亦称蛋白聚糖，蛋白聚糖存在于所有哺乳动物中，是细胞间质、细胞质膜的重要组成成分。There are four members of the Syndecans family, namely Syndecan-1, Syndecan-2, Syndecan-3, and Syndecan-4, among which Syndecan-1 (hereinafter referred to as CD138) is the most widely studied. CD138 is a proteoglycan that regulates cellular behavior by interacting with various effectors such as heparin-binding growth factor and soluble matrix components. CD138 molecule is a member of the transmembrane cohesin family, which is composed of cytoplasmic segment, transmembrane segment and extracellular segment. CD138 is a very complex macromolecular complex with a molecular weight of about 85-92kD, composed of core protein A type of sugar conjugate formed by covalently bonding with unbranched glycosaminoglycan chains, also known as proteoglycan, which exists in all mammals and is an important component of the intercellular substance and plasma membrane.

对于CD138的分子结构，主要是根据其cDNA的序列分析而来，CD138分子属I型跨膜蛋白，有一个N端信息肽、一个含糖胺聚糖附着处的膜外区、一个疏水跨膜区和一个短的C端胞浆区组成。CD138的跨膜区和胞浆区是高度保守的，胞浆区肽链较短，由13个氨基酸组成，与跨膜区紧密相连，CD138在C末端尾都有一个4肽序列，目前胞浆区部分的功能还不太清楚。The molecular structure of CD138 is mainly based on the sequence analysis of its cDNA. The CD138 molecule is a type I transmembrane protein, which has an N-terminal information peptide, an extramembrane region containing glycosaminoglycan attachment, and a hydrophobic transmembrane protein. domain and a short C-terminal cytoplasmic domain. The transmembrane and cytoplasmic regions of CD138 are highly conserved. The cytoplasmic region has a short peptide chain consisting of 13 amino acids and is closely connected with the transmembrane region. CD138 has a 4-peptide sequence at the C-terminal tail. Currently, the cytoplasmic The function of the area part is still unclear.

CD138是一种跨膜蛋白聚糖，可表达在各种来源的肿瘤细胞表面，包括骨髓瘤、卵巢癌、乳腺癌、肝癌、何杰金氏病及与某些HIV有关的淋巴瘤。当正常细胞恶变后，细胞膜表面CD138分子的表达常发生改变，并且与肿瘤的恶性程度、预后有一定相关性。CD138是一种粘附分子，它在肿瘤细胞膜表面表达减少或者缺失，使细胞的生长、分化等行为发生紊乱，导致了瘤细胞大量繁殖，表现出极强的侵袭活性和转移特性。CD138 is a transmembrane proteoglycan expressed on the surface of tumor cells of various origins, including myeloma, ovarian cancer, breast cancer, liver cancer, Hodgkin's disease, and certain HIV-related lymphomas. When normal cells become malignant, the expression of CD138 molecules on the cell membrane surface often changes, and it is related to the degree of malignancy and prognosis of the tumor. CD138 is an adhesion molecule. Its reduced or absent expression on the surface of tumor cell membranes disrupts cell growth, differentiation, and other behaviors, resulting in massive proliferation of tumor cells, showing strong invasive activity and metastatic properties.

CD138与肿瘤的发生、发展有一定的相关性，并与肿瘤的分型、分化、分期有一定关系。因此我们可根据癌组织中CD138表达程度来判断肿瘤分型，并采取相应治疗手段。CD138可促进细胞与细胞，细胞与基质之间的粘附作用，其具有抑制肿瘤细胞生长、侵袭和转移的特性，可用于肿瘤的免疫或基因治疗。随着分子生物学的不断发展，癌基因研究的不断深入，CD138在肿瘤诊断、病情进展、转移潜能和预后的评价等方面具有很大的潜在价值。CD138 has a certain correlation with the occurrence and development of tumors, and has a certain relationship with the type, differentiation and stage of tumors. Therefore, we can judge the tumor type according to the expression level of CD138 in the cancer tissue, and take corresponding treatment measures. CD138 can promote cell-cell, cell-matrix adhesion, it has the property of inhibiting tumor cell growth, invasion and metastasis, and can be used for tumor immunity or gene therapy. With the continuous development of molecular biology and the deepening of oncogene research, CD138 has great potential value in tumor diagnosis, disease progression, metastasis potential and prognosis evaluation.

发明内容Contents of the invention

如上所述，对CD138的分析研究越来越多，基于这一背景，本发明的目的是构建抗CD138单克隆抗体的重链可变区的核苷酸序列和轻链可变区的核苷酸序列，同上提供制备上述核苷酸序列的方法，以及上述核苷酸序列的应用。As mentioned above, there are more and more analytical studies on CD138. Based on this background, the purpose of the present invention is to construct the nucleotide sequence of the heavy chain variable region and the nucleotide sequence of the light chain variable region of the anti-CD138 monoclonal antibody acid sequence, the method for preparing the above-mentioned nucleotide sequence and the application of the above-mentioned nucleotide sequence are provided above.

为实现上述目的，本发明用CD138蛋白设计合成的多肽和U266细胞作为抗原，免疫小鼠后，经检测得到相应抗体表达呈阳性的小鼠，取表达呈阳性小鼠的脾脏，分离脾细胞后，融合脾细胞和骨髓瘤细胞，在HAT培养基中培养后，检测得到抗体表达阳性克隆，提取阳性杂交瘤细胞总RNA，以总RNA中的mRNA为模板，反转录得到相应抗体基因的cDNA，再通过PCR扩增获得相应抗体重链可变区和轻链可变区，由此实现本发明。In order to achieve the above purpose, the present invention uses the polypeptide designed and synthesized by CD138 protein and U266 cells as antigens. After immunizing the mice, the mice with positive expression of corresponding antibodies are obtained through detection. The spleens of the mice with positive expression are taken, and after the splenocytes are separated , fusion of splenocytes and myeloma cells, cultured in HAT medium, positive antibody expression clones were detected, total RNA of positive hybridoma cells was extracted, and the mRNA in the total RNA was used as a template to reverse transcribe the cDNA of the corresponding antibody gene , and then obtain the corresponding antibody heavy chain variable region and light chain variable region by PCR amplification, thereby realizing the present invention.

本发明提供了下述各项：The present invention provides the following:

抗CD138单克隆抗体可变区序列，包含六种核苷酸序列，所述核苷酸序列分别为58208（1）-VH、58208（1）-VL、58208（2）-VH、58208（2）-VL、59166-VH和59166-VL。Anti-CD138 monoclonal antibody variable region sequence, including six nucleotide sequences, the nucleotide sequences are 58208(1)-VH, 58208(1)-VL, 58208(2)-VH, 58208(2) )-VL, 59166-VH and 59166-VL.

优选的，所述核苷酸序列58208（1）-VH和58208（1）-VL由保藏号为480CT5.4.3的杂交瘤细胞株制备而成，所述核苷酸序列58208（2）-VH和58208（2）-VL由保藏号为480CT13.4.3.2的杂交瘤细胞株制备而成，所述核苷酸序列59166-VH和59166-VL由保藏号为587CT7.3.6.5的杂交瘤细胞株制备而成。Preferably, the nucleotide sequences 58208(1)-VH and 58208(1)-VL are prepared from a hybridoma cell line with a deposit number of 480CT5.4.3, and the nucleotide sequences 58208(2)-VH and 58208(2)-VL were prepared from the hybridoma cell line with the deposit number 480CT13.4.3.2, and the nucleotide sequences 59166-VH and 59166-VL were prepared from the hybridoma cell line with the deposit number 587CT7.3.6.5 Cell lines were prepared.

优选的，还包括与上述六种核苷酸序列具有相同氨基酸序列产物的核苷酸序列。Preferably, nucleotide sequences having the same amino acid sequence products as the above six nucleotide sequences are also included.

优选的，还包括：经过一个或者几个碱基替换、缺失或添加后仍具有与所述核苷酸序列产生的氨基酸序列具有相同活性的核苷酸序列。Preferably, it also includes: a nucleotide sequence that still has the same activity as the amino acid sequence generated by the nucleotide sequence after one or several base substitutions, deletions or additions.

