CN101967493A - Prokaryotic expression vector and application thereof - Google Patents

Abstract

Translated fromChinese

一种原核表达载体及其应用，载体包含有(a)促进目标蛋白可溶性表达或/和二硫键正确折叠的分子伴侣和(b)能在C末端肽键断裂的内含肽的编码核苷酸序列。该载体通过分子伴侣促进高效可溶性表达目标蛋白并使目标蛋白保持天然生物活性，而且，通过在一定条件下有效诱导内含肽的自我剪切作用使目标蛋白与融合蛋白分离后，再分离、纯化目标蛋白，可以经济高效地获得保持有天然生物活性的目标蛋白。本发明还涉及包含上述载体的宿主细胞及上述载体在重组表达生物活性蛋白中的应用。

A prokaryotic expression vector and its application, the vector contains (a) a molecular chaperone that promotes the soluble expression of the target protein or/and the correct folding of the disulfide bond and (b) the encoding nucleoside of the intein that can break the C-terminal peptide bond acid sequence. The carrier promotes high-efficiency soluble expression of the target protein through molecular chaperones and maintains the natural biological activity of the target protein. Moreover, the target protein is separated from the fusion protein by effectively inducing the self-cleavage of intein under certain conditions, and then separated and purified. Target protein, which can cost-effectively obtain target protein that maintains natural biological activity. The present invention also relates to the host cell containing the above-mentioned vector and the application of the above-mentioned vector in the recombinant expression of biologically active protein.

Description

Translated fromChinese

一种原核表达载体及其应用A kind of prokaryotic expression vector and its application

技术领域technical field

本发明属于生物工程技术领域。具体的说，本发明涉及一种原核表达载体及包含该载体的宿主细胞与该载体在重组表达生物活性蛋白中的应用。The invention belongs to the technical field of bioengineering. Specifically, the present invention relates to a prokaryotic expression vector, a host cell containing the vector and the application of the vector in recombinantly expressing biologically active proteins.

技术背景technical background

大肠杆菌是目前生产重组外源蛋白的主要工程菌。然而，目前利用原核系统表达生产重组蛋白，重组蛋白常出现以不溶的包含体(inclusion body)的形式存在、表达量低、生物活性不高等问题。将分子伴侣(molecular chaperone或chaperone；也称融合伴侣，fusion partner或融合标签，fusion tag)和目标蛋白连接成为一个融合蛋白进行表达是增加目标蛋白可溶性表达的常用策略之一。目前最常用的分子伴侣有谷胱甘肽转移酶(GST)、硫氧还蛋白(Trx)、麦芽糖结合蛋白(MBP)、NusA蛋白、二硫键还原酶及异构酶(Dsb家族，如DsbA、DsbC)、小泛素修饰蛋白(SUMO)及热休克蛋白(heat shock protein，HSP)等(Esposito D，Chatterjee DK.Curr Opin Biotechnol.2006，17：353-358；Betiku E.Biotechnology and Molecular Biology Review.2006，1：66-75)。对生产重组蛋白而言，不仅要获得高产量可溶性重组蛋白，更重要的是要保证重组蛋白的生物学活性。特别对于许多含二硫键的蛋白质，正确的二硫键折叠在维持蛋白质的结构和活性方面起着重要的作用。在原核表达系统中，融合表达不仅可以提高蛋白表达量及可溶性，部分分子伴侣还可以帮助目标蛋白正确折叠，从而保持较好生物活性。如，二硫键还原酶及异构酶(Dsb家族，如DsbA、DsbC)、硫氧还蛋白(Trx)及小泛素修饰蛋白(SUMO)等能促进表达的目标蛋白的二硫键的正确连接，从而使表达的目标蛋白保持天然生物活性(Marblestone JG et al.Protein Sci.2006，15：182-189；Panavas T et al.Methods Mol Biol.2009，497：303-317；Yasukawa T et al，J Biol Chem.1995，270：25328-25331；Lauber T et al，Protein Expr Purif.2001.22：108-112)。为纯化的方便，可在目标蛋白融合相应亲和纯化标签(affinity purification tags)，如6×his-tag、GST、MBP、FLAG，BAP、Strep-II及CBP等(Esposito D，Chatterjee DK.Curr Opin Biotechnol.2006，17：353-358)。然而，融合表达不足之处是在融合表达后往往要通过特异的蛋白酶，如肠激酶、fXa等的酶切或化学裂解除去融合标签和分子伴侣。这就可能需要二次纯化，并且引入酶切的蛋白酶价格昂贵，会带来成本的增加，并且可能导致非特异性切割(谢浩等.生物化学与生物物理进展.2009，36：1364-1369)。另外，还存在切除融合标签后目标蛋白重新聚集形成沉淀现象。Escherichia coli is currently the main engineering bacteria for the production of recombinant foreign proteins. However, prokaryotic systems are currently used to express and produce recombinant proteins, and recombinant proteins often appear in the form of insoluble inclusion bodies, low expression levels, and low biological activity. Linking molecular chaperone (molecular chaperone or chaperone; also known as fusion partner, fusion partner or fusion tag, fusion tag) and target protein into a fusion protein for expression is one of the common strategies to increase the soluble expression of target protein. The most commonly used molecular chaperones are glutathione transferase (GST), thioredoxin (Trx), maltose binding protein (MBP), NusA protein, disulfide bond reductase and isomerase (Dsb family, such as DsbA , DsbC), small ubiquitin-modified protein (SUMO) and heat shock protein (heat shock protein, HSP), etc. (Esposito D, Chatterjee DK. Curr Opin Biotechnol. 2006, 17: 353-358; Betiku E. Review. 2006, 1:66-75). For the production of recombinant proteins, it is not only necessary to obtain high-yield soluble recombinant proteins, but more importantly, to ensure the biological activity of recombinant proteins. Especially for many disulfide bond-containing proteins, correct disulfide bond folding plays an important role in maintaining protein structure and activity. In prokaryotic expression systems, fusion expression can not only improve protein expression and solubility, but some molecular chaperones can also help the target protein to fold correctly, thereby maintaining better biological activity. For example, disulfide bond reductase and isomerase (Dsb family, such as DsbA, DsbC), thioredoxin (Trx) and small ubiquitin-modified protein (SUMO) can promote the correct disulfide bond of the expressed target protein. Linked, so that the expressed target protein maintains natural biological activity (Marblestone JG et al. Protein Sci.2006, 15: 182-189; Panavas T et al. Methods Mol Biol. , J Biol Chem.1995, 270:25328-25331; Lauber T et al, Protein Expr Purif.2001.22:108-112). For the convenience of purification, the target protein can be fused with corresponding affinity purification tags (affinity purification tags), such as 6×his-tag, GST, MBP, FLAG, BAP, Strep-II and CBP, etc. (Esposito D, Chatterjee DK.Curr Opin Biotechnol. 2006, 17: 353-358). However, the disadvantage of fusion expression is that fusion tags and molecular chaperones are usually removed by specific proteases, such as enterokinase, fXa, etc., after digestion or chemical cleavage. This may require secondary purification, and the introduction of cleavage proteases is expensive, which will increase the cost and may lead to non-specific cleavage (Xie Hao et al. Advances in Biochemistry and Biophysics. 2009, 36: 1364-1369) . In addition, there is also the phenomenon that the target protein re-aggregates to form a precipitate after the fusion tag is cut off.

