幽门螺杆菌CagA和VacA重组蛋白的表达、纯化及抗原性检测
发布时间：2019-05-13 18:17
【摘要】：目的：构建幽门螺杆菌(Helicobacter pylori, Hp)细胞毒素相关基因A(Cytotoxin associated gene A, CagA)和空泡细胞毒素(Vacuolating cytotoxin A, VacA)的重组质粒,并在大肠杆菌中表达获得基因重组蛋白,为检出幽门螺杆菌致病株和运用于临床检测Hp的感染奠定基础。 方法：挑选CagA基因和VacA基因的优势抗原表位片段,收集H. pylori标准株NCTC11639,提取基因组DNA, PCR扩增目的基因片断,将其分别克隆到克隆载体pGEM-T和pMD18-T载体并测序,构建重组质粒pGEM-T/CagA和pMD18-T/VacA,将鉴定正确的重组质粒进行双酶切,把目的基因克隆入表达载体pET-16b,构建重组表达载体pET16b/CagA与pET16b/VacA。转化至大肠杆菌E. Coli Rosetta后诱导表达,经SDS-PAGE鉴定、镍亲和层析纯化,并透析复性后,ELISA法检测重组蛋白CagA、VacA的免疫原性。 结果：PCR扩增结果表明分别得到了大小约为1962bp和2256bp的目的片段；构建的重组质粒经酶切鉴定和测序证明其中插入片段分别为CagA和VacA的目的基因,测序结果与Genbank上登录序列比对后,结果完全一致；SDS-PAGE分析显示,在IPTG诱导下,重组工程菌表达了一相对分子量(Mr)约为75KDa和87KDa的目的蛋白条带,表达量占细菌总蛋白的20%,镍亲和层析纯化率约90%；目的蛋白在菌体细胞内主要以可溶性蛋白和包涵体形式存在,经间接ELISA法检测重组蛋白CagA、VacA具有一定免疫原性。 结论：成功构建了pET16b/CagA、pET16b/VacA2个原核表达重组体,将其分别转化至大肠杆菌后,分别表达出了相对分子量(Mr)约为75KDa和87KDa的CagA和VacA重组蛋白,间接ELISA法检测重组蛋白CagA、VacA具有较好的免疫反应性,其中CagA蛋白的免疫反应性较VacA的强些,为H.pylori蛋白质疫苗的研制和以基因工程抗原为基础快速诊断试剂盒的研究打下了基础。
[Abstract]:Objective: to construct the recombinant plasmid of (Helicobacter pylori, Hp) cytotoxicity related gene A (Cytotoxin associated gene A, CagA) and vacuole toxin (Vacuolating cytotoxin A, VacA) of HP and express the recombinant protein in E. coli. It lays a foundation for detection of HP pathogenic strains and clinical detection of Hp infection. Methods: the dominant epitope fragments of CagA gene and VacA gene were selected, and the genomic DNA, PCR amplified gene fragments were extracted from H. pylori standard strain NCTC11639, and cloned into clone vector pGEM-T and pMD18-T vector and sequenced, respectively. The recombinant plasmid pGEM-T/CagA and pMD18-T/VacA, were constructed and the correct recombinant plasmid was identified by double enzyme digestion. The target gene was cloned into the expression vector pET-16b, to construct the recombinant expression vector pET16b/CagA and pET16b/VacA.. After transformed into E. coli E. Coli Rosetta, the expression was induced and identified by SDS-PAGE and purified by nickel affinity chromatography. After dialysis and renaturation, the immunogenicity of the recombinant protein CagA,VacA was detected by ELISA method. Results: the results of PCR amplification showed that the target fragments were about 1962bp and 2256bp, respectively. The constructed recombinant plasmid was identified by enzyme digestion and sequencing to prove that the inserted fragment was the target gene of CagA and VacA, respectively. the sequencing results were in good agreement with the login sequence on Genbank. SDS-PAGE analysis showed that under the induction of IPTG, the recombinant engineering bacteria expressed a target protein band with relative molecular weight (Mr) of 75KDa and 87KDa, which accounted for 20% of the total bacterial protein and 90% of the purification rate of nickel affinity chromatography. The target protein mainly exists in the form of soluble protein and inclusion body in bacterial cells. The recombinant protein CagA,VacA detected by indirect ELISA method has certain immunogenicity. Conclusion: pET16b/CagA,pET16b/VacA2 prokaryotic expression recombinant was successfully constructed and transformed into E. coli respectively. CagA and VacA recombinant proteins with relative molecular weight (Mr) of 75KDa and 87KDa were expressed respectively. The recombinant protein CagA, was detected by indirect ELISA method. VacA has better immunoreactivity, in which CagA protein is more reactive than VacA, which lays a foundation for the development of H.pylori protein vaccine and the study of rapid diagnostic kit based on genetic engineering antigen.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378;Q93
本文编号：2476086
[Abstract]:Objective: to construct the recombinant plasmid of (Helicobacter pylori, Hp) cytotoxicity related gene A (Cytotoxin associated gene A, CagA) and vacuole toxin (Vacuolating cytotoxin A, VacA) of HP and express the recombinant protein in E. coli. It lays a foundation for detection of HP pathogenic strains and clinical detection of Hp infection. Methods: the dominant epitope fragments of CagA gene and VacA gene were selected, and the genomic DNA, PCR amplified gene fragments were extracted from H. pylori standard strain NCTC11639, and cloned into clone vector pGEM-T and pMD18-T vector and sequenced, respectively. The recombinant plasmid pGEM-T/CagA and pMD18-T/VacA, were constructed and the correct recombinant plasmid was identified by double enzyme digestion. The target gene was cloned into the expression vector pET-16b, to construct the recombinant expression vector pET16b/CagA and pET16b/VacA.. After transformed into E. coli E. Coli Rosetta, the expression was induced and identified by SDS-PAGE and purified by nickel affinity chromatography. After dialysis and renaturation, the immunogenicity of the recombinant protein CagA,VacA was detected by ELISA method. Results: the results of PCR amplification showed that the target fragments were about 1962bp and 2256bp, respectively. The constructed recombinant plasmid was identified by enzyme digestion and sequencing to prove that the inserted fragment was the target gene of CagA and VacA, respectively. the sequencing results were in good agreement with the login sequence on Genbank. SDS-PAGE analysis showed that under the induction of IPTG, the recombinant engineering bacteria expressed a target protein band with relative molecular weight (Mr) of 75KDa and 87KDa, which accounted for 20% of the total bacterial protein and 90% of the purification rate of nickel affinity chromatography. The target protein mainly exists in the form of soluble protein and inclusion body in bacterial cells. The recombinant protein CagA,VacA detected by indirect ELISA method has certain immunogenicity. Conclusion: pET16b/CagA,pET16b/VacA2 prokaryotic expression recombinant was successfully constructed and transformed into E. coli respectively. CagA and VacA recombinant proteins with relative molecular weight (Mr) of 75KDa and 87KDa were expressed respectively. The recombinant protein CagA, was detected by indirect ELISA method. VacA has better immunoreactivity, in which CagA protein is more reactive than VacA, which lays a foundation for the development of H.pylori protein vaccine and the study of rapid diagnostic kit based on genetic engineering antigen.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378;Q93
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