沙门菌OmpD介导的rBCG口服疫苗的制备与鉴定
发布时间：2018-10-22 07:25
【摘要】： 背景 重组活菌疫苗是以某些减毒或者无毒力的活菌为载体,将病原体的保护性抗原基因插入细菌的基因组或者质粒DNA中并使之高效表达的一种新型疫苗。重组卡介苗(Recombinant Bacillus Calmette-Guerin,rBCG)就是重组活菌疫苗中具有代表性的新型疫苗。它是以BCG为工程菌,借助分子生物学技术对其进行基因改造,利用其活疫苗特性,在机体内表达各种疾病的相关抗原,从而达到预防和治疗多种疾病的目的。本研究小组曾成功将屋尘螨抗原Der p2基因转入BCG中表达,制备出抗原Der p2-rBCG。经静脉和腹腔注射接种后,在小鼠体内诱导了Der p2特异性的Th1优势应答。因此我们推测可以通过抗原rBCG疫苗来治疗对某些特定抗原过敏的疾病。 目前分枝杆菌几乎全部是以皮下注射的方式接种。但是研究发现,如果短期内重复皮下注射接种rBCG会引起局部严重的迟发性变态反应(DTH),因此我们考虑采用口服途径接种rBCG。而且,我们前期的研究发现给小鼠口服Der p2-rBCG,同样可以诱导抗原特异性的Th1应答[1, 2]。然而,BCG分枝杆菌并不是肠道定植菌,与肠黏膜的亲和力低,大量口服还会引起肠道正常菌群失调及口咽部感染,从而影响免疫效果。文献回顾发现,沙门菌外膜蛋白Omp D介导了沙门菌与肠黏膜上皮的黏附,因此我们设想此外膜蛋白能够介导rBCG与肠道黏膜的高亲和力结合,从而制备出具有肠道高亲和力的rBCG口服疫苗。 目的 利用分子生物学及基因工程技术,构建能以串联和并联形式、在细菌表面表达Der p2和Omp D抗原的两种rBCG口服疫苗,为体外实验和临床应用提供基础。 实验方法和结果 1. Omp D蛋白的表达及纯化 以鼠伤寒沙门菌Salmonella typhimurium基因组为模板,通过PCR来扩增Omp D基因,与pMD-18T克隆载体连接后测序,结果与Genbank公布的Omp D基因序列完全一致。将Omp D基因克隆入原核表达载体pET-28a(+),经PCR和酶切鉴定后,阳性质粒命名为pET28a(+)-Omp D,并经IPTG进行诱导表达。SDS-PAGE分析证实成功表达了与预期分子量一致的Omp D蛋白。Western-blot证实该蛋白可与抗6×His-mAb发生特异性反应。可溶性分析显示该蛋白以包涵体形式存在于沉淀中,经Ni+-NTA亲和色谱法纯化获得了目的蛋白,且纯度达90%以上。 2.兔Omp D多克隆抗体的制备及鉴定 重组蛋白经变性、复性后,按100μg/kg的纯化蛋白加等量弗氏完全佐剂(FCA)乳化后接种于兔颈背部皮内,间隔2w后再进行第二、三次免疫;第三次免疫结束2w后以等量重组蛋白追加免疫1次,1w后收集血液,分离血清后ELISA检测特异性抗体效价达到1:10 000以上。Western-blot检测表明,该抗体能与Omp D蛋白发生特异性结合,其特异性与敏感性均较好。 3.两种rBCG口服疫苗的构建与鉴定 为了获得Der p2-Omp D融合基因,我们重新设计引物,经PCR法分别扩增获得新的Der p2和Omp D基因,测序正确后将两者克隆入原核表达载体pProEX HTb,获得pProEX HTb-Der p2-Omp D质粒。①串联表达:将Der p2-Omp D融合基因亚克隆入穿梭胞壁表达载体pCW,经酶切鉴定阳性命名为pCW-Der p2-Omp D质粒。将此重组质粒电穿导入BCG感受态细胞,构建可胞壁串联表达Der p2-Omp D融合蛋白的rBCG;②并联表达:构建pCW-Der p2与pCW-Omp D两种质粒,并将二者同时电穿导入BCG感受态细胞,以构建胞壁并联表达Der p2和Omp D蛋白的rBCG。经潮霉素抗性筛选的阳性克隆,均采用以下三种方式进行鉴定:PCR特异性扩增目的基因片段;用兔抗Der p2多克隆抗体与兔抗Omp D多克隆抗体对阳性克隆分别进行斑点免疫杂交法和间接免疫荧光法鉴定。证实两种rBCG口服疫苗构建成功,并能与特异性抗体发生反应。 结论 1.在E.coli表达系统中成功表达并纯化出Omp D蛋白。该蛋白能够与6×His-mAb发生特异性反应,免疫新西兰兔后获得Omp D多克隆抗体,通过ELISA检测抗体效价达到1:10 000以上。该抗体能与Omp D蛋白发生特异性结合,其特异性与敏感性均较好。 2.采用基因工程手段制备出以胞壁形式串联和并联表达Der p2和Omp D蛋白的两种rBCG口服疫苗,并通过PCR、斑点免疫杂交法及间接免疫荧光法分别进行鉴定。证实了两种rBCG口服疫苗构建成功。
[Abstract]:Background The recombinant live bacteria vaccine is one kind of organism which takes some attenuated or non-toxic live bacteria as the carrier, inserts the protective antigen gene of the pathogen into the genome of bacteria or the plasmid DNA and makes it highly effective. Recombinant Bacillus Calmette-Guerin (rBCG) is representative of recombinant live vaccine The invention relates to a novel vaccine, which takes BCG as engineering bacteria and uses the molecular biology technology to transform the gene, utilizes the characteristic of the live vaccine, and expresses the relevant antigens of various diseases in the organism, thus achieving the prevention and treatment of various diseases. The research team successfully transferred the Der p2 gene into BCG to prepare the antigen Der p2-rBCG. After intravenous and intraperitoneal injection, Der p2-specific Th1 was induced in mice. We think we can treat some specific antigens by antigen rBCG vaccine Min's disease. Currently, mycobacteria are almost entirely A subcutaneous injection. However, it has been found that if repeated subcutaneous injection of rBCG in the short term results in local severe delayed allergy (DTH), we consider the use of a mouth In addition, we found that oral Der p2-rBCG can also induce antigen-specific T in mice. "h1 response[1,2]. However, Mycobacterium BCG is not an intestinal colonization bacterium, and has low affinity with intestinal mucosa, and a large amount of oral administration may also cause normal flora imbalance and oropharyngeal infection in the intestinal tract." The literature review found that the Salmonella outer membrane protein Omp D mediates the adhesion of Salmonella to the intestinal mucosa epithelium, so we assume that the membrane proteins can mediate the high affinity binding of rBCG to the intestinal mucosa, thus preparing a high affinity for the intestinal tract. r BCG oral vaccine aims to construct two kinds of rBCG which can express Der p2 and Omp D antigen in series and parallel in serial and parallel forms by using molecular biology and genetic engineering