内含肽(intein)是存在于前体蛋白中的一段序列，在前体蛋白转化为成熟蛋白质的过程中，依靠自我剪切作用从前体蛋白中释放出来，同时将两端的蛋白质外显肽(extein)连接在一起。内含肽经诱导能够介导N端或C端单侧肽键断裂。在蛋白质工程方面，内含肽具有自我剪切特性的这种特性可以实现目标蛋白与亲和标签分离的目的。如将编码亲和标签、内含肽及目标蛋白的基因序列连接在一起，在合适的表达系统中表达出一个亲和标签(Tag)-内含肽-目标蛋白的三联体，再利用亲和层析吸附标签而截留融合蛋白，随后在理化因素作用下(如pH、温度的变化或者巯基化合物的加入)该三联体从内含肽的N-端或者C-端发生自我剪切释放目标蛋白。新英格兰生物实验室(New England Biolabs，NEB)根据此原理已经构建IMPACT^TM，如pTWIN1表达载体，该系统不需用蛋白酶酶切。IMPACT^TM系统表达的目标蛋白与内含肽及几丁质结合蛋白形成融合蛋白，通过几丁质柱亲和纯化融合蛋白。然后诱导纯化融合蛋白中的内含肽的肽键裂解活性，在几丁质介质上将目标蛋白释放出来，而内含肽与几丁质结合蛋白(CBD)仍结合在几丁质介质上，达到单柱分离纯化蛋白的目的。这种通过引入内含肽自我剪切亲和纯化而避免了外源蛋白酶的加入以及后期对蛋白酶的清除步骤。而且内含肽介导的裂解仅发生在剪接位点，与目标蛋白上的蛋白酶敏感位点无关，也避免了蛋白酶对目标蛋白的降解(Chong S et al.Gene.1997，192：277-281；Sharma SS et al.J Biotechnol.2006，125：48-56)。Intein is a sequence that exists in the precursor protein. During the process of converting the precursor protein into a mature protein, it is released from the precursor protein by self-cleavage, and at the same time, the protein exteins at both ends ( extein) together. Inteins are induced to mediate N-terminal or C-terminal unilateral peptide bond cleavage. In terms of protein engineering, the self-cleavage property of inteins can achieve the purpose of separating target proteins from affinity tags. For example, link the gene sequence encoding affinity tag, intein and target protein together, express a triplet of affinity tag (Tag)-intein-target protein in a suitable expression system, and then use affinity Chromatography adsorbs the label to trap the fusion protein, and then under the action of physical and chemical factors (such as pH, temperature change or the addition of sulfhydryl compounds), the triplet undergoes self-cleavage from the N-terminal or C-terminal of the intein to release the target protein . Based on this principle, New England Biolabs (NEB) has constructed IMPACT^TM , such as pTWIN1 expression vector, which does not require protease digestion. The target protein expressed by the IMPACT^TM system forms a fusion protein with intein and chitin-binding protein, and the fusion protein is affinity-purified through a chitin column. Then induce the peptide bond cleavage activity of the intein in the purified fusion protein, and release the target protein on the chitin medium, while the intein and the chitin-binding protein (CBD) are still bound on the chitin medium, To achieve the purpose of single-column separation and purification of protein. The introduction of intein self-cleavage affinity purification avoids the addition of exogenous proteases and the subsequent removal of proteases. Moreover, intein-mediated cleavage only occurs at the splicing site and has nothing to do with the protease-sensitive site on the target protein, and also avoids the degradation of the target protein by proteases (Chong S et al. Gene.1997, 192: 277-281 ; Sharma SS et al. J Biotechnol. 2006, 125:48-56).

但目前通过内含肽介导生产重组蛋白均是先分离、纯化融合蛋白，然后再对在亲和介质上的融合蛋白(如用镍柱亲和带his-tag的融合蛋白，几丁质介质亲和带CBD的融合蛋白)进行诱导剪切，以获得目标蛋白(Chong S et al.Gene.1997，192：277-281；Sharma SS et al.J Biotechnol.2006，125：48-56)。该法往往造成在融合蛋白表达过程中及其分离纯化过程中发生的提前剪切的目标蛋白大量损失。同时，内含肽也不能有效提高目标蛋白的产量、可溶性表达及促进其二硫键的有效折叠。However, at present, the production of recombinant proteins mediated by intein is to first isolate and purify the fusion protein, and then analyze the fusion protein on the affinity medium (such as the fusion protein with his-tag with nickel column affinity, chitin medium). Affinity fusion protein with CBD) to obtain target protein (Chong S et al. Gene.1997, 192: 277-281; Sharma SS et al. J Biotechnol. 2006, 125: 48-56). This method often results in a large loss of the prematurely cleaved target protein that occurs during the expression of the fusion protein and its separation and purification. At the same time, inteins cannot effectively increase the yield, soluble expression and promote the effective folding of the target protein.