制备以上所述的抗CD138单克隆抗体可变区序列的方法，包括以下步骤：The method for preparing the variable region sequence of the anti-CD138 monoclonal antibody described above comprises the following steps:

（1）杂交瘤细胞的制备：首先用CD138蛋白设计合成的多肽以及U266细胞作为抗原来免疫雌性健康的BALB/c小鼠，挑选出免疫后抗体表达呈阳性的小鼠，取其脾脏细胞，然后将分离的小鼠脾脏细胞与骨髓瘤细胞融合，形成杂交瘤细胞；(1) Preparation of hybridoma cells: Firstly, the polypeptide designed and synthesized by CD138 protein and U266 cells were used as antigens to immunize healthy female BALB/c mice, and the mice with positive antibody expression after immunization were selected, and the spleen cells were collected. The isolated mouse spleen cells were then fused with myeloma cells to form hybridoma cells;

（2）单克隆细胞的筛选：将上述杂交瘤细胞在HAT培养基中培养，对ELISA检测呈阳性的单克隆细胞进行ELISA复筛，然后将筛选出的ELISA阳性单克隆细胞进行WB复筛，再将WB阳性细胞进行亚克隆，经过2次亚克隆，筛选出能够稳定分泌抗体的单克隆细胞，将阳性的单克隆细胞扩大培养后定株、冻存；(2) Screening of monoclonal cells: culture the above-mentioned hybridoma cells in HAT medium, perform ELISA re-screening on the monoclonal cells that are positive in the ELISA test, and then perform WB re-screening on the screened ELISA-positive monoclonal cells, Then subclone the WB positive cells, after 2 times of subcloning, screen out the monoclonal cells that can stably secrete antibodies, expand the positive monoclonal cells, determine the strains, and freeze them;

（3）杂交瘤细胞抗CD138单克隆抗体的获得：首先鉴定步骤（2）所制备的单克隆细胞的亚类亚型，然后将阳性的单克隆细胞注射到小鼠体内进行腹水生产，再将产生的腹水通过层析纯化后得到抗CD138单克隆抗体；(3) Obtaining anti-CD138 monoclonal antibody of hybridoma cells: first identify the subtype of the monoclonal cells prepared in step (2), then inject the positive monoclonal cells into mice for ascites production, and then inject The produced ascites was purified by chromatography to obtain anti-CD138 monoclonal antibody;

（4）抗CD138单克隆抗体重链和轻链可变区基因的克隆：提取步骤（3）中所用的单克隆细胞的总RNA，然后获得mRNA，再以所述的mRNA为模板，反转录得到cDNA，最后克隆得到抗CD138单克隆抗体的重链可变区和轻链可变区。(4) Cloning of heavy chain and light chain variable region genes of anti-CD138 monoclonal antibody: extract the total RNA of the monoclonal cells used in step (3), then obtain mRNA, and then use the mRNA as a template to reverse The cDNA was recorded, and finally the heavy chain variable region and the light chain variable region of the anti-CD138 monoclonal antibody were cloned.

一种表达载体，含有以上任意一项所述的核苷酸序列。An expression vector containing the nucleotide sequence described in any one of the above.

一种表达宿主，载有以上所述的表达载体。An expression host carrying the above-mentioned expression vector.

核苷酸序列在制备抗体上的应用，其特征在于，上述核苷酸序列包括：58208（1）-VH、58208（1）-VL、58208（2）-VH、58208（2）-VL、59166-VH和59166-VL。The application of nucleotide sequences in the preparation of antibodies is characterized in that the above nucleotide sequences include: 58208(1)-VH, 58208(1)-VL, 58208(2)-VH, 58208(2)-VL, 59166-VH and 59166-VL.

优选的，上述抗体包括：重组抗体、ScFv抗体、人源化抗体、嵌合抗体、双特异性抗体、单域抗体。Preferably, the above-mentioned antibodies include: recombinant antibodies, ScFv antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, and single domain antibodies.

本发明提供了抗CD138单克隆抗体可变区序列，及其克隆测序方法，其可用于用于制备重组抗体、ScFv抗体、人源化抗体、嵌合抗体、双特异性抗体、单域抗体等；并且在肿瘤诊断、病情进展、转移潜能和预后的评价等方面具有很大的潜在价值。The present invention provides the variable region sequence of anti-CD138 monoclonal antibody, and its cloning and sequencing method, which can be used to prepare recombinant antibody, ScFv antibody, humanized antibody, chimeric antibody, bispecific antibody, single domain antibody, etc. ; And it has great potential value in the evaluation of tumor diagnosis, disease progression, metastasis potential and prognosis.

附图说明Description of drawings

图1.抗CD138单克隆抗体（480CT5.4.3）免疫印迹实验图；Figure 1. Western blot diagram of anti-CD138 monoclonal antibody (480CT5.4.3);

其中抗体浓度依次分别为8ug/ml,4ug/ml,2ug/ml；The antibody concentrations were 8ug/ml, 4ug/ml, and 2ug/ml in turn;

图2.抗CD138单克隆抗体（480CT13.4.3.2）免疫印迹实验图；Figure 2. Western blot diagram of anti-CD138 monoclonal antibody (480CT13.4.3.2);

图3.抗CD138单克隆抗体（587CT7.3.6.5）免疫印迹实验图；Figure 3. Western blot diagram of anti-CD138 monoclonal antibody (587CT7.3.6.5);

其中抗体浓度1-4依次分别为8ug/ml,4ug/ml,2ug/ml,1ug/ml；The antibody concentrations 1-4 are 8ug/ml, 4ug/ml, 2ug/ml, 1ug/ml respectively;

图4.保藏号为480CT5.4.3的杂交瘤细胞制备得到的单克隆抗体可变区VH和VL的RT-PCR产物琼脂糖凝胶电泳图；Figure 4. Agarose gel electrophoresis of RT-PCR products of the variable region VH and VL of the monoclonal antibody prepared by hybridoma cells with the preservation number 480CT5.4.3;

其中泳道1为DL2000DNA Marker，泳道2为VH基因，泳道3为VL基因，泳道4为RT-PCR扩增VH基因阴性对照，泳道5为RT-PCR扩增VL基因阴性对照；Whereinswimming lane 1 is DL2000DNA Marker,swimming lane 2 is VH gene,swimming lane 3 is VL gene,swimming lane 4 is RT-PCR amplification VH gene negative control,swimming lane 5 is RT-PCR amplification VL gene negative control;

图5.480CT13.4.3.2克隆抗体可变区VH和VL的RTPCR产物琼脂糖凝胶电泳图；Figure 5. Agarose gel electrophoresis of the RTPCR product of the variable region VH and VL of the 480CT13.4.3.2 cloned antibody;

其中泳道1为DL2000DNA Marker，泳道2为VH基因，泳道3为VL基因，泳道4为RT-PCR扩增VH基因阴性对照，泳道5为RT-PCRLane 1 is DL2000 DNA Marker,lane 2 is VH gene,lane 3 is VL gene,lane 4 is RT-PCR amplified VH gene negative control, andlane 5 is RT-PCR

扩增VL基因阴性对照；Amplified VL gene negative control;

图6.587CT7.3.6.5克隆抗体可变区VH和VL的RT-PCR产物琼脂糖凝胶电泳图；Figure 6. Agarose gel electrophoresis of the RT-PCR product of the variable region VH and VL of the 587CT7.3.6.5 cloned antibody;

其中泳道1为DL2000DNA Marker，泳道2为VH基因；泳道3为VL基因，泳道4为RT-PCR扩增VH基因阴性对照，泳道5为RT-PCR扩增VL基因阴性对照；Whereinswimming lane 1 is DL2000DNA Marker, andswimming lane 2 is VH gene;Swimming lane 3 is VL gene, andswimming lane 4 is RT-PCR amplification VH gene negative control, andswimming lane 5 is RT-PCR amplification VL gene negative control;

图7.480CT5.4.3克隆抗体可变区VH和VL目的片段构建到pMD18-T载体上酶切鉴定琼脂糖凝胶电泳图；Figure 7. The agarose gel electrophoresis diagram of the variable region VH and VL target fragments of the 480CT5.4.3 cloned antibody constructed on the pMD18-T vector for enzyme digestion and identification;

其中泳道1为DL5000DNA Marker，泳道2、3为pMD18-T/VH的2个克隆，泳道4、5为pMD18-T/VL的2个克隆；Among them,lane 1 is DL5000DNA Marker,lanes 2 and 3 are two clones of pMD18-T/VH, andlanes 4 and 5 are two clones of pMD18-T/VL;

图8.480CT13.4.3.2克隆抗体可变区VH和VL目的片段，构建到pMD18-T载体上酶切鉴定琼脂糖凝胶电泳图；Figure 8. 480CT13.4.3.2 Cloned antibody variable region VH and VL target fragments, constructed on the pMD18-T vector for enzyme digestion and identification of agarose gel electrophoresis;

其中泳道1为DL5000DNA Marker，泳道2、3为pMD18-T/VH的2个克隆，泳道4、5为pMD18-T/VL的2个克隆；Among them,lane 1 is DL5000DNA Marker,lanes 2 and 3 are two clones of pMD18-T/VH, andlanes 4 and 5 are two clones of pMD18-T/VL;

图9.587CT7.3.6.5克隆抗体可变区VH和VL目的片段，构建到pMD18-T载体上酶切鉴定琼脂糖凝胶电泳图；Figure 9. 587CT7.3.6.5 Cloned antibody variable region VH and VL target fragments, constructed on the pMD18-T vector for enzyme digestion and identification of agarose gel electrophoresis;

其中泳道1为DL5000DNA Marker，泳道2、3为pMD18-T/VH的2个克隆，泳道4、5为pMD18-T/VL的2个克隆。Among them,lane 1 is DL5000 DNA Marker,lanes 2 and 3 are two clones of pMD18-T/VH, andlanes 4 and 5 are two clones of pMD18-T/VL.