technology. Oral vaccine, body The external test and clinical application provide the basis. The expression of Omp D protein and the purification of Omp D protein were carried out by PCR to amplify the Omp D gene. The Omp D gene was cloned into prokaryotic expression vector pET28a (+), amplified by PCR and enzyme digestion. The positive plasmid was named pET28a (+)-Omp D and induced by IPTG. Da. SDS-PAGE analysis demonstrated successful expression of the Omp D protein consistent with the desired molecular weight. Stern-blot confirmed that the protein could be specifically reacted with anti-6 anti-His-mAb. Soluble analysis showed that the protein existed in the form of inclusion bodies in the form of inclusion bodies. Purification by Ni +-NTA Affinity Chromatography The target protein was obtained and purity up to more than 90%. The preparation and identification of the polyclonal antibody of rabbit Omp D were denatured and renatured, and purified by 100. m u.g/ kg. Protein plus equal amount of Freund's complete adjuvant (FCA) was emulsified and then inoculated into the dorsal skin of rabbit's neck, after 2w, the second and third immunization were carried out; after the third immunization, 2w was followed by the same amount of recombinant protein. After immunization for 1 time, blood was collected after 1w, and the ELISA was used to detect specific antibody titer by more than 1: 10,000 after separation of serum. Wes Pattern-blot analysis showed that the antibody can In order to obtain the Der p2-Omp D fusion gene, we re-designed the primers to amplify the new Der p2 and Omp D by PCR. After correct sequencing, the gene was cloned into prokaryotic expression vector pProX HTb to obtain pProX HTb-Der p2-Omp D plasmid. The recombinant plasmid pCW-Der p2 and pCW-Der p2-Omp D fusion protein were constructed. Both plasmids of Omp D were introduced into BCG cell line at the same time to construct rBCG for the parallel expression of Der p2 and Omp D protein in cell wall. The positive clones screened by hygromycin resistance were identified in three ways: PCR-specific amplification target gene Fragments; Rabbit anti-Der p2 polyclonal antibody and rabbit anti-Omp D polyclonal antibody to yang Chick Two kinds of rB were confirmed by dot immunhybridization and indirect immunofluorescence. CG oral vaccine was successfully constructed and could react with specific antibodies. Conclusion 1. Omp D protein was successfully expressed and purified in E. coli expression system. White can specifically react with the 6-way His-mAb to immunize New Zealand rabbits to obtain O the antibody titer of the antibody can be more than 1: 10,000 by ELISA, the specificity and sensitivity of the antibody can be better than that of the Omp D protein, Method for the preparation of a series and parallel expression of D in the form of a cell wall
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
[Abstract]:Background The recombinant live bacteria vaccine is one kind of organism which takes some attenuated or non-toxic live bacteria as the carrier, inserts the protective antigen gene of the pathogen into the genome of bacteria or the plasmid DNA and makes it highly effective. Recombinant Bacillus Calmette-Guerin (rBCG) is representative of recombinant live vaccine The invention relates to a novel vaccine, which takes BCG as engineering bacteria and uses the molecular biology technology to transform the gene, utilizes the characteristic of the live vaccine, and expresses the relevant antigens of various diseases in the organism, thus achieving the prevention and treatment of various diseases. The research team successfully transferred the Der p2 gene into BCG to prepare the antigen Der p2-rBCG. After intravenous and intraperitoneal injection, Der p2-specific Th1 was induced in mice. We think we can treat some specific antigens by antigen rBCG vaccine Min's disease. Currently, mycobacteria are almost entirely A subcutaneous injection. However, it has been found that if repeated subcutaneous injection of rBCG in the short term results in local severe delayed