钩虫抗凝肽是从吸血寄生钩虫发现的一类具有显著抗凝活性的多肽，通常由80个氨基酸组成，含5对二硫键，可能开发为抗凝抗栓新药或试剂(彭礼飞等.广东医学院学报.2006，24：624-627)。其中，本发明人课题组发现的源自犬钩虫的抗凝肽AcaNAP10具有很强的抗凝活性，是凝血途径启动阶段关键凝血因子-组织因子与活化的凝血因子VII复合物(TF/fVIIa)及凝血途径放大阶段关键因子-活化的凝血因子XI(fXIa)的高效抑制剂(Li Det al.Biochem Biophys Res Commun.2010，392：155-159)，是目前唯一既能抑制TF/fVIIa又能抑制fXIa的抗凝物质，其作为抗凝抗栓新药或试剂开发值得期待。Hookworm anticoagulant peptides are a class of peptides with significant anticoagulant activity discovered from blood-sucking parasitic hookworms. They usually consist of 80 amino acids and contain 5 pairs of disulfide bonds. They may be developed as new anticoagulant and antithrombotic drugs or reagents (Peng Lifei et al. Guangdong Journal of Medical College. 2006, 24:624-627). Among them, the anticoagulant peptide AcaNAP10 derived from H. caninum found by the inventor's research group has strong anticoagulant activity, and is a key coagulation factor-tissue factor and activated coagulation factor VII complex (TF/fVIIa) in the initiation stage of the coagulation pathway. and the key factor in the amplification stage of the coagulation pathway - an efficient inhibitor of activated coagulation factor XI (fXIa) (Li De et al. Biochem Biophys Res Commun. 2010, 392: 155-159), is currently the only one that can both inhibit TF/fVIIa and Anticoagulant substances that inhibit fXIa are worth looking forward to as new anticoagulant and antithrombotic drugs or reagents.

为了重组表达商用蛋白，特别是希望通过原核表达系统高效地表达可溶性、保持天然生物活性的目标蛋白，减少分离目标蛋白与融合标签和分子伴侣所带来的生产成本以及较大量的获得目标蛋白，本发明人研制了一种原核表达载体，并提供了对表达的融合蛋白首先进行有效诱导剪切使目标蛋白与融合蛋白分离，然后再对与融合蛋白分离的目标蛋白进行分离纯化的方法，并进一步揭示了该表达载体在表达含二硫键的蛋白，特别是钩虫抗凝肽AcaNAP10的应用。In order to recombinantly express commercial proteins, it is especially hoped to efficiently express soluble target proteins that maintain natural biological activity through prokaryotic expression systems, reduce the production costs caused by the separation of target proteins, fusion tags and molecular chaperones, and obtain large quantities of target proteins. The inventors have developed a prokaryotic expression vector, and provided a method for effectively inducing shearing of the expressed fusion protein to separate the target protein from the fusion protein, and then separating and purifying the target protein separated from the fusion protein, and It further reveals the application of the expression vector in expressing proteins containing disulfide bonds, especially hookworm anticoagulant peptide AcaNAP10.

发明内容Contents of the invention

本发明的目的在于提供一种原核表达载体及其应用。该载体能通过分子伴侣促进高效可溶性地表达目标蛋白并使目标蛋白，特别是含有二硫键的目标蛋白保持天然生物活性。应用该载体表达的目标蛋白能通过内含肽的自我剪切作用实现目标蛋白与融合蛋白经济高效地分离，分离后的目标蛋白进一步分离纯化，可经济高效获得大量目的蛋白。The purpose of the present invention is to provide a prokaryotic expression vector and its application. The carrier can promote efficient and soluble expression of the target protein through molecular chaperones and maintain the natural biological activity of the target protein, especially the target protein containing a disulfide bond. The target protein expressed by this vector can be economically and efficiently separated from the fusion protein through the self-cleavage of intein, and the separated target protein can be further separated and purified to obtain a large amount of target protein economically and efficiently.

本发明提供了一种原核表达载体，构建本发明中所述表达载体的出发载体可为基因工程领域中所述各种原核表达载体，如pET系列表达载体。通过基因工程常规方法可把能促进目标蛋白可溶性表达或/和二硫键正确折叠的分子伴侣和能在C端自我剪切的内含肽编码核苷酸序列连接入出发载体中。该载体通过分子伴侣促进高效可溶性表达目标蛋白或/和目标蛋白的二硫键正确折叠以提高目标蛋白产量并保持天然活性，并通过融合在分子伴侣的内含肽的C末端的自我剪切作用使目标蛋白与融合蛋白分离。所述的分子伴侣与内含肽之间可由或长或短一段序列(编码柔性氨基酸区)连接。该载体也可基于根据纯化需要在融合伴侣中设计亲和标签，如6×his-tag、GST、MBP、FLAG，BAP、Strep-II、CBD及CBP等(Esposito D，Chatterjee DK.Curr Opin Biotechnol.2006，17：353-358)，用于简单快捷亲和层析除去还没有剪切完的融合蛋白，如可用几丁质柱亲和除去带CBD的没有剪切完的融合蛋白。常见的能促进目标蛋白可溶性表达或/和二硫键正确折叠地方融合的融合伴侣有谷胱甘肽转移酶(GST)、硫氧还蛋白(Trx)、麦芽糖结合蛋白(MBP)、NusA蛋白、二硫键还原酶及异构酶(Dsb家族，如DsbA、DsbC)、小泛素修饰蛋白(SUMO)及热休克蛋白(heat shock protein，HSP)等，但不仅限于上述蛋白。能在C端自我剪切的内含肽包括mini Ssp DnaB(Mathys S et al.Gene.1999，231：1-13)及Sce VMA(Chong S et al.Gene.1997，192：277-281)等，以mini Ssp DnaB为佳，但不仅限于上述蛋白。The present invention provides a prokaryotic expression vector. The starting vector for constructing the expression vector in the present invention can be various prokaryotic expression vectors described in the field of genetic engineering, such as pET series expression vectors. The molecular chaperone that can promote the soluble expression of the target protein or/and the correct folding of the disulfide bond and the intein coding nucleotide sequence that can be self-cleaved at the C-terminus can be linked into the starting vector by conventional methods of genetic engineering. The carrier promotes high-efficiency soluble expression of the target protein or/and the correct folding of the disulfide bond of the target protein through the molecular chaperone to increase the yield of the target protein and maintain natural activity, and through the self-cleavage of the C-terminus of the intein fused to the molecular chaperone Separate the protein of interest from the fusion protein. The molecular chaperone and the intein may be connected by a long or short sequence (coding flexible amino acid region). The vector can also be based on the design of affinity tags in the fusion partner according to purification needs, such as 6×his-tag, GST, MBP, FLAG, BAP, Strep-II, CBD and CBP, etc. (Esposito D, Chatterjee DK. Curr Opin Biotechnol .2006, 17: 353-358), used for simple and fast affinity chromatography to remove uncut fusion protein, for example, a chitin column can be used to affinity remove uncut fusion protein with CBD. Common fusion partners that can promote the soluble expression of the target protein or/and the correct folding of disulfide bonds include glutathione transferase (GST), thioredoxin (Trx), maltose binding protein (MBP), NusA protein, Disulfide bond reductase and isomerase (Dsb family, such as DsbA, DsbC), small ubiquitin-modified protein (SUMO) and heat shock protein (heat shock protein, HSP), etc., but not limited to the above-mentioned proteins. Inteins capable of self-cleavage at the C-terminal include mini Ssp DnaB (Mathys S et al. Gene. 1999, 231: 1-13) and Sce VMA (Chong S et al. Gene. 1997, 192: 277-281) etc., preferably mini Ssp DnaB, but not limited to the above proteins.