具体实施方式Detailed ways

下面结合附图及实施例对本发明做进一步描述：The present invention will be further described below in conjunction with accompanying drawing and embodiment:

实施例1.抗CD138单克隆抗体的制备与鉴定Example 1. Preparation and Identification of Anti-CD138 Monoclonal Antibody

1、杂交瘤细胞制备1. Preparation of hybridoma cells

1.1动物免疫1.1 Animal immunity

以CD138蛋白设计合成的多肽以及U266细胞作为抗原同时各免疫3只6-8周龄的雌性健康的BALB/c小鼠，3次免疫后，7d采血并测血清中抗体效价，每周采血一次，选择1：4000稀释时ELISA且检测OD值大于1.0的血清，同时血清WB检测呈阳性的BALB/c小鼠用于融合，融合之前的3d，用不加佐剂的抗原腹腔加强免疫注射，注射剂量为60ug/只。Use CD138 protein designed and synthesized peptides and U266 cells as antigens to immunize 3 female healthy BALB/c mice aged 6-8 weeks respectively. After 3 times of immunization, blood was collected 7 days and the antibody titer in serum was measured, and blood was collected every week Once, select the serum with ELISA at 1:4000 dilution and detect OD value greater than 1.0, and select BALB/c mice with positive serum WB test for fusion, 3 days before fusion, intraperitoneal booster injection with antigen without adjuvant, The injection dose is 60ug/only.

1.2收集B淋巴细胞1.2 Collection of B lymphocytes

追加免疫后3d，取小鼠血液，离心后血清留作阳性对照；在无菌条件下取其脾脏，并将脾脏放在10ml预温不完全培养基中，剥去周围结缔组织，置100目不锈钢网中，用注射器的内芯研磨，边研磨边滴加不完全培养基冲洗；收集过滤后的细胞悬液于离心管中，离心，弃上清液，用不完全培养基悬浮细胞，取1×10⁸个细胞，置室温待用。3 days after the booster immunization, the blood of the mice was taken, and the serum after centrifugation was kept as a positive control; the spleen was taken under sterile conditions, and the spleen was placed in 10ml of pre-warmed incomplete medium, the surrounding connective tissue was peeled off, and placed in a 100-mesh In the stainless steel mesh, use the inner core of the syringe to grind, add incomplete medium dropwise to wash while grinding; collect the filtered cell suspension in a centrifuge tube, centrifuge, discard the supernatant, suspend the cells with incomplete medium, take 1×10⁸ cells, set aside at room temperature.

1.3小鼠骨髓瘤细胞的制备1.3 Preparation of mouse myeloma cells

融合前10d,将骨髓瘤细胞从液氮中取出，迅速放入37℃水浴中，在10min内至冷冻液完全溶解；离心，弃上清液，置IMDM完全培养基中，37℃，5%CO₂培养；融合前将对数生长期小鼠骨髓瘤细胞收集到离心管中，计数，取2×10⁷-3×10⁷个细胞，离心弃上清液，用不完全培养基悬浮细胞，置室温待用。10 days before fusion, the myeloma cells were taken out of the liquid nitrogen, quickly placed in a 37°C water bath, and the frozen liquid was completely dissolved within 10 minutes; centrifuged, discarded the supernatant, and placed in IMDM complete medium, 37°C, 5% CO₂ culture; before fusion, collect the mouse myeloma cells in the logarithmic growth phase into a centrifuge tube, count, take 2×10⁷ -3×10⁷ cells, centrifuge to discard the supernatant, and suspend the cells with incomplete medium , set aside at room temperature.

1.4细胞融合1.4 Cell Fusion

融合前将50%PEG置37℃，5%CO₂细胞培养箱中调整温度，将2×10⁷-3×10⁷个骨髓瘤细胞悬液和1×10⁸个脾脏B淋巴细胞悬液移至一个50ml离心管中，补加30ml不完全培养基，在转速1500r/min的条件下离心10min，弃去上清液，轻轻弹击管底，使细胞团松散，一边均匀地转动离心管，一边将1ml预温的PEG融合剂在60s内，沿管壁慢慢加入细胞中，边加边转动离心管，轻摇离心管30s，静置60s，然后立即加入20ml不完全培养液，具体加法是第一分钟加1ml,第二分钟加4ml，随后的三分钟之内将剩余液体加完，使PEG稀释而失去促融作用，在37℃水浴中静置10min，1500r/min，离心10min，弃去上清，加入含有HAT的完全培养基，制成细胞悬液，铺到96孔细胞培养板上，置37℃，5%CO₂细胞培养箱中。Before fusion, place 50% PEG in a 37°C, 5% CO₂ cell incubator to adjust the temperature, andpipette 2×10⁷ -3×10⁷ myeloma cell suspension and 1×10⁸ spleen B lymphocyte suspension Add 30ml of incomplete medium to a 50ml centrifuge tube, centrifuge at 1500r/min for 10min, discard the supernatant, lightly flick the bottom of the tube to loosen the cell clusters, and rotate the centrifuge tube evenly , while slowly adding 1ml of pre-warmed PEG fusion agent into the cells along the tube wall within 60s, turn the centrifuge tube while adding, shake the centrifuge tube gently for 30s, let stand for 60s, then immediately add 20ml of incomplete culture medium, specifically Addition is to add 1ml in the first minute, add 4ml in the second minute, and add the remaining liquid within the next three minutes to dilute the PEG and lose the effect of promoting fusion. Let it stand in a water bath at 37°C for 10min, centrifuge at 1500r/min for 10min , discard the supernatant, add complete medium containing HAT to make a cell suspension, spread it on a 96-well cell culture plate, and place it in a 37°C, 5% CO₂ cell culture incubator.

1.5筛选阳性单克隆细胞1.5 Screen positive monoclonal cells

融合的杂交瘤细胞能在HAT培养基中存活和增殖；在37℃，5%CO₂细胞培养箱中培养7-10d；用抗原包被酶标板，37℃包被2h，然后用2%BSA封闭；吸取96孔板中生长克隆的细胞上清加到封闭好的酶标板中，37℃孵育1h；洗涤5遍后，加入HRP标记的羊抗鼠IgG，37℃孵育1h；洗涤后加入TMB显色液，然后加2ml硫酸终止反应，在酶标仪上进行读数；将ELISA检测阳性的克隆挑入24孔细胞培养板培养，3d后进行ELISA复筛，将筛选出的ELISA阳性克隆进行WB复筛，WB阳性细胞进行亚克隆，经过2次亚克隆，筛选出能够稳定分泌抗体的单克隆细胞，将阳性的单克隆扩大培养后定株、冻存。Fused hybridoma cells can survive and proliferate in HAT medium; culture at 37°C in a 5% CO₂ cell incubator for 7-10 days; coat the microtiter plate with antigen, coat at 37°C for 2 hours, and then use 2% Blocked with BSA; pipette the cell supernatant of the growing clone in the 96-well plate and add it to the sealed microtiter plate, incubate at 37°C for 1 hour; after washing 5 times, add HRP-labeled goat anti-mouse IgG, and incubate at 37°C for 1 hour; after washing Add TMB chromogenic solution, then add 2ml of sulfuric acid to terminate the reaction, and read on a microplate reader; pick the positive clones detected by ELISA into 24-well cell culture plates for culture, and perform ELISA re-screening after 3 days, and screen the ELISA positive clones WB re-screening was carried out, and WB positive cells were subcloned. After subcloning twice, monoclonal cells capable of stably secreting antibodies were screened out, and the positive monoclonal cells were expanded and cultured to establish strains and cryopreserved.

2、单克隆抗体的生物学鉴定2. Biological identification of monoclonal antibodies

将定株的单克隆细胞的细胞上清液用美国BD公司MouseMonoclonalAntibody IsotypingKit鉴定单抗的亚类亚型，然后将阳性的单克隆细胞注射到小鼠体内进行腹水生产，生产的腹水通过层析纯化后的得到抗体，采用Western Blot鉴定抗体的特异性，ELISA鉴定单抗的亲和力，具体操作如下：Use the mouseMonoclonalAntibody IsotypingKit of BD Company in the United States to identify the subtype of the monoclonal antibody in the cell supernatant of the monoclonal cells determined by the strain, and then inject the positive monoclonal cells into the mice for ascites production, and the produced ascites is purified by chromatography After obtaining the antibody, use Western Blot to identify the specificity of the antibody, and ELISA to identify the affinity of the monoclonal antibody. The specific operations are as follows:

2.1Western blot检测抗体的特异性2.1 The specificity of Western blot detection antibody

取已处理的细胞裂解液，在凝胶上进行垂直SDS-PAGE，120v90min，电泳结束后，取下凝胶，置于PVDF膜上，将蛋白用半干法转染到PVDF膜上，10v,120min；将转印后的PVDF膜在5%脱脂奶粉中室温封闭2h；将抗体溶解于3ml封闭液中，4℃过夜；用洗涤液洗涤3次，每次5min，用封闭液来稀释HRP标记的羊抗鼠IgG，室温下孵育2h；再用洗涤液洗涤3次，将化学发光试剂加到PVDF膜上，到暗室曝光到X胶片上，通过显影和定影，将结果反映在胶片上；扫描胶片，分析结果，其结果参照图1-图3。Take the treated cell lysate and perform vertical SDS-PAGE on the gel, 120v90min, after the electrophoresis, remove the gel, place it on the PVDF membrane, transfect the protein to the PVDF membrane by semi-dry method, 10v, 120min; block the transferred PVDF membrane in 5% skimmed milk powder at room temperature for 2h; dissolve the antibody in 3ml of blocking solution, overnight at 4°C; wash 3 times with washing solution, 5min each time, and dilute the HRP marker with blocking solution Goat anti-mouse IgG, incubate at room temperature for 2 hours; then wash with washing solution for 3 times, add chemiluminescence reagent to PVDF membrane, expose to X film in dark room, and reflect the result on the film by developing and fixing; scan Film, analysis results, the results refer to Figure 1-Figure 3.