allergy (DTH), we consider the use of a mouth In addition, we found that oral Der p2-rBCG can also induce antigen-specific T in mice. "h1 response[1,2]. However, Mycobacterium BCG is not an intestinal colonization bacterium, and has low affinity with intestinal mucosa, and a large amount of oral administration may also cause normal flora imbalance and oropharyngeal infection in the intestinal tract." The literature review found that the Salmonella outer membrane protein Omp D mediates the adhesion of Salmonella to the intestinal mucosa epithelium, so we assume that the membrane proteins can mediate the high affinity binding of rBCG to the intestinal mucosa, thus preparing a high affinity for the intestinal tract. r BCG oral vaccine aims to construct two kinds of rBCG which can express Der p2 and Omp D antigen in series and parallel in serial and parallel forms by using molecular biology and genetic engineering technology. Oral vaccine, body The external test and clinical application provide the basis. The expression of Omp D protein and the purification of Omp D protein were carried out by PCR to amplify the Omp D gene. The Omp D gene was cloned into prokaryotic expression vector pET28a (+), amplified by PCR and enzyme digestion. The positive plasmid was named pET28a (+)-Omp D and induced by IPTG. Da. SDS-PAGE analysis demonstrated successful expression of the Omp D protein consistent with the desired molecular weight. Stern-blot confirmed that the protein could be specifically reacted with anti-6 anti-His-mAb. Soluble analysis showed that the protein existed in the form of inclusion bodies in the form of inclusion bodies. Purification by Ni +-NTA Affinity Chromatography The target protein was obtained and purity up to more than 90%. The preparation and identification of the polyclonal antibody of rabbit Omp D were denatured and renatured, and purified by 100. m u.g/ kg. Protein plus equal amount of Freund's complete adjuvant (FCA) was emulsified and then inoculated into the dorsal skin of rabbit's neck, after 2w, the second and third immunization were carried out; after the third immunization, 2w was followed by the same amount of recombinant protein. After immunization for 1 time, blood was collected after 1w, and the ELISA was used to detect specific antibody titer by more than 1: 10,000 after separation of serum. Wes Pattern-blot analysis showed that the antibody can In order to obtain the Der p2-Omp D fusion gene, we re-designed the primers to amplify the new Der p2 and Omp D by PCR. After correct sequencing, the gene was cloned into prokaryotic expression vector pProX HTb to obtain pProX HTb-Der p2-Omp D plasmid. The recombinant plasmid pCW-Der p2 and pCW-Der p2-Omp D fusion protein were constructed. Both plasmids of Omp D were introduced into BCG cell line at the same time to construct rBCG for the parallel expression of Der p2 and Omp D protein in cell wall. The positive clones screened by hygromycin resistance were identified in three ways: PCR-specific amplification target gene Fragments; Rabbit anti-Der p2 polyclonal antibody and rabbit anti-Omp D polyclonal antibody to yang Chick Two kinds of rB were confirmed by dot immunhybridization and indirect immunofluorescence. CG oral vaccine was successfully constructed and could react with specific antibodies. Conclusion 1. Omp D protein was successfully expressed and purified in E. coli expression system. White can specifically react with the 6-way His-mAb to immunize New Zealand rabbits to obtain O the antibody titer of the antibody can be more than 1: 10,000 by ELISA, the specificity and sensitivity of the antibody can be better than that of the Omp D protein, Method for the preparation of a series and parallel expression of D in the form of a cell wall
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前8条
1 史皆然,李元,戚好文,李别虎,柏银兰,范雄林,薛莹;屋尘螨抗原Derp2基因的克隆和表达[J];第四军医大学学报;2002年13期
2 史皆然;师长宏;吴昌归;李元;戚好文;李别虎;范雄林;;分泌表达Der p2的重组BCG的构建与鉴定[J];解放军医学杂志;2006年08期
3 李秋根;张志大;熊国亮;刘乾中;柳U，
本文编号：2286494
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2286494.html
最近更新
教材专著