利用发明载体高效融合表达的目标蛋白与融合蛋白的通过自我剪切分离，可在0℃～40℃下，较佳的是在低温条件下，如0℃～20℃下，pH为6.0～8.0的缓冲液中进行。在低温下进行自我剪切能避免或减少对目的蛋白的降解。与融合蛋白分离后的目标蛋白的分离、纯化方法有：可以根据目标蛋白的大小、带电荷及等电点等理化性质，用相应的常规纯化技术进行分离纯化；为纯化方便，也可在表达的目标蛋白中设计亲和标签，如在目标蛋白的N末端或C末端融合His-tag后，可用镍亲和层析进行快速方便纯化目标蛋白；还可利用目标蛋白的抗体进行亲和纯化。对于还没有切割完的融合蛋白需要除去，可在融合伴侣中设计不同于目标蛋白带有的亲和标签的亲和标签，通过亲和层析除去没有切割完的融合蛋白，也可以根据分子伴侣及融合蛋白的大小、带电荷及等电点等理化性质，用常规的层析技术、分子筛技术等除去没有切割完的融合蛋白。The self-shearing separation of the target protein expressed by the efficient fusion of the inventive vector and the fusion protein can be carried out at 0°C to 40°C, preferably at low temperature, such as at 0°C to 20°C, with a pH of 6.0 to 8.0 in the buffer solution. Self-shearing at low temperature can avoid or reduce the degradation of the target protein. The separation and purification methods of the target protein after separation from the fusion protein include: according to the physical and chemical properties such as the size, charge and isoelectric point of the target protein, the corresponding conventional purification technology can be used for separation and purification; for the convenience of purification, it can also be expressed in Design an affinity tag in the target protein, for example, after fusing the His-tag at the N-terminal or C-terminal of the target protein, nickel affinity chromatography can be used to quickly and conveniently purify the target protein; antibodies to the target protein can also be used for affinity purification. For fusion proteins that have not been cleaved to be removed, an affinity tag that is different from the affinity tag carried by the target protein can be designed in the fusion partner, and the fusion protein that has not been cleaved can be removed by affinity chromatography. And the physical and chemical properties such as the size, charge and isoelectric point of the fusion protein, use conventional chromatography technology, molecular sieve technology, etc. to remove the fusion protein that has not been cut.

本发明的一个应用在于应用发明的表达载体经济高效地获得保持天然生物活性的目标蛋白，特别是含二硫键的生物活性蛋白，如水蛭素、钩虫抗凝肽、蛋白酶抑制剂等。钩虫抗凝肽AcaNAP10(SEQ ID NO.1)是目前唯一对凝血途径启动阶段关键凝血因子-组织因子与活化的凝血因子VII复合物(TF/fVIIa)及凝血途径放大阶段关键因子-活化的凝血因子XI(fXIa)的均有抑制作用的高效抑制剂。应用本发明载体表达AcaNAP10或其保守性变异多肽、或它们的活性片段、或它们的活性衍生物，可以作为抗凝抗栓药物或制剂进行开发应用。One application of the present invention is to use the inventive expression vector to economically and efficiently obtain target proteins that maintain natural biological activity, especially biologically active proteins containing disulfide bonds, such as hirudin, hookworm anticoagulant peptide, protease inhibitors, etc. Hookworm anticoagulant peptide AcaNAP10 (SEQ ID NO.1) is currently the only key coagulation factor in the initiation phase of the coagulation pathway - tissue factor and activated coagulation factor VII complex (TF/fVIIa) and the key factor in the amplification phase of the coagulation pathway - activated coagulation Highly potent inhibitor of factor XI (fXIa). Using the vector of the present invention to express AcaNAP10 or its conservative variant polypeptide, or their active fragments, or their active derivatives can be developed and applied as anticoagulant and antithrombotic drugs or preparations.

本发明的其它方面由于本文技术内容的公开，对本领域技术人员是易于理解并实施的。例如，利用线性连接技术或PCR技术等，把仅含目标蛋白编码核苷酸序列连接入本发明载体，可以获得没有带任何自身之外的氨基酸残基序列的目标蛋白；一种涉及分离纯化自我剪切后的目标蛋白的方法，可根据目标蛋白性质进行分离、纯化等；利用本发明特点可以涉及一种基于pET表达载体之外的其它原核表达载体出发构建的具有本发明特点的原核表达载体；一种用含有本发明表达载体表达生物活性蛋白的遗传工程化的宿主细胞等。Other aspects of the present invention are easily understood and implemented by those skilled in the art due to the disclosure of the technical contents herein. For example, by using linear ligation technology or PCR technology, etc., only the target protein coding nucleotide sequence is connected to the carrier of the present invention, and the target protein without any amino acid residue sequence other than itself can be obtained; one method involves the separation and purification of self The method of shearing the target protein can be separated and purified according to the properties of the target protein; the use of the characteristics of the present invention can involve a prokaryotic expression vector with the characteristics of the present invention constructed based on other prokaryotic expression vectors other than the pET expression vector ; A genetically engineered host cell expressing a biologically active protein containing the expression vector of the present invention, etc.

