沙門菌OmpD介導的rBCG口服疫苗的制備與鑒定
發(fā)布時間：2018-10-22 07:25
【摘要】： 背景 重組活菌疫苗是以某些減毒或者無毒力的活菌為載體,將病原體的保護性抗原基因插入細菌的基因組或者質粒DNA中并使之高效表達的一種新型疫苗。重組卡介苗(Recombinant Bacillus Calmette-Guerin,rBCG)就是重組活菌疫苗中具有代表性的新型疫苗。它是以BCG為工程菌,借助分子生物學技術對其進行基因改造,利用其活疫苗特性,在機體內表達各種疾病的相關抗原,從而達到預防和治療多種疾病的目的。本研究小組曾成功將屋塵螨抗原Der p2基因轉入BCG中表達,制備出抗原Der p2-rBCG。經靜脈和腹腔注射接種后,在小鼠體內誘導了Der p2特異性的Th1優(yōu)勢應答。因此我們推測可以通過抗原rBCG疫苗來治療對某些特定抗原過敏的疾病。 目前分枝桿菌幾乎全部是以皮下注射的方式接種。但是研究發(fā)現(xiàn),如果短期內重復皮下注射接種rBCG會引起局部嚴重的遲發(fā)性變態(tài)反應(DTH),因此我們考慮采用口服途徑接種rBCG。而且,我們前期的研究發(fā)現(xiàn)給小鼠口服Der p2-rBCG,同樣可以誘導抗原特異性的Th1應答[1, 2]。然而,BCG分枝桿菌并不是腸道定植菌,與腸黏膜的親和力低,大量口服還會引起腸道正常菌群失調及口咽部感染,從而影響免疫效果。文獻回顧發(fā)現(xiàn),沙門菌外膜蛋白Omp D介導了沙門菌與腸黏膜上皮的黏附,因此我們設想此外膜蛋白能夠介導rBCG與腸道黏膜的高親和力結合,從而制備出具有腸道高親和力的rBCG口服疫苗。 目的 利用分子生物學及基因工程技術,構建能以串聯(lián)和并聯(lián)形式、在細菌表面表達Der p2和Omp D抗原的兩種rBCG口服疫苗,為體外實驗和臨床應用提供基礎。 實驗方法和結果 1. Omp D蛋白的表達及純化 以鼠傷寒沙門菌Salmonella typhimurium基因組為模板,通過PCR來擴增Omp D基因,與pMD-18T克隆載體連接后測序,結果與Genbank公布的Omp D基因序列完全一致。將Omp D基因克隆入原核表達載體pET-28a(+),經PCR和酶切鑒定后,陽性質粒命名為pET28a(+)-Omp D,并經IPTG進行誘導表達。SDS-PAGE分析證實成功表達了與預期分子量一致的Omp D蛋白。Western-blot證實該蛋白可與抗6×His-mAb發(fā)生特異性反應�？扇苄苑治鲲@示該蛋白以包涵體形式存在于沉淀中,經Ni+-NTA親和色譜法純化獲得了目的蛋白,且純度達90%以上。 2.兔Omp D多克隆抗體的制備及鑒定 重組蛋白經變性、復性后,按100μg/kg的純化蛋白加等量弗氏完全佐劑(FCA)乳化后接種于兔頸背部皮內,間隔2w后再進行第二、三次免疫;第三次免疫結束2w后以等量重組蛋白追加免疫1次,1w后收集血液,分離血清后ELISA檢測特異性抗體效價達到1:10 000以上。Western-blot檢測表明,該抗體能與Omp D蛋白發(fā)生特異性結合,其特異性與敏感性均較好。 3.兩種rBCG口服疫苗的構建與鑒定 為了獲得Der p2-Omp D融合基因,我們重新設計引物,經PCR法分別擴增獲得新的Der p2和Omp D基因,測序正確后將兩者克隆入原核表達載體pProEX HTb,獲得pProEX HTb-Der p2-Omp D質粒。①串聯(lián)表達:將Der p2-Omp D融合基因亞克隆入穿梭胞壁表達載體pCW,經酶切鑒定陽性命名為pCW-Der p2-Omp D質粒。將此重組質粒電穿導入BCG感受態(tài)細胞,構建可胞壁串聯(lián)表達Der p2-Omp D融合蛋白的rBCG;②并聯(lián)表達:構建pCW-Der p2與pCW-Omp D兩種質粒,并將二者同時電穿導入BCG感受態(tài)細胞,以構建胞壁并聯(lián)表達Der p2和Omp D蛋白的rBCG。經潮霉素抗性篩選的陽性克隆,均采用以下三種方式進行鑒定:PCR特異性擴增目的基因片段;用兔抗Der p2多克隆抗體與兔抗Omp D多克隆抗體對陽性克隆分別進行斑點免疫雜交法和間接免疫熒光法鑒定。證實兩種rBCG口服疫苗構建成功,并能與特異性抗體發(fā)生反應。 結論 1.在E.coli表達系統(tǒng)中成功表達并純化出Omp D蛋白。該蛋白能夠與6×His-mAb發(fā)生特異性反應,免疫新西蘭兔后獲得Omp D多克隆抗體,通過ELISA檢測抗體效價達到1:10 000以上。該抗體能與Omp D蛋白發(fā)生特異性結合,其特異性與敏感性均較好。 2.采用基因工程手段制備出以胞壁形式串聯(lián)和并聯(lián)表達Der p2和Omp D蛋白的兩種rBCG口服疫苗,并通過PCR、斑點免疫雜交法及間接免疫熒光法分別進行鑒定。證實了兩種rBCG口服疫苗構建成功。
[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
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：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
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
【參考文獻】
相關期刊論文 前8條
1 史皆然,李元,戚好文,李別虎,柏銀蘭,范雄林,薛瑩;屋塵螨抗原Derp2基因的克隆和表達[J];第四軍醫(yī)大學學報;2002年13期
2 史皆然;師長宏;吳昌歸;李元;戚好文;李別虎;范雄林;;分泌表達Der p2的重組BCG的構建與鑒定[J];解放軍醫(yī)學雜志;2006年08期
3 李秋根;張志大;熊國亮;劉乾中;柳U，
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