【作者】 高峰；

【导师】 吴利先；

【作者基本信息】 大理学院 ， 病原生物学， 2011， 硕士

【摘要】 目的构建幽门螺杆菌vacA-hpaA融合基因；构建大肠杆菌-双歧杆菌穿梭表达质粒pGEX-vacA-hpaA;分析重组质粒pGEX-vacA-hpaA在大肠杆菌BL21中的表达;电穿孔转化两歧双歧杆菌(Bb),构建幽门螺杆菌重组Bb-vacA-hpaA候选疫苗。方法以质粒pQE-vacA、pET-hpaA为模板,PCR扩增获得vacA和hpaA编码基因序列；依次构建重组质粒pQE-hpaA, pQE-vacA-hpaA,以重组质粒pQE-vacA-hpaA为模板,PCR扩增构建融合基因vacA-hpaA。将融合基因vacA-hpaA定向连接至大肠杆菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-vacA-hpaA。电穿孔将pGEX-vacA-hpaA导入BL21, SDS-PAGE分析pGEX-vacA-hpaA在大肠杆菌BL21中的表达；Western blot鉴定表达蛋白的抗原性。最后将pGEX-vacA-hpaA电转化导入Bb,构建幽门螺杆菌重组Bb-vacA-hpaA候选疫苗。结果琼脂糖凝胶电泳证实vacA-hpaA融合基因全长1500bp左右,测序结果显示其与预期结果一致。双酶切证实vacA-hpaA融合基因成功插入pGEX-1λT质粒,成功构建重组质粒pGEX-vacA-hpaA并成功转入大肠杆菌BL21。SDS-PAGE结果显示重组质粒在大肠杆菌BL21中经IPTG诱导4h表达了约85KDa的目的蛋白,Western blot分析显示该蛋白能分别与兔抗VacA和兔抗HpaA免疫血清发生特异性结合。最后PCR及双酶切鉴定证实重组质粒pGEX-vacA-hpaA成功转入两歧双歧杆菌Bb,成功获得rBb-vacA-hpaA候选疫苗。结论1.成功构建vacA-hpaA融合基因。2.成功构建幽门螺杆菌穿梭表达载体pGEX-vacA-hpaA。3.幽门螺杆菌穿梭表达质粒pGEX-vacA-hpaA能在大肠杆菌BL21中经IPTG诱导表达,而且所表达的重组蛋白同时具有VacA和HpaA蛋白单独的抗原性。4.成功构建幽门螺杆菌rBb-vacA-hpaA候选疫苗。更多还原

【Abstract】 ObjectiveTo compose the vacA-hpaA fusion gene of Helicobacter pylori; To construct the Escherichia coil-Bifidobacterium shuttle plasmid pGEX-vacA-hpaA; To analyse the plasmid pGEX-vacA-hpaA induced by IPTG in BL21; To transform the recombinant plasmid into Bifidobacterium bifidum by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.MethodsAmplified the vacA and hpaA genes by PCR from plasmids pQE-vacA and pET-hpaA; Constructed recombinant plasmids pQE-hpaA and pQE-vacA-hpaA in order, and amplified the vacA-hpaA fusion gene by PCR from recombinant plasmid pQE-vacA-hpaA. Linked the vacA-hpaA fusion gene to Escherichia coil-Bifidobacterium shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA. Introduced the recombinant plasmid pGEX-vacA-hpaA into BL21 by electroporation, analysed the target protein induced by IPTG in BL21 for 4h by SDS-PAGE and Western blot.Finally introduced the recombinant plasmid pGEX-vacA-hpaA into Bb by electroporation to construct the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori.ResultsAgarose gel electrophoresis identified the vacA-hpaA fusion gene was about 1500bp, sequence analysis confirmed the sequence of the vacA-hpaA fusion gene was in accordance with the expected result. Double-digestion demonstrated the vacA-hpaA fusion gene was correctly cloned into the multiple cloning site of Escherichia coil-Bifidobacterium expression shuttle plasmid pGEX-1λT to construct recombinant plasmid pGEX-vacA-hpaA, and the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into BL21. SDS-PAGE showed the recombinant plasmid pGEX-vacA-hpaA was able to express a 85Kda target protein in E.coil BL21 induced by IPTG for 4h, Western blot showed the target protein could combine specifically with the rabbit VacA immune surum and the rabbit HpaA immune surum. Amplifying the plasmid from the rBb selected in MRS medium with 100μg/ml AMP by PCR and double-digestion demonstrated that the recombinant plasmid pGEX-vacA-hpaA was successfully introduced into Bb and the recombinant candidate vaccine Bb-vacA-hpaA of Helicobacter pylori was constructed successfully.Conclusion1. Amplified the vacA-hpaA fusion gene successfully.2. Constructed the recombinant plasmid pGEX-vacA-hpaA successfully.3. The recombinant plasmid pGEX-vacA-hpaA was able to express the target protein in E.coil BL21 induced by IPTG, and the target protein had specific antigenicity of both VacA and HpaA.4. Constructed the Helicobacter pylori recombinant candidate vaccine Bb-vacA-hpaA successfully.更多还原