因此，本发明载体一方面通过分子伴侣提高表达目标蛋白的产量、可溶性及保持目的蛋白天然生物活性；另外一方面，通过内含肽的自我剪切实现目标蛋白与融合蛋白经济高效地分离，减少使用肠激酶、fXa等的酶切或化学裂解除去融合标签和分子伴侣及进行二次纯化带来的成本；第三方面，通过内含肽的自我剪切作用实现目标蛋白与融合蛋白经济高效地分离后，再对与融合蛋白分离后的目标蛋白进行进一步的分离纯化，可以减少在融合蛋白表达过程中及其分离纯化过程中发生的提前剪切的目标蛋白大量损失。Therefore, on the one hand, the carrier of the present invention improves the yield and solubility of the expressed target protein and maintains the natural biological activity of the target protein through the molecular chaperone; Use enterokinase, fXa, etc. to digest or chemically cleavage the fusion tag and molecular chaperone and carry out the cost of secondary purification; the third aspect is to realize the economical and efficient separation of the target protein and the fusion protein through the self-cleavage of intein. After separation, further separation and purification of the target protein after separation from the fusion protein can reduce the loss of a large amount of prematurely sheared target protein during the expression process of the fusion protein and its separation and purification process.

附图说明Description of drawings

图1表达载体pICET32质粒图。Fig. 1 Plasmid map of expression vector pICET32.

图2表达载体pICET32完整核酸序列。Fig. 2 The complete nucleic acid sequence of the expression vector pICET32.

图3在大肠杆菌BL21(DE3)宿主中中，用pICET32表达含有5对二硫键的钩虫抗凝肽AcaNAP10(SEQ ID NO.1)的SDS-PAGE电泳图。Fig. 3 is in Escherichia coli BL21 (DE3) host, uses pICET32 to express the hookworm anticoagulant peptide AcaNAP10 (SEQ ID NO.1) containing 5 pairs of disulfide bonds SDS-PAGE electrophoresis.

图4在大肠杆菌BL21(DE3)宿主中中，用pICET32表达含有3对二硫键的Kunitz型丝氨酸蛋白酶抑制剂AduKuI4的SDS-PAGE电泳图。Fig. 4 is an SDS-PAGE electrophoresis image of expressing Kunitz-type serine protease inhibitor AduKuI4 containing 3 pairs of disulfide bonds with pICET32 in Escherichia coli BL21 (DE3) host.

具体实施方式Detailed ways

下面结合具体实例，进一步阐述本发明。应理解，这些实施仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如分子克隆实验指南(第3版)：([美]J.萨姆布鲁克，D.W拉塞尔著.黄培堂等译，科学出版社，2002年)，精编分子生物学实验指南(第5版)([美国]F.M.奥斯伯，R.布伦特编.金由辛，包慧中，赵丽云译.科学出版社，2008年)所述条件，或按照制造厂商所建议的条件。Below in conjunction with specific example, further set forth the present invention. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions such as Molecular Cloning Experiment Guideline (3rd Edition): ([US] J. Sambrook, D.W Russell. Translated by Huang Peitang, etc., published by Science Society, 2002), Molecular Biology Experiment Guide (5th Edition) ([USA] F.M. Osper, R. Brent edited. Jin Youxin, Bao Huizhong, Zhao Liyun translated. Science Press, 2008 year), or as recommended by the manufacturer.

实施例1表达载体pICET32的构建The construction ofembodiment 1 expression vector pICET32

以pET32a出发载体，pET32a具有T7高效启动子，含硫氧还蛋白TrxA，是目前常用促进蛋白可溶性高效表达载体之一。设计上游引物DP1：5’-CATGGCCATATGAAAATCGAAGAAGGTAAAC-3’(下划线即斜体部分为限制性内切酶Msc I酶切位点)及下游引物DP2：5’-GTCCATGGCCGTTGTGTACAATGATGTCATTC-3’用pfu酶从表达载体pTWIN1(NEB公司产品)扩增获得几丁质结合域(CBD)及内含肽mini SspDnaB编码核苷酸序列，以此产物为模板，用上游引物DP1与新设计引物5’-GTACACAACGGCCATGGACATCATCATCATCATCATGGATCC-3’扩增获得第二轮产物。通过此轮扩增引入限制性内切酶NcoI酶切位点及6×his tag编码序列。以第二轮扩增为模板，用引物DP1与新设计引物5’-TGCTCGAGTAAGCTTTGAATTCGGATCCATGATGATGATG-3’(下划线即斜体部分为限制性内切酶Xho I酶切位点)扩增获得第三轮产物。通过第三轮扩增引入限制性内切酶BamH I、EcoR I、Hind III及Xho I酶切位点。第三轮产物经Msc I及Xho I双酶切后，连接入同样经Msc I及Xho I酶切的pET32a载体。连接的载体转入E.coli DH5α，构建质粒经酶切及测序鉴定正确，成功获得构建目标载体pICET32。The starting vector is pET32a, which has a high-efficiency T7 promoter and contains thioredoxin TrxA. It is one of the commonly used vectors to promote high-efficiency protein soluble expression. Design upstream primer DP1: 5'-CATGGCCA TATGAAAATCGAAGAAGGTAAAC-3' (the underlined italic part is the restriction endonuclease Msc I restriction site) and downstream primer DP2: 5'-GTCCATGGCCGTTGTGTACAATGATGTCATTC-3' from the expression vector with pfu enzyme pTWIN1 (product of NEB Company) was amplified to obtain the chitin-binding domain (CBD) and the intein mini SspDnaB coding nucleotide sequence, and this product was used as a template to use the upstream primer DP1 and the newly designed primer 5'-GTACACAACGGCCATGGACATCATCATCATCATCATGGATCC-3' Amplification yields the second-round product. A restriction endonuclease NcoI cutting site and a 6×his tag coding sequence were introduced through this round of amplification. Using the second round of amplification as a template, use the primer DP1 and the newly designed primer 5'-TGCTCGAG TAAGCTTTGAATTCGGATCCATGATGATGATG-3' (the underlined part in italics is the restriction endonuclease Xho I site) to amplify to obtain the third round product . Restriction endonucleases BamH I, EcoR I, Hind III and Xho I were introduced by the third round of amplification. After the product of the third round was digested with Msc I and Xho I, it was ligated into the pET32a vector which was also digested with Msc I and Xho I. The connected vector was transferred into E.coli DH5α, and the constructed plasmid was confirmed to be correct by enzyme digestion and sequencing, and the target vector pICET32 was successfully constructed.