2.2单克隆抗体亲和常数测定2.2 Determination of monoclonal antibody affinity constant

用包被液将抗原稀释，分别加入酶标板，100微升/孔，37℃2h，PBS洗涤5次，拍干，加2%BSA封闭液200微升/孔，4℃过夜，洗涤5次，拍干；将抗体从4ug/ml开始进行梯度稀释后加入到包被好的酶标板中，100微升/孔，37℃1h，洗涤5次，拍干；加入HRP标记的羊抗小鼠IgG抗体，工作液100微升/孔，37℃1h，洗涤5次，拍干；加入TMB显色液，37℃反应5-10min，加终止液50微升/孔，立即在酶标仪上以450nm波长测定OD值，按照公式计算出单克隆抗体亲和常数。Dilute the antigen with the coating solution, add 100 microliters/well to the microtiter plate, 37°C for 2 hours, wash 5 times with PBS, pat dry, add 200 microliters/well of 2% BSA blocking solution, overnight at 4°C, wash 5times 1 time, pat dry; the antibody was serially diluted from 4ug/ml and then added to the coated microtiter plate, 100 μl/well, 37 ℃ 1h, washed 5 times, pat dry; add HRP-labeled goat antibody Mouse IgG antibody, workingsolution 100 μl/well, 37°C for 1 hour, wash 5 times, pat dry; add TMB chromogenic solution, react at 37°C for 5-10 minutes, add stop solution 50 μl/well, immediately in the enzyme labeling The OD value was measured with a wavelength of 450nm on the instrument, and the monoclonal antibody affinity constant was calculated according to the formula.

3、抗CD138单克隆抗体重链和轻链可变区基因的克隆3. Cloning of heavy chain and light chain variable region genes of anti-CD138 monoclonal antibody

所用细胞株为采用上述方法获得的能分泌高亲和力、高特异性的抗CD138或CD138抗体的杂交瘤细胞株，对应的保藏编号以及其分泌的抗体分子亚型如表1所示：The cell line used is a hybridoma cell line capable of secreting high-affinity and high-specificity anti-CD138 or CD138 antibody obtained by the above method, and the corresponding deposit number and the subtype of the antibody molecule secreted by it are shown in Table 1:

表1Table 1

保藏编号Deposit number抗体分子亚型Antibody subtype480CT5.4.3480CT5.4.3IgG1IgG1480CT13.4.3.2480CT13.4.3.2IgM、IgkIgM, Igk587CT7.3.6.5587CT7.3.6.5IgMIgM

取对数生长期的3种杂交瘤细胞（每种杂交瘤细胞取2×10⁶个左右），用QIAGEN公司的试剂盒RNeasy MiniKit（货号：QIAGEN-74106）提取总RNA，取少量用nanodrop定量及1%非变性琼脂糖凝胶电泳检测，再用SuperScript.IIIFirst-Strand Synthesis System for RT-PCR试剂盒（货号：Invitrogen-18080-051）反转录cDNA，用特异引物扩增抗CD138抗体基因的轻链或重链可变区，将含有相应重链可变区或轻链可变区片段的PCR反应产物经1%琼脂糖凝胶电泳，切胶分离目的片段；通过胶回收试剂盒（捷瑞-GK2042）纯化目的产物后，1%琼脂糖凝胶电泳鉴定目的片段的纯度；然后，将回收得到的相应重链可变区和轻链可变区克隆至测序载体pMD18-T，并进行测序，对测序结果进行同源性和结构分析。Take 3 kinds of hybridoma cells in the logarithmic growth phase (about 2×10⁶ cells for each hybridoma cell), use QIAGEN’s kit RNeasy MiniKit (product number: QIAGEN-74106) to extract total RNA, and take a small amount to quantify with nanodrop and 1% non-denaturing agarose gel electrophoresis detection, and then use the SuperScript.IIIFirst-Strand Synthesis System for RT-PCR kit (Cat. No.: Invitrogen-18080-051) to reverse transcribe the cDNA, and use specific primers to amplify the anti-CD138 antibody gene The light chain or heavy chain variable region, the PCR reaction products containing the corresponding heavy chain variable region or light chain variable region fragments were subjected to 1% agarose gel electrophoresis, and the target fragments were separated by gel cutting; Gel recovery kit ( Jerry-GK2042) to purify the target product, 1% agarose gel electrophoresis to identify the purity of the target fragment; then, the recovered corresponding heavy chain variable region and light chain variable region were cloned into the sequencing vector pMD18-T, and Sequencing was performed, and homology and structure analysis was performed on the sequencing results.

3.1引物设计：3.1 Primer design:

根据克隆480CT5.4.3亚型的可变区序列，分别在信号区和恒定区合成5’和3’端引物用于扩增CD138抗体重链可变区，引物序列如下：According to the variable region sequence of the cloned 480CT5.4.3 subtype, the 5' and 3' end primers were synthesized in the signal region and the constant region respectively to amplify the heavy chain variable region of the CD138 antibody. The primer sequences are as follows:

VHF（5’-ACTAGTCGACATGGVTTGGSTGTGGAMCTTGCYATTCCT-3’），含有SalⅠ酶切位点；VHF (5'-ACTAGTCGAC ATGGVTTGGSTGTGGAMCTTGCYATTCCT-3'), containing a SalⅠ restriction site;

VHR（5’-CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG-3’），含有HindⅢ酶切位点；VHR (5'-CCCAAGCTT CCAGGGRCCARKGGATARACIGRTGG-3'), containing HindⅢ restriction site;

根据克隆480CT5.4.3亚型的可变区序列，分别在信号区和恒定区合成5’和3’端引物用于扩增CD138抗体轻链可变区，引物序列如下：According to the variable region sequence of the cloned 480CT5.4.3 subtype, the 5' and 3' end primers were synthesized in the signal region and the constant region respectively to amplify the light chain variable region of the CD138 antibody. The primer sequences are as follows:

LHF（5’-ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3’），含有SalⅠ酶切位点；LHF (5'-ACTAGTCGAC ATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3'), containing a SalⅠ restriction site;

LHR（5’-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3’），含有HindIII酶切位点；LHR (5'-CCCAAGCTT ACTGGATGGTGGGAAGATGGA-3'), containing HindIII restriction site;

根据480CT13.4.3.2亚型的可变区的序列，在信号区和恒定区合成5’和3’端引物用于扩增CD138抗体重链可变区，引物序列如下：According to the sequence of the variable region of the 480CT13.4.3.2 subtype, synthesize 5' and 3' end primers in the signal region and constant region to amplify the heavy chain variable region of the CD138 antibody. The primer sequences are as follows:

VHF（5’-ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT-3’），含有SalⅠ酶切位点；VHF (5'-ACTAGTCGAC ATGGGATGGAGCTRTATCATSYTCTT-3'), containing a SalⅠ restriction site;

VHR（5’-CCCAAGCTTACGAGGGGGAAGACATTTGGGAA-3’），含有HindIII酶切位点；VHR (5'-CCCAAGCTT ACGAGGGGGAAGACATTTGGGAA-3'), containing HindIII restriction site;

根据480CT13.4.3.2亚型的可变区的序列，在信号区和恒定区合成5’和3’端引物用于扩增CD138抗体轻链可变区，引物序列如下：According to the sequence of the variable region of the 480CT13.4.3.2 subtype, synthesize 5' and 3' end primers in the signal region and constant region to amplify the light chain variable region of the CD138 antibody. The primer sequences are as follows:

LHF（5’-ACTAGTCGACATGGTRTCCWCASCTCAGTTCCTTG-3’），含有SalⅠ酶切位点；LHF (5'-ACTAGTCGAC ATGGTRTCCWCASCTCAGTTCCTTG-3'), containing SalⅠ restriction site;

LHR（5’-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3’），含有HindIII酶切位点；LHR (5'-CCCAAGCTT ACTGGATGGTGGGAAGATGGA-3'), containing HindIII restriction site;

根据587CT7.3.6.5亚型的可变区的序列，在信号区和恒定区合成5’和3’端引物用于扩增CD20抗体重链可变区，引物序列如下：According to the sequence of the variable region of the 587CT7.3.6.5 subtype, the 5' and 3' end primers were synthesized in the signal region and the constant region to amplify the heavy chain variable region of the CD20 antibody. The primer sequences are as follows:

