幽門螺桿菌CagA和VacA重組蛋白的表達(dá)、純化及抗原性檢測
發(fā)布時間：2019-05-13 18:17
【摘要】：目的：構(gòu)建幽門螺桿菌(Helicobacter pylori, Hp)細(xì)胞毒素相關(guān)基因A(Cytotoxin associated gene A, CagA)和空泡細(xì)胞毒素(Vacuolating cytotoxin A, VacA)的重組質(zhì)粒,并在大腸桿菌中表達(dá)獲得基因重組蛋白,為檢出幽門螺桿菌致病株和運用于臨床檢測Hp的感染奠定基礎(chǔ)。 方法：挑選CagA基因和VacA基因的優(yōu)勢抗原表位片段,收集H. pylori標(biāo)準(zhǔn)株NCTC11639,提取基因組DNA, PCR擴(kuò)增目的基因片斷,將其分別克隆到克隆載體pGEM-T和pMD18-T載體并測序,構(gòu)建重組質(zhì)粒pGEM-T/CagA和pMD18-T/VacA,將鑒定正確的重組質(zhì)粒進(jìn)行雙酶切,把目的基因克隆入表達(dá)載體pET-16b,構(gòu)建重組表達(dá)載體pET16b/CagA與pET16b/VacA。轉(zhuǎn)化至大腸桿菌E. Coli Rosetta后誘導(dǎo)表達(dá),經(jīng)SDS-PAGE鑒定、鎳親和層析純化,并透析復(fù)性后,ELISA法檢測重組蛋白CagA、VacA的免疫原性。 結(jié)果：PCR擴(kuò)增結(jié)果表明分別得到了大小約為1962bp和2256bp的目的片段；構(gòu)建的重組質(zhì)粒經(jīng)酶切鑒定和測序證明其中插入片段分別為CagA和VacA的目的基因,測序結(jié)果與Genbank上登錄序列比對后,結(jié)果完全一致；SDS-PAGE分析顯示,在IPTG誘導(dǎo)下,重組工程菌表達(dá)了一相對分子量(Mr)約為75KDa和87KDa的目的蛋白條帶,表達(dá)量占細(xì)菌總蛋白的20%,鎳親和層析純化率約90%；目的蛋白在菌體細(xì)胞內(nèi)主要以可溶性蛋白和包涵體形式存在,經(jīng)間接ELISA法檢測重組蛋白CagA、VacA具有一定免疫原性。 結(jié)論：成功構(gòu)建了pET16b/CagA、pET16b/VacA2個原核表達(dá)重組體,將其分別轉(zhuǎn)化至大腸桿菌后,分別表達(dá)出了相對分子量(Mr)約為75KDa和87KDa的CagA和VacA重組蛋白,間接ELISA法檢測重組蛋白CagA、VacA具有較好的免疫反應(yīng)性,其中CagA蛋白的免疫反應(yīng)性較VacA的強些,為H.pylori蛋白質(zhì)疫苗的研制和以基因工程抗原為基礎(chǔ)快速診斷試劑盒的研究打下了基礎(chǔ)。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378;Q93
本文編號：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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378;Q93
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