pICET32质粒图见附图1，核酸序列见附图2。该载体是基于pET32a出发的构建的载体，具有原核表达系统所需的基础元件。具有T7高效启动子，含有分子伴侣硫氧还蛋白(TrxA)促进目标蛋白高效可溶性表达并使目标蛋白保持天然生物活性，通过含肽mini Ssp DnaB的自我剪切内可实现目标蛋白与融合蛋白分离。为纯化需要，在内含肽中融合了亲和标签几丁质结合域(CBD)，在硫氧还蛋白及Ssp DnaB相互之间连接区段均有相应柔性区。在mini Ssp DnaB编码序列的3’端依次为多克隆位点、6×his tag编码序列、多克隆位点及6×his tag编码序列。也可通过PCR实现目标蛋白与内含肽的无缝连接，使获得的目标蛋白不带本身之外的氨基酸序列。The plasmid map of pICET32 is shown in Figure 1, and the nucleic acid sequence is shown in Figure 2. The vector is a constructed vector based on pET32a, and has basic elements required for a prokaryotic expression system. It has a T7 high-efficiency promoter, and contains molecular chaperone thioredoxin (TrxA) to promote efficient soluble expression of the target protein and maintain the natural biological activity of the target protein. The target protein can be separated from the fusion protein through self-cleavage of the peptide mini Ssp DnaB . For purification needs, an affinity tag chitin-binding domain (CBD) is fused to the intein, and there are corresponding flexible regions in the connecting segments between thioredoxin and Ssp DnaB. At the 3' end of the coding sequence of mini Ssp DnaB are multiple cloning site, 6×his tag coding sequence, multiple cloning site and 6×his tag coding sequence. The seamless connection between the target protein and intein can also be realized by PCR, so that the obtained target protein does not contain amino acid sequences other than itself.

实施例2用pICET32表达钩虫抗凝肽AcaNAP10及其分离与纯化Example 2 Expression of hookworm anticoagulant peptide AcaNAP10 by pICET32 and its isolation and purification

根据犬钩虫抗凝肽AcaNAP10(SEQ ID NO.1)编码序列设计引物从犬钩虫成虫cDNA中扩增编码核酸序列，设计出引物序列为：N10-3：5’-GAGGATCCAATCCAAGCTGTGGTGAG-3’(含内切酶位点BamH I)；N11-2：5’-CGAAGCTTGGTCATTTTCTATTAGGG-3’(含内切酶位点Hind III)。PCR产物回收纯化后，用BamH I和Hind III进行双酶切，与经同样双酶切的表达质粒pICET32过夜连接。连接产物转化至E.coli DH5α感受态细胞中，在含氨苄青霉素的培养基中培养。经PCR及测序鉴定的重组克隆质粒命名为AcaNAP10/pICET32。重组表达质粒转化至大肠杆菌BL21(DE3)感受态细胞。在含氨苄青霉素的LB培养液中培养含重组表达质粒AcaNAP10/pICET32宿主菌BL21(DE3)，1.0mmol/L终浓度的IPTG诱导。离心收集菌体，经超声破碎后收集上清，以1∶4(上清∶缓冲液)的比例加入不同pH值(分别为pH6.0、pH6.4、pH6.8、pH7.2、pH7.6、pH8.0)的10×磷酸盐缓冲液置于4℃、16℃、30℃、40℃裂解，分别于48小时内每间隔6小时取样进行SDS-PAGE观察裂解效果，用软件Quantity One 4.52分析裂解效率。取相应裂解上清用Ni-IDA亲和纯化，经几丁质柱亲和除去还没有裂解的融合蛋白，流穿液即为目标蛋白，可除咪唑浓缩后备用。纯化目标蛋白用PT及aPTT法检测体外抗凝活性(方法参照Li D et al.Biochem Biophys Res Commun.2010，392：155-159)。同时，用pTWIN1载体表达AcaNAP10，按照说明书制备重组AcaNAP10，制备好的蛋白用作对照。用Bradford蛋白浓度测定试剂盒测定纯化蛋白浓度。According to the coding sequence of hookworm anticoagulant peptide AcaNAP10 (SEQ ID NO.1), primers were designed to amplify the coding nucleic acid sequence from the adult cDNA of hookworm, and the designed primer sequence was: N10-3: 5'-GAGGATCCAATCCAAGCTGTGGTGAG-3' (included Dicer site BamH I); N11-2: 5'-CGAAGCTTGGTCATTTTCTATTAGGG-3' (including endonuclease site Hind III). After the PCR product was recovered and purified, it was digested with BamH I and Hind III, and then ligated overnight with the expression plasmid pICET32 that had undergone the same double digestion. The ligation product was transformed into E.coli DH5α competent cells and cultured in a medium containing ampicillin. The recombinant cloned plasmid identified by PCR and sequencing was named AcaNAP10/pICET32. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3) competent cells. The host strain BL21(DE3) containing the recombinant expression plasmid AcaNAP10/pICET32 was cultivated in LB medium containing ampicillin, and induced by IPTG at a final concentration of 1.0mmol/L. The bacterial cells were collected by centrifugation, and the supernatant was collected after sonication, and added with different pH values (respectively pH6.0, pH6.4, pH6.8, pH7.2, pH7 .6, pH8.0) in 10× phosphate buffer solution at 4°C, 16°C, 30°C, and 40°C for lysis, and samples were taken every 6 hours within 48 hours for SDS-PAGE to observe the lysis effect, using the software Quantity One 4.52 analyzes lysis efficiency. The corresponding lysed supernatant was affinity-purified with Ni-IDA, and the fusion protein that had not been lysed was removed by chitin column affinity, and the flow-through was the target protein, which could be concentrated by removing imidazole for later use. The purified target protein was tested for anticoagulant activity in vitro by PT and aPTT methods (refer to Li D et al. Biochem Biophys Res Commun. 2010, 392: 155-159). At the same time, the pTWIN1 vector was used to express AcaNAP10, and the recombinant AcaNAP10 was prepared according to the instructions, and the prepared protein was used as a control. The concentration of purified protein was determined with Bradford Protein Concentration Assay Kit.