VHF（5’-GGGAATTCATGRAATGSASCTGGGTYWTYCTCTT-3’），含有EcoRⅠ酶切位点；VHF (5'-GGGAATTC ATGRAATGSASCTGGGTYWTYCTCTT-3'), containing EcoRI restriction site;

VHR（5’-CCCAAGCTTACGAGGGGGAAGACATTTGGGAA-3’），含有HindIII酶切位点；VHR (5'-CCCAAGCTT ACGAGGGGGAAGACATTTGGGAA-3'), containing HindIII restriction site;

根据587CT7.3.6.5亚型的可变区的序列，在信号区和恒定区合成5’和3’端引物用于扩增CD20抗体轻链可变区，引物序列如下：According to the sequence of the variable region of the 587CT7.3.6.5 subtype, 5' and 3' end primers were synthesized in the signal region and constant region to amplify the light chain variable region of the CD20 antibody. The primer sequences are as follows:

LHF（5’-ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3’），含有SalⅠ酶切位点；LHF (5'-ACTAGTCGAC ATGAAGTTGCCTGTTAGGCTGTTGGTGCT-3'), containing a SalⅠ restriction site;

LHR（5’-CCCAAGCTTACTGGATGGTGGGAAGATGGA-3’），含有HindIII酶切位点。LHR (5'-CCCAAGCTT ACTGGATGGTGGGAAGATGGA-3'), contains a HindIII restriction site.

3.2杂交瘤细胞总RNA提取3.2 Extraction of total RNA from hybridoma cells

用QIAGEN公司的试剂盒（货号：74106）提取总RNA，具体步骤如下：Total RNA was extracted with a kit from QIAGEN (Catalog No.: 74106), and the specific steps were as follows:

收集对数生长期杂交瘤细胞，各2×10⁶个，800rpm/mim离心5min，去上清，沉淀即细胞，保存于-80℃，或直接用于RNA提取；Collect hybridoma cells in the logarithmic growth phase, each 2×¹⁰⁶ , centrifuge at 800rpm/mim for 5min, remove the supernatant, pellet the cells, store at -80°C, or directly use for RNA extraction;

每份细胞（2×10⁶）样品中加入350ul RTL溶液，涡旋30s；Add 350ul RTL solution to each cell (2×10⁶ ) sample, vortex for 30s;

向以上每体系中加入70%乙醇，用1mL移液枪轻轻吹打均匀；Add 70% ethanol to each of the above systems, and gently blow evenly with a 1mL pipette gun;

将上述体系混合液转入RNeasy spin column，8000xg离心15s，去滤液；Transfer the above system mixture into RNeasy spin column, centrifuge at 8000xg for 15s, and remove the filtrate;

向上述RNeasy spin column中各加入700uL RW1溶液，8000xg离心15s，去滤液；Add 700uL RW1 solution to each of the above RNeasy spin columns, centrifuge at 8000xg for 15s, and remove the filtrate;

向上述RNeasy spin column中各加入500uL RPE溶液，8000xg离心15s，去滤液；Add 500uL RPE solution to each of the above RNeasy spin columns, centrifuge at 8000xg for 15s, and remove the filtrate;

向上述RNeasy spin column中各加入500uL RPE溶液，8000xg离心2min，去滤液；Add 500uL RPE solution to each of the above RNeasy spin columns, centrifuge at 8000xg for 2min, and remove the filtrate;

将RNeasy spin column换到无RNA酶的2ml收集管中，全速离心1min；Change the RNeasy spin column to a RNase-free 2ml collection tube and centrifuge at full speed for 1min;

将RNeasy spin column换到无RNA酶的的1.5mlEP管中，加入30-50uL无RNA酶的H₂O，8000xg离心1min，洗脱RNA。Change the RNeasy spin column to an RNase-free 1.5ml EP tube, add 30-50uL RNase-free H₂ O, centrifuge at 8000xg for 1min, and elute the RNA.

3.3反转录PCR3.3 Reverse transcription PCR

用Invitrgen的RT-PCR第一链合成SuperScript.III试剂盒（货号18080-051），以杂交瘤细胞总RNA为模板，反转录cDNA，具体步骤如下：Use Invitrgen’s RT-PCR First Strand Synthesis SuperScript.III Kit (Catalog No. 18080-051), and use the total RNA of hybridoma cells as a template to reverse transcribe cDNA. The specific steps are as follows:

3.3.1引物与模板变性：3.3.1 Primer and template denaturation:

按表2配成10uLA体系，放置65℃水浴中加热5min，随后冰浴中放置1min，让引物与RNA模板充分结合。Prepare a 10uLA system according to Table 2, place it in a water bath at 65°C for 5 minutes, and then place it in an ice bath for 1 minute to allow the primers to fully combine with the RNA template.

表2Table 2

成分Element体积(μL)Volume (μL)RNARNAx(100ng<x<1ug)x(100ng<x<1ug)引物Oligo(dT)20Primer Oligo(dT)2011dNTPdNTP11无RNA酶H₂ORNase-free H₂ O8-x8-x

3.3.2复性：3.3.2 Refolding:

按表3，配成10uLB体系，然后加入A体系，在50℃的水浴中加热50min；再转到85℃的水浴中加热5min。According to Table 3, make up 10uLB system, then add system A, heat in a water bath at 50°C for 50 minutes; then transfer to a water bath at 85°C and heat for 5 minutes.

表3table 3

成分Element体积(μL)Volume (μL)10×RT buffer10×RT buffer2225mM MgCl₂25mM_MgCl2440.1M DTT0.1M DTT22RNase OUTRNase OUT11SuperScriptIII RTSuperScript III RT11

3.3.3RNA消化：3.3.3 RNA digestion:

向上述体系中加入1uL RNaseH,37℃,20min。Add 1uL RNaseH to the above system, 37°C, 20min.

3.4抗体可变区特异引物PCR3.4 Antibody variable region-specific primer PCR

按表4和表5中的体系，以反转录得到的cDNA为模板，用特异引物合成抗体重链和轻链可变区。According to the system in Table 4 and Table 5, the cDNA obtained by reverse transcription was used as a template, and the antibody heavy chain and light chain variable regions were synthesized with specific primers.

表4Table 4

成分Element体积(μL)Volume (μL)H₂OH₂ O37.437.410×pfu buffer(Mg²⁺)10×pfu buffer(Mg²⁺ )55dNTP(10mM)dNTP (10mM)11pfupfu0.40.4taqtaq0.20.2Sense primer(20uM)Sense primer(20uM)11Anti-sense primer(20uM)Anti-sense primer(20uM)11cDNAcDNA44

表5table 5

3.5PCR产物克隆、测序3.5PCR product cloning and sequencing

将上述PCR得到的重链可变区和轻链可变区核苷酸片段产物胶回收后，分别构建到测序载体上，并测序。具体步骤如下：The heavy chain variable region and light chain variable region nucleotide fragment products obtained by the above PCR were gel-recovered, respectively constructed on sequencing vectors, and sequenced. Specific steps are as follows:

3.5.1胶回收PCR产物：3.5.1 Gel recovery of PCR products:

PCR产物跑1%琼脂糖凝胶电泳后，割胶回收目的条带，用捷瑞胶回收试剂盒（货号GK-2042）回收。步骤如下：After the PCR product was run on 1% agarose gel electrophoresis, the target band was recovered by tapping the gel, and recovered with the Jerry Gel Recovery Kit (Catalog No. GK-2042). Proceed as follows:

每100mg琼脂糖凝胶加400uL结合液B，60℃水浴锅中放置至凝胶块完全融化;Add 400uL of binding solution B for every 100mg of agarose gel, place in a 60°C water bath until the gel block is completely melted;

将上述混合液转移至套有2mL收集管的GenClean柱中，室温放置2min，6000rpm离心1min，去废液;Transfer the above mixed solution to a GenClean column with a 2mL collection tube, place it at room temperature for 2 minutes, centrifuge at 6000rpm for 1 minute, and remove the waste liquid;

加入500uL洗涤液,12000rpm，室温离心1min，去废液;Add 500uL washing solution, centrifuge at room temperature for 1min at 12000rpm, and remove the waste solution;

重复上述步骤;Repeat the above steps;

将GenClean柱放回收集管，12000rpm，室温离心1min;Put the GenClean column back into the collection tube, centrifuge at 12000rpm for 1min at room temperature;

将GenClean柱放至1.5mLEP管，加入30-50uL洗脱液，37℃放置2min，12000rpm，室温离心1min,来洗脱目的片段。Put the GenClean column into a 1.5mLEP tube, add 30-50uL eluent, place at 37°C for 2min, centrifuge at 12000rpm for 1min at room temperature, to elute the target fragment.

