幽门螺杆菌重组Bb-vacA-hpaA候选疫苗的构建
发布时间：2018-07-24 21:08
【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。
[Abstract]:objective
To construct vacA-hpaA fusion gene of Helicobacter pylori, construct E. coli - Bifidobacterium shuttle expression plasmid pGEX-vacA-hpaA, analyze the expression of recombinant plasmid pGEX-vacA-hpaA in Escherichia coli BL21, electroporation transformation of Bifidobacterium Bifidobacterium (Bb), and construct a recombinant Bb-vacA-hpaA candidate vaccine for Helicobacter pylori.
Method
The plasmid pQE-vacA and pET-hpaA were used as templates to amplify the sequence of vacA and hpaA encoding gene, and the recombinant plasmid pQE-hpaA, pQE-vacA-hpaA, recombinant plasmid pQE-vacA-hpaA as the template, and PCR amplification and construction of the fusion gene vacA-hpaA. to connect the fusion gene vacA-hpaA directed to the Escherichia coli shuttle expression vector pGEX-1 lambda, were constructed by PCR amplification. To construct recombinant plasmid pGEX-vacA-hpaA. electroporation, pGEX-vacA-hpaA was introduced into BL21, SDS-PAGE was used to analyze the expression of pGEX-vacA-hpaA in Escherichia coli BL21; Western blot was used to identify the antigenicity of the expressed protein. Finally, pGEX-vacA-hpaA electrical conversion was transferred into Bb to construct a recombinant Bb-vacA-hpaA candidate vaccine for Helicobacter pylori.
Result
The agarose gel electrophoresis confirmed that the vacA-hpaA fusion gene was about 1500bp in length. The sequencing results showed that it was in agreement with the expected results. The double enzyme cut confirmed that the vacA-hpaA fusion gene was successfully inserted into the pGEX-1 lambda T plasmid, successfully constructed the recombinant plasmid pGEX-vacA-hpaA and successfully transferred to the Escherichia coli BL21.SDS-PAGE results to show that the recombinant plasmid was in Escherichia coli BL21. The target protein of about 85KDa was expressed by 4H by IPTG, and Western blot analysis showed that the protein could be specifically combined with Rabbit anti VacA and Rabbit anti HpaA immune sera. Finally, the identification of the recombinant plasmid pGEX-vacA-hpaA successfully transferred to Bifidobacterium Bifidobacterium Bb, and the rBb-vacA-hpaA candidate vaccine was successfully obtained by the identification of the Rabbit anti VacA and the Rabbit anti HpaA immune sera.
conclusion
1. the vacA-hpaA fusion gene was successfully constructed.
2. The shuttle expression vector pGEX-vacA-hpaA was successfully constructed.
3. Helicobacter pylori shuttle expression plasmid pGEX-vacA-hpaA can be induced by IPTG in Escherichia coli BL21, and the recombinant protein expressed at the same time has the antigenicity of VacA and HpaA protein alone.
4. Successful construction of candidate vaccine for Helicobacter pylori rBb-vacA-hpaA.
【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
[Abstract]:objective
To construct vacA-hpaA fusion gene of Helicobacter pylori, construct E. coli - Bifidobacterium shuttle expression plasmid pGEX-vacA-hpaA, analyze the expression of recombinant plasmid pGEX-vacA-hpaA in Escherichia coli BL21, electroporation transformation of Bifidobacterium Bifidobacterium (Bb), and construct a recombinant Bb-vacA-hpaA candidate vaccine for Helicobacter pylori.
Method
The plasmid pQE-vacA and pET-hpaA were used as templates to amplify the sequence of vacA and hpaA encoding gene, and the recombinant