结果表明：重组质粒pICET32/AcaNAP10在大肠杆菌中得到高效可溶性表达，95％以上目标蛋白(融合蛋白)为可溶蛋白，融合蛋白量可达宿主总蛋白的40％左右(图3泳道1)。在每升LB培养基中，获得可溶性融合蛋白量可达250mg以上。用pICET32制备的AcaNAP10的可溶性及产出量均比pTWIN1显著提高。纯化获得了N末端带6×his tag的AcaNAP10，蛋白分子量大小约为20Kda(形成了二聚体)(图3泳道3)，没有剪切及目标蛋白剪切后的融合蛋白大小分别约为49kDa，39kDa(图3泳道1)，与理论预测相符。在LB培养基中，最终获得的纯化rAcaNAP10量可达70mg/L以上。用pICET32及pTWIN1表达的蛋白在表达及宿主菌分离、裂解破碎过程均发生了提前自我剪切。如图2泳道1，pICET32在诱导表达及收集宿主菌进行破碎裂解的过程中，有超过三分之一的融合蛋白已发生自我剪切。在4℃放置12小时后，有80％以上的融合蛋白已完成自我剪切裂解，在4℃放置18小时的融合蛋白已基本上完成自我剪切裂解(图3泳道2)，在4℃放置36小时的融合蛋白几乎全部完成自我剪切裂解。在4℃中进行自我剪切，放置至48小时内的目标蛋白及融合伴侣均没有明显降解。随机进行5次蛋白表达纯化及活性检测对照表明，用pICET32载体制备的重组AcaNAP10(4℃，pH6.4磷酸盐缓冲液，自我剪切36小时后纯化蛋白)延长2倍PT所需浓度为30.5±4.2nM，而用pTWIN1载体制备的重组AcaNAP10延长2倍PT所需浓度为58.3±3.8nM，即用pICET32载体制备的重组AcaNAP10活性显著要高。The results showed that: the recombinant plasmid pICET32/AcaNAP10 was highly soluble expressed in Escherichia coli, more than 95% of the target protein (fusion protein) was a soluble protein, and the amount of the fusion protein could reach about 40% of the total host protein (Figure 3, lane 1). In every liter of LB medium, the amount of soluble fusion protein can reach more than 250 mg. The solubility and yield of AcaNAP10 prepared with pICET32 were significantly higher than those of pTWIN1. AcaNAP10 with 6×his tag at the N-terminus was purified and the molecular weight of the protein was about 20Kda (dimer was formed) (lane 3 in Figure 3). , 39kDa (Figure 3 lane 1), consistent with theoretical predictions. In LB medium, the amount of purified rAcaNAP10 finally obtained can reach more than 70mg/L. The proteins expressed by pICET32 and pTWIN1 were self-cleaved in advance during the process of expression, isolation of host bacteria, lysis and fragmentation. As shown inLane 1 of Figure 2, during the process of inducing expression of pICET32 and collecting the host bacteria for fragmentation and lysis, more than one-third of the fusion protein had undergone self-cleavage. After being placed at 4°C for 12 hours, more than 80% of the fusion protein has completed self-cleavage and cleavage, and the fusion protein that has been placed at 4°C for 18 hours has basically completed self-cleavage and cleavage (Figure 3 lane 2). The 36-hour fusion protein was almost completely self-cleaved and cleaved. Self-cleavage was carried out at 4°C, and the target protein and fusion partner were not significantly degraded within 48 hours. Five times random protein expression purification and activity detection control showed that the recombinant AcaNAP10 prepared with pICET32 vector (4°C, pH 6.4 phosphate buffer, purified protein after self-shearing for 36 hours) required a concentration of 30.5 to prolong the 2-fold PT ±4.2nM, while the recombinant AcaNAP10 prepared with the pTWIN1 vector required a 2-fold PT concentration of 58.3±3.8nM, that is, the activity of the recombinant AcaNAP10 prepared with the pICET32 vector was significantly higher.

实施例3丝氨酸蛋白酶抑制剂AduKuI4的表达及其分离纯化Example 3 Expression of serine protease inhibitor AduKuI4 and its separation and purification

AduKuI4是发明人课题组从十二指肠钩虫分离出来的一种Kunitz型丝氨酸蛋白酶抑制剂，含有3对二硫键，其成熟肽氨基酸序列为：RNPHRKGRCGDDPAETGGECPDPETKYTYKFGDCHEVKYCGEQETRNLFDSYEKCSGKCVIF。根据AduKuI4成熟肽编码序列，设计上游引物：5’-TAGGATCCCGCAATCCTCACAGAAAG  -3’及下游引物：5’-CCAAGCTTAGAAGATCACGCACTTTCC-3’，从十二指肠钩虫成虫cDNA中扩增获得成熟肽编码序列。扩增产物连接入表达载体成功构建pICET32/AduKuI4表达质粒。质粒转化至大肠杆菌BL21(DE3)，经诱导培养，离心收集菌体，超声破碎后收集上清，以1∶4(上清∶缓冲液)的比例加入不同pH值(分别为pH6.0、pH6.4、pH6.8、pH7.2、pH7.6、pH8.0)的10×磷酸盐缓冲液置于4℃、16℃、30℃、40℃裂解，分别于48小时内每间隔6小时取样进行SDS-PAGE观察裂解效果。取相应裂解上清用Ni-NTA亲和纯化，经几丁质柱亲和除去还没有裂解的融合蛋白，流穿液即为目标蛋白。AduKuI4 is a Kunitz-type serine protease inhibitor isolated from Ancylostoma duodenale by the inventor's research group. It contains 3 pairs of disulfide bonds. The amino acid sequence of its mature peptide is: RNPHRKGRCGDDPAETGGECPDPETKYTYKFGDCHEVKYCGEQETRNLFDSYEKCSGKCVIF. According to the mature peptide coding sequence of AduKuI4, the upstream primer: 5'-TAGGATCCCGCAATCCTCACAGAAAG -3' and the downstream primer: 5'-CCAAGCTTAGAAGATCACGCACTTTCC-3' were designed, and the mature peptide coding sequence was amplified from the adult cDNA of Ancylostoma duodenale. The amplified product was ligated into the expression vector to successfully construct the pICET32/AduKuI4 expression plasmid. The plasmid was transformed into Escherichia coli BL21(DE3), induced and cultured, the cells were collected by centrifugation, and the supernatant was collected after sonication, and added with different pH values (respectively pH6.0, pH6.0, pH6.4, pH6.8, pH7.2, pH7.6, pH8.0) 10× phosphate buffer at 4°C, 16°C, 30°C, 40°C for lysing, and within 48 hours at intervals of 6 Samples were taken every hour for SDS-PAGE to observe the lysis effect. The corresponding lysed supernatant was affinity-purified with Ni-NTA, and the uncleaved fusion protein was affinity removed through a chitin column, and the flow-through was the target protein.