3.5.2酶切3.5.2 Digestion

酶切体系如表6和表7所示：The enzyme digestion system is shown in Table 6 and Table 7:

表6Table 6

成分Element体积（μL）Volume (μL)质粒plasmidX(1ug)X(1ug)10×FD buffer10×FDbuffer55酶1Enzyme 111酶2Enzyme 211H₂OH₂ O43-X43-X

表7Table 7

成分Element体积（μL）Volume (μL)抗体目的片段Antibody Target Fragment171710×FD buffer10×FDbuffer22酶1Enzyme 10.50.5酶2Enzyme 20.50.5

酶切条件：37℃,30min；85℃，5min。Digestion conditions: 37°C, 30min; 85°C, 5min.

跑胶回收质粒酶切体系中长片段，抗体目的片段酶切体系直接用于连接。Run the gel to recover the long fragments in the plasmid enzyme digestion system, and the antibody target fragment enzyme digestion system is directly used for ligation.

3.5.3连接3.5.3 Connection

连接体系如表8所示:The connection system is shown in Table 8:

表8Table 8

连接条件：16℃,4h。Connection conditions: 16°C, 4h.

3.5.4转化3.5.4 Conversion

向连接体系加入100uLDH5α感受态细胞，冰浴30min;Add 100uLDH5α competent cells to the connection system, ice bath for 30min;

42℃热击90s，再冰浴3min;Heat shock at 42°C for 90s, then ice bath for 3min;

加入500uLLB培养基，37℃,120rpm，复苏50min；Add 500uLLB medium, 37°C, 120rpm, recover for 50min;

4000rpm离心2min，去上清，留100uL上清重悬细胞沉淀，Centrifuge at 4000rpm for 2min, remove the supernatant, leave 100uL supernatant to resuspend the cell pellet,

涂相应抗性LB平板。待平皿中液体吸收后，倒置平皿，于37℃培养12-16h可出现菌落；Coat the corresponding resistant LB plates. After the liquid in the plate is absorbed, invert the plate and incubate at 37°C for 12-16 hours to form colonies;

3.5.5菌落PCR鉴定3.5.5 Colony PCR identification

每块转化板上挑4-6个菌落，点到相应抗性LB平板后，做菌落PCR鉴定，PCR反应产物跑1%琼脂糖凝胶检测有无目的长度片段，检测结果参见图4-图6。Pick 4-6 colonies from each transformation plate, point to the corresponding resistant LB plate, and perform colony PCR identification. The PCR reaction product is run on 1% agarose gel to detect whether there is a fragment of the target length. The test results are shown in Figure 4-figure 6.

菌落PCR体系如表9和表10所示：The colony PCR system is shown in Table 9 and Table 10:

表9Table 9

成分Element体积（μL）Volume (μL)H₂OH₂ O15.815.810×pfu buffer(Mg²⁺)10×pfu buffer(Mg²⁺ )22dNTP(10mM)dNTP (10mM)11taqtaq0.20.2Sense primer(20uM)Sense primer(20uM)0.50.5Anti-sense primer(20uM)Anti-sense primer(20uM)0.50.5克隆clone1个1

表10Table 10

3.5.6提质粒3.5.6 Plasmid extraction

挑菌落PCR鉴定为阳性的单克隆摇菌，20%甘油保菌后，用Biomiga-PlasmidMiniprepkit(货号为BIOMEGA-PD1211-02)提质粒。具体步骤如下：Pick the monoclonal shaking bacteria identified as positive by colony PCR, and use Biomiga-Plasmid Miniprepkit (Cat. No. BIOMEGA-PD1211-02) to extract the plasmid after preserving the bacteria with 20% glycerol. Specific steps are as follows:

取4mLLB新鲜菌液，室温下1000rpm离心1min，吸去上清，收集菌体；Take 4 mL of LB fresh bacterial liquid, centrifuge at 1000 rpm for 1 min at room temperature, suck off the supernatant, and collect the bacterial cells;

加入250uL Buffer A1(已加入RNase A)，涡旋震荡充分悬浮细菌细胞；Add 250uL Buffer A1 (RNase A has been added), vortex and shake to fully suspend the bacterial cells;

加入250uL BufferB1，轻轻反转10次以混合均匀，静置5min至溶液粘稠而澄清；Add 250uL BufferB1, gently invert 10 times to mix evenly, let stand for 5min until the solution is viscous and clear;

加入350uL Buffer N1,立即反转多次，至溶液充分混匀，此时出现白色絮状沉淀；Add 350uL Buffer N1, invert several times immediately until the solution is fully mixed, and white flocculent precipitate appears at this time;

将离心管转至高速离心机，在室温下13000rpm离心10min（若上清中有白色沉淀，可再次离心）;Transfer the centrifuge tube to a high-speed centrifuge and centrifuge at 13000rpm for 10min at room temperature (if there is white precipitate in the supernatant, centrifuge again);

小心吸取离心后的上清液至带有收集管的离心柱中（避免吸起沉淀），室温下13000rpm离心1min，倒掉收集管中的废液，将离心柱重新放回收集管中;Carefully draw the supernatant after centrifugation into a spin column with a collection tube (avoid sucking up the precipitate), centrifuge at 13,000 rpm for 1 min at room temperature, pour off the waste liquid in the collection tube, and put the spin column back into the collection tube;

向DNA柱中加入500uL Buffer KB,室温下离心1min，倒掉收集管中的废液，将离心柱重新放回收集管中;Add 500uL Buffer KB to the DNA column, centrifuge for 1min at room temperature, pour off the waste liquid in the collection tube, and put the spin column back into the collection tube;

向离心柱中加入500uL DNA洗涤液(已加入无水乙醇)，室温下，13,000rpm离心1min，倒掉收集管中废液，将离心柱重新放回到收集管中。重复此步骤一次;Add 500uL of DNA washing solution (absolute ethanol has been added) to the spin column, centrifuge at 13,000rpm for 1min at room temperature, discard the waste liquid in the collection tube, and put the spin column back into the collection tube. Repeat this step once;

将离心柱放回高速离心机中，13000rpm室温下开盖离心5-10min，以彻底去除残留的乙醇;Put the spin column back into the high-speed centrifuge, and centrifuge at room temperature at 13000 rpm for 5-10 minutes to completely remove residual ethanol;

将离心柱转移至一个新的1.5mL离心管中，向DNA柱的正中间加入50uL60℃预热的洗脱液,室温放置2min，13000rpm离心1min，洗脱质粒DNA。Transfer the spin column to a new 1.5mL centrifuge tube, add 50uL 60°C preheated eluent to the middle of the DNA column, place at room temperature for 2min, and centrifuge at 13000rpm for 1min to elute the plasmid DNA.

3.5.7酶切鉴定3.5.7 Enzyme digestion identification

将提取的质粒DNA用相应限制性内切酶酶切鉴定，体系如表11所示：The extracted plasmid DNA was digested with corresponding restriction endonucleases and identified, and the system is shown in Table 11:

表11Table 11

成分Element体积（uL）Volume (uL)质粒plasmidX(500ng)X (500ng)10×FD buffer10×FDbuffer22酶1Enzyme 10.50.5酶2Enzyme 20.50.5H₂OH₂ O17-X17-X

酶切条件：37℃,30min；85℃，5min,最后跑1%琼脂糖凝胶电泳鉴定是否有目的条带。Digestion conditions: 37°C, 30min; 85°C, 5min, and finally run 1% agarose gel electrophoresis to identify the target band.

3.5.8质粒PCR鉴定3.5.8 Plasmid PCR identification

酶切鉴定阳性的质粒再PCR鉴定是否有目的条带，反应体系如表12和表13所示：The positive plasmid identified by restriction enzyme digestion is then identified by PCR to see if there is a target band. The reaction system is shown in Table 12 and Table 13:

表12Table 12

成分Element体积（uL）Volume (uL)H₂OH₂ O14.814.810×pfu buffer(Mg²⁺)10×pfu buffer(Mg²⁺ )22dNTP(10mM)dNTP (10mM)11taqtaq0.20.2Sense primer(20uM)Sense primer(20uM)0.50.5Anti-sense primer(20uM)Anti-sense primer(20uM)0.50.5酶切鉴定阳性质粒Enzyme digestion to identifypositive plasmids11

表13Table 13

PCR产物跑1%琼脂糖凝胶电泳，其结果参照图7-图9；若有阳性条带，则进行测序。Run 1% agarose gel electrophoresis on the PCR product, and refer to Figure 7-9 for the results; if there is a positive band, perform sequencing.

3.6测序比对和结构分析3.6 Sequence comparison and structure analysis

保藏号480CT5.4.3测序比对和结构分析：Accession number 480CT5.4.3 Sequence comparison and structural analysis:

VH链通过FR-IGMT和CDR-IGMT分析显示：VH chain analysis by FR-IGMT and CDR-IGMT shows:

CDR1：GGGTATACCTTCACAGACTATGGACDR1: GGGTATACCTTTCACAGACTATGGA

CDR2：ATAAACACCTACACTGGAGCCCCACDR2: ATAAACACCTACACTGGAGCCCCA

CDR3：GCAAAATCGTATGGGTGGTATTTTGATGTGCDR3: GCAAAATCGTATGGGTGGTATTTTGATGTG

VH基因属于VGAM3.8VH9家族The VH gene belongs to the VGAM3.8VH9 family

VL链通过FR-IGMT和CDR-IGMT分析显示：VL chain analysis by FR-IGMT and CDR-IGMT showed:

CDR1：CAGAGCATTCTACATAGTAATGGAAACACCTATCDR1: CAGAGCATTCTACATAGTAATGGAAACACCTAT

CDR2：CTGATCTACAAAGTTTCCCDR2: CTGATCTACAAAGTTTCC

CDR3：TTTCAAGGTTCACATGTTCCGTGGACGCDR3: TTTCAAGGTTCACATGTTCCGTGGACG

VL基因属于IGKV1亚群The VL gene belongs to the IGKV1 subgroup

保藏号480CT13.4.3.2测序比对和结构分析：Accession No. 480CT13.4.3.2 Sequence comparison and structural analysis:

VH链通过FR-IGMT和CDR-IGMT分析显示：VH chain analysis by FR-IGMT and CDR-IGMT shows:

CDR1：GGCTACACCTTCACCAGCTACTGGCDR1: GGCTACACCTTCACCAGCTACTGG

CDR2：ATTGGAGAGATTCATCCTAATAGTGGTAATATTCDR2: ATTGGAGAGATTCATCCTAATAGTGGTAATATT

CDR3：GCAAGACTGGGACGTGACTACCDR3: GCAAGACTGGGACGTGACTAC

VH基因属于VJ558VH1家族The VH gene belongs to the VJ558VH1 family

VL链通过FR-IGMT和CDR-IGMT分析显示：VL chain analysis by FR-IGMT and CDR-IGMT showed:

CDR1：CAGGATATTAGCAATTATCDR1: CAGGATATTAGCAATTAT

CDR2：CTGATCTACTACACATCACDR2: CTGATCTACTACACATCA

CDR3：CAGCAGTATAGTAAGCGTCCGTGGACGCDR3: CAGCAGTATAGTAAGCGTCCGTGGACG

VL基因属于IGKV10亚群The VL gene belongs to the IGKV10 subgroup

保藏号587CT7.3.6.5测序比对和结构分析：Accession number 587CT7.3.6.5 Sequence comparison and structural analysis:

VH链通过FR-IGMT和CDR-IGMT分析显示：VH chain analysis by FR-IGMT and CDR-IGMT shows:

CDR1：GGATACACATTCACTGACTACTACCDR1: GGATACACATTCACTGACTACTAC

CDR2：ATTAATCCTTACAATGGTGATACTCDR2: ATTAATCCTTACAATGGTGATACT

CDR3：GCAAGAGGGGATGGCCTTGCTTACCDR3: GCAAGAGGGGATGGCCTTGCTTAC

VH基因属于Igh-VJ558VH1家族The VH gene belongs to the Igh-VJ558VH1 family

VL链通过FR-IGMT和CDR-IGMT分析显示：VL chain analysis by FR-IGMT and CDR-IGMT showed:

CDR1：CAGAGCATTGTACACAGTAATGGAAACACCTATCDR1: CAGAGCATTGTACACAGTAATGGAAACACCTAT

CDR2：AGGGTTTCCCDR2: AGGGTTTCC

CDR3：TTTCAAGGTACACATGTTCCGCTCACGCDR3: TTTCAAGGTACACATGTTCCGCTCACG

VL基因属于IGKV1亚群The VL gene belongs to the IGKV1 subgroup

本发明提供了一种表达载体，表达载体包括了本发明所述的任意一个核苷酸序列，本发明优选的表达载体是质粒，除了抗体链编码序列之外，载体将包括用于在预期宿主细胞中编码序列的适当转录和翻译的必要的调节序列。本发明的表达载体包含抗CD138单克隆抗体可变区序列。The present invention provides an expression vector. The expression vector includes any one of the nucleotide sequences described in the present invention. The preferred expression vector of the present invention is a plasmid. In addition to the antibody chain coding sequence, the vector will include Regulatory sequences necessary for proper transcription and translation of coding sequences in a cell. The expression vector of the present invention comprises the variable region sequence of the anti-CD138 monoclonal antibody.

本发明所使用的表达宿主，含有上述的表达载体。该表达宿主是宿主细胞，宿主细胞可以是任何适合的类型。在优选的实施方式中，宿主细胞即被转染的细胞是真核细胞，更优选的是脊椎动物细胞，最优选的是哺乳动物细胞。宿主细胞可以使用标准技术和转染条件利用表达载体来转染，这是本技术领域中惯用的。The expression host used in the present invention contains the above-mentioned expression vector. The expression host is a host cell, which can be of any suitable type. In a preferred embodiment, the host cell, ie the cell to be transfected, is a eukaryotic cell, more preferably a vertebrate cell, most preferably a mammalian cell. Host cells can be transfected with expression vectors using standard techniques and transfection conditions, as is routine in the art.

本发明提供的核苷酸序列，例如：58208（1）-VH、58208（1）-VL、58208（2）-VH、58208（2）-VL、59166-VH和59166-VL可应用在制备抗体上，制备抗体包括：重组抗体、ScFv抗体、人源化抗体、嵌合抗体、双特异性抗体、单域抗体。The nucleotide sequences provided by the present invention, for example: 58208(1)-VH, 58208(1)-VL, 58208(2)-VH, 58208(2)-VL, 59166-VH and 59166-VL can be used in the preparation of In terms of antibodies, the preparation of antibodies includes: recombinant antibodies, ScFv antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, and single domain antibodies.

以上所述，是结合实施例对发明的详细阐述，应理解的是，这些实施例仅用于说明本发明而不是对本发明的限制，在本发明的构思前题下对本发明制备方法的简单改进，对本发明可变区核苷酸序列的利用都属于本发明要求保护的范围。The above is a detailed elaboration of the invention in conjunction with the examples. It should be understood that these examples are only used to illustrate the present invention rather than to limit the present invention. The simple improvement of the preparation method of the present invention under the concept of the present invention , the utilization of the nucleotide sequence of the variable region of the present invention all falls within the scope of protection claimed by the present invention.

58208（1）-VH核苷酸序列为：The 58208(1)-VH nucleotide sequence is:

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAGACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGTCTGGATAAACACCTACACTGGAGCCCCAACATTTGCTGATGACTTCAAGGGACGGTTTGCCCTGTCATTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAAATCGTATGGGTGGTATTTTGATGTGTGGGGCGCAGGGACCACGGTCACCGTCTCCTCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAGACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGTCTGGATAAACACCTACACTGGAGCCCCAACATTTGCTGATGACTTCAAGGGACGGTTTGCCCTGTCATTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCAAAATCGTATGGGTGGTATTTTGATGTGTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA

58208（2）-VH核苷酸序列为：The 58208(2)-VH nucleotide sequence is:

CAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGCGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTCATCCTAATAGTGGTAATATTAACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACTGGGACGTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCACAGGTCCAACTGCAGCAGCCTGGGTCTGTGCTGGTGAGGCCTGGAGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGCGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTCATCCTAATAGTGGTAATATTAACTACAATGAGAAGTTCAAGGGCAAGGCCACACTGACTGTAGACACATCCTCCAGCACAGCCTACGTGGATCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACTGGGACGTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA

59166-VH核苷酸序列为：The 59166-VH nucleotide sequence is:

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGGCTTTAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGAAATTAATCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCTAGTACAGCCTACATGGAGCTCCGGAGCCTGACATCTGAGGACTCTGCAGTCTATTATTGTGCAAGAGGGGATGGCCTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAACCTGGGGCTTTAGTGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTGACTACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGAAATTAATCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCTAGTACAGCCTACATGGAGCTCCGGAGCCTGACATCTGAGGACTCTGCAGTCTATTATTGTGCAAGAGGGGATGGCCTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

58208（1）-VH编码氨基酸序列为：58208(1)-VH encodes the amino acid sequence as:

QIQLVQSGPELKKPGETVKISCKASGYTFTDYGMNWVKQAPGKGLKWMVWINTYTGAPTFADDFKGRFALSLETSASTAYLQINNLKNEDTATYFCAKSYGWYFDVWGAGTTVTVSS QIQLVQSGPELKKPGETVKISCKASGYTFTDYGMNWVKQAPGKGLKWMVWINTYTGAPTFADDFKGRFALSLETSASTAYLQINNLKNEDTATYFCAKSYGWYFDVWGAGTTVTVSS

58208（2）-VH编码氨基酸序列为：The 58208(2)-VH encoded amino acid sequence is:

QIQLVQSGPELKKPGETVKISCKASGYTFTDYGMNWVKQAPGKGLKWMVWINTYTGAPTFADDFKGRFALSLETSASTAYLQINNLKNEDTATYFCAKSYGWYFDVWGAGTTVTVSSQIQLVQSGPELKKPGETVKISCKASGYTFTDYGMNWVKQAPGKGLKWMVWINTYTGAPTFADDFKGRFALSLETSASTAYLQINNLKNEDTATYFCAKSYGWYFDVWGAGTTVTVSS

59166-VH编码氨基酸序列为：The amino acid sequence encoded by 59166-VH is:

EVQLQQSGPELVKPGALVKMSCKASGYTFTDYYMHWVKQSHGKSLEWIGEINPYNGDTFYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGDGLAYWGQGTLVTVSAEVQLQQSGPELVKPGALVKMSCKASGYTFTDYYMHWVKQSHGKSLEWIGEINPYNGDTFYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARGDGLAYWGQGTLVTVSA

58208（1）-VL核苷酸序列为：The 58208(1)-VL nucleotide sequence is:

GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTCTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAATAGATTTTCTGGGGTCCCAGACAGGTTCAGTGGAAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTCTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAATAGATTTTCTGGGGTCCCAGACAGGTTCAGTGGAAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC

58208（2）-VL核苷酸序列为：The 58208(2)-VL nucleotide sequence is:

GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTTTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGATATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGCGTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTTTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGATATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTACTACACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGCGTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC

59166-VL核苷酸序列为：The 59166-VL nucleotide sequence is:

GATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACACAGTAATGGAAACACCTATTTATATTGGTACCTGCAGAAAGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACACAGTAATGGAAACCTATTATATTGGTACCTGCAGAAA

CCAGGCCAGTCTCCAAAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATATGGGAGTTTATTACTGCTTTCAAGGTACACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACCCAGGCCAGTCTCCAAAGCTCCTGATCTACAGGGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAAGATCAGCAGAGTGGAGGCTGAGGATATGGGAGTTTATTACTGCTTTCAAGGTACACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC

58208（1）-VL编码氨基酸序列为：The amino acid sequence encoded by 58208(1)-VL is:

DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPWTFGGGTKLEIK  DVLMTQTPLSLPVSLGDQASISCRSSQSILHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGSHVPWTFGGGTKLEIK

58208（2）-VL编码氨基酸序列为：The amino acid sequence encoded by 58208(2)-VL is:

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKRPWTFGGGTKLEIKDIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKRPWTFGGGTKLEIK

59166-VL编码氨基酸序列为：The amino acid sequence encoded by 59166-VL is:

DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLYWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDMGVYYCFQGTHVPLTFGAGTKLELKDVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLYWYLQKPGQSPKLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDMGVYYCFQGTHVPLTFGAGTKLELK

 the

Claims (9)

Translated fromChinese

1.抗CD138单克隆抗体可变区序列，其特征在于，包含六种核苷酸序列，所述核苷酸序列分别为58208（1）-VH、58208（1）-VL、58208（2）-VH、58208（2）-VL、59166-VH和59166-VL。1. The variable region sequence of the anti-CD138 monoclonal antibody is characterized in that it contains six nucleotide sequences, the nucleotide sequences are respectively 58208(1)-VH, 58208(1)-VL, and 58208(2) -VH, 58208(2)-VL, 59166-VH, and 59166-VL.2.根据权利要求1所述的抗CD138单克隆抗体可变区序列，其特征在于，所述核苷酸序列58208（1）-VH和58208（1）-VL由保藏号为480CT5.4.3的杂交瘤细胞株制备而成，所述核苷酸序列58208（2）-VH和58208（2）-VL由保藏号为480CT13.4.3.2的杂交瘤细胞株制备而成，所述核苷酸序列59166-VH和59166-VL由保藏号为587CT7.3.6.5的杂交瘤细胞株制备而成。2. The anti-CD138 monoclonal antibody variable region sequence according to claim 1, characterized in that, the nucleotide sequences 58208(1)-VH and 58208(1)-VL are obtained from the depository number 480CT5.4.3 Prepared from a hybridoma cell line, the nucleotide sequences 58208(2)-VH and 58208(2)-VL are prepared from a hybridoma cell line with a preservation number of 480CT13.4.3.2, and the nucleotide sequences The sequences 59166-VH and 59166-VL are prepared from the hybridoma cell line with the deposit number 587CT7.3.6.5.3.根据权利要求1所述的抗CD138单克隆抗体可变区序列，其特征在于，还包括：与上述六种核苷酸序列具有相同氨基酸序列产物的核苷酸序列。3. The anti-CD138 monoclonal antibody variable region sequence according to claim 1, further comprising: a nucleotide sequence having the same amino acid sequence product as the above six nucleotide sequences.4.根据权利要求3所述的抗CD138单克隆抗体可变区序列，其特征在于，还包括：经过一个或者几个碱基替换、缺失或添加后仍具有与所述核苷酸序列产生的氨基酸序列具有相同活性的核苷酸序列。4. the anti-CD138 monoclonal antibody variable region sequence according to claim 3, is characterized in that, also comprises: after one or several base substitutions, deletions or additions, still have the sequence produced with the nucleotide sequence An amino acid sequence has the same activity as a nucleotide sequence.5.制备权利要求1至4任意一项所述的抗CD138单克隆抗体可变区序列的方法，其特征在于，包括以下步骤：5. The method for preparing the variable region sequence of the anti-CD138 monoclonal antibody according to any one of claims 1 to 4, characterized in that it comprises the following steps:（1）杂交瘤细胞的制备：首先用CD138蛋白设计合成的多肽以及U266细胞作为抗原来免疫雌性健康的BALB/c小鼠，挑选出免疫后抗体表达呈阳性的小鼠，取其脾脏细胞，然后将分离的小鼠脾脏细胞与骨髓瘤细胞融合，形成杂交瘤细胞；(1) Preparation of hybridoma cells: Firstly, the polypeptide designed and synthesized by CD138 protein and U266 cells were used as antigens to immunize healthy female BALB/c mice, and the mice with positive antibody expression after immunization were selected, and the spleen cells were collected. The isolated mouse spleen cells were then fused with myeloma cells to form hybridoma cells;（2）单克隆细胞的筛选：将上述杂交瘤细胞在HAT培养基中培养，对ELISA检测呈阳性的单克隆细胞进行ELISA复筛，然后将筛选出的ELISA阳性单克隆细胞进行WB复筛，再将WB阳性细胞进行亚克隆，经过2次亚克隆，筛选出能够稳定分泌抗体的单克隆细胞，将阳性的单克隆细胞扩大培养后定株、冻存；(2) Screening of monoclonal cells: culture the above-mentioned hybridoma cells in HAT medium, perform ELISA re-screening on the monoclonal cells that are positive in the ELISA test, and then perform WB re-screening on the screened ELISA-positive monoclonal cells, Then subclone the WB positive cells, after 2 times of subcloning, screen out the monoclonal cells that can stably secrete antibodies, expand the positive monoclonal cells, determine the strains, and freeze them;（3）杂交瘤细胞抗CD138单克隆抗体的获得：首先鉴定步骤（2）所制备的单克隆细胞的亚类亚型，然后将阳性的单克隆细胞注射到小鼠体内进行腹水生产，再将产生的腹水通过层析纯化后得到抗CD138单克隆抗体；(3) Obtaining anti-CD138 monoclonal antibody of hybridoma cells: first identify the subtype of the monoclonal cells prepared in step (2), then inject the positive monoclonal cells into mice for ascites production, and then inject The produced ascites was purified by chromatography to obtain anti-CD138 monoclonal antibody;（4）抗CD138单克隆抗体重链和轻链可变区基因的克隆：提取步骤（3）中所用的单克隆细胞的总RNA，然后获得mRNA，再以所述的mRNA为模板，反转录得到cDNA，最后克隆得到抗CD138单克隆抗体的重链可变区和轻链可变区。(4) Cloning of heavy chain and light chain variable region genes of anti-CD138 monoclonal antibody: extract the total RNA of the monoclonal cells used in step (3), then obtain mRNA, and then use the mRNA as a template to reverse The cDNA was recorded, and finally the heavy chain variable region and the light chain variable region of the anti-CD138 monoclonal antibody were cloned.6.一种表达载体，其特征在于，含有权利要求1至4任意一项所述的核苷酸序列。6. An expression vector, characterized in that it contains the nucleotide sequence according to any one of claims 1-4.7.一种表达宿主，其特征在于，载有权利要求6所述的表达载体。7. An expression host, characterized in that it carries the expression vector according to claim 6.8.核苷酸序列在制备抗体上的应用，其特征在于，上述核苷酸序列包括：58208（1）-VH、58208（1）-VL、58208（2）-VH、58208（2）-VL、59166-VH和59166-VL。8. The application of nucleotide sequences in the preparation of antibodies, characterized in that the above nucleotide sequences include: 58208(1)-VH, 58208(1)-VL, 58208(2)-VH, 58208(2)- VL, 59166-VH, and 59166-VL.9.根据权利要求8所述的核苷酸序列在制备抗体上的应用，其特征在于，上述抗体包括：重组抗体、ScFv抗体、人源化抗体、嵌合抗体、双特异性抗体、单域抗体。9. The application of the nucleotide sequence according to claim 8 in the preparation of antibodies, characterized in that said antibodies include: recombinant antibodies, ScFv antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single domain antibodies Antibody.

Images