结果表明：AduKuI4得到高效可溶表达，目标蛋白(融合蛋白)几乎全部可溶，可溶融合蛋白总量可达细菌总蛋白量50％以上。从裂解下来的融合伴侣可以看出，在诱导表达及细菌破碎过程中即有50％以上可溶蛋白已发生提前自我剪切(图4泳道2)。各温度下均能诱导剪切，24小时后，融合蛋白均绝大部分剪切(图4泳道3、4分别为宿主菌上清在4℃及40℃下经内含肽自我剪切后的裂解情况)，取相应裂解上清用Ni-IDA亲和纯化，经几丁质柱亲和除去还没有裂解完的融合蛋白，获得目标蛋白，大小约为8kDa，与理论预测相符(图4泳道5、6、7)。在LB培养基中，最终获得的纯化rAduKuI4量可达80mg/L以上，即AduKuI4得到高效可溶表达。The results show that: AduKuI4 is highly soluble expressed, the target protein (fusion protein) is almost completely soluble, and the total amount of soluble fusion protein can reach more than 50% of the total bacterial protein. From the cleaved fusion partners, it can be seen that more than 50% of the soluble protein has undergone premature self-cleavage during the process of induced expression and bacterial disruption (Figure 4, lane 2). Cutting can be induced at all temperatures, and after 24 hours, most of the fusion protein is cut (lanes 3 and 4 in Figure 4 are the supernatant of the host bacteria at 4°C and 40°C after self-shearing by intein lysis), the corresponding lysis supernatant was affinity-purified with Ni-IDA, and the fusion protein that had not been cleaved was removed by chitin column affinity to obtain the target protein, which was about 8 kDa in size, which was consistent with the theoretical prediction (Fig. 4Swimming lane 5, 6, 7). In LB medium, the amount of purified rAduKuI4 finally obtained can reach more than 80 mg/L, that is, AduKuI4 is efficiently and solublely expressed.

序列表sequence listing

<110>广东医学院<110>Guangdong Medical College

<120>一种原核表达载体及其应用<120> A prokaryotic expression vector and its application

 the

<160>1<160>1

 the

<210>1<210>1

<211>80<211>80

<212>PRT<212>PRT

<213>犬钩虫(Ancylostoma caninum)<213>Ancylostoma caninum

 the

<400>2<400>2

Asn Pro Ser Cys Gly Glu Asn Glu Arg His Asp Glu Cys Ser ArgAsn Pro Ser Cys Gly Glu Asn Glu Arg His Asp Glu Cys Ser Arg

                  5                  10                   155 10 15

Lys Glu Cys Asp Pro Lys Cys Lys Tyr Asp Gly Thr Glu Glu LysLys Glu Cys Asp Pro Lys Cys Lys Tyr Asp Gly Thr Glu Glu Lys

                 20                   25                   3020 25 30

Asp Asp Glu Lys Pro Val Val Cys Leu Thr Arg Val Cys Tyr GlyAsp Asp Glu Lys Pro Val Val Cys Leu Thr Arg Val Cys Tyr Gly

                 35                   40                   4535 40 45

Asp Cys Ile Cys Arg Asp Gly Phe Leu Arg Asn Lys Asn Gly AlaAsp Cys Ile Cys Arg Asp Gly Phe Leu Arg Asn Lys Asn Gly Ala

                 50                   55                   6050 55 60

Cys Val Lys Ala Glu Asp Cys Glu Leu Asp Asn MET Glu Phe IleCys Val Lys Ala Glu Asp Cys Glu Leu Asp Asn MET Glu Phe Ile

                 65                   70                   7565 70 75

Tyr Pro Asn Arg LysTyr Pro Asn Arg Lys

                 8080

Claims (10)

1. prokaryotic expression carrier is characterized in that containing:

(a) promote the target protein solubility expression or/and the coding nucleotide sequence of the molecular chaperones that disulfide linkage correctly folds;

(b) can be at the coding nucleotide sequence of the intein of C-terminal peptide bond rupture.

2. according to the described a kind of prokaryotic expression carrier of claim 1, it is characterized in that host cell is intestinal bacteria.

3. according to the described a kind of prokaryotic expression carrier of claim 1, it is characterized in that described molecular chaperones can promote the target protein solubility expression or/and disulfide linkage is correctly folding, makes the target protein of expression maintain natural bioactive; Describedly include the peptide bond cracking that Toplink is effectively induced C-terminal under certain condition, target protein is separated with fusion rotein.

4. according to the described a kind of prokaryotic expression carrier of claim 3, it is characterized in that described molecular chaperones comprises Trx, disulfide bond reduction enzyme and isomerase, little ubiquitin modified protein, NusA and glutathione-S-transferase; Described intein comprises mini Ssp DnaB, Sce VMA etc., is good to select mini Ssp DnaB for use.

5. according to the application of the described a kind of prokaryotic expression carrier of claim 1, it is characterized in that, the target protein of expressing is the albumen or the polypeptide of biologically active, the biological activity protein or the polypeptide that particularly contain disulfide linkage comprise hookworm coagulate peptide resistant and conservative property thereof variation polypeptide or their active fragments or their reactive derivative.

6. according to the described a kind of prokaryotic expression carrier of claim 2, it is characterized in that a kind of host cell includes the described carrier of claim 5.

7. according to the application of the described a kind of prokaryotic expression carrier of claim 5, it is characterized in that it is under 0 ℃～40 ℃ that target protein effectively separates with fusion rotein, preferable under 0 ℃～20 ℃, pH carries out in 6.0～8.0 the damping fluid.

8. according to the application of the described a kind of prokaryotic expression carrier of claim 7, it is characterized in that, the separation of the target protein of separating from fusion rotein, purification process comprise with the affinitive layer purification fusion affinity tag, as target protein with His-tag, or with the antibody affinity purification target protein of preparation, or carry out chromatography purification according to the physico-chemical property of target protein.

9. according to the application of the described a kind of prokaryotic expression carrier of claim 5, it is characterized in that,

(a) in suitable medium, cultivate the described host cell of claim 6;

(b), carry out effectively separating of target protein and fusion rotein according under the described condition of claim 7;

(c) carry out the isolation and purification of target protein according to the described method of claim 8.

10. according to the application of claim 5 prokaryotic expression carrier, it is characterized in that target protein comprises polypeptide or its conservative property variation polypeptide or their active fragments or their reactive derivative of being made up of SEQ ID NO.1 aminoacid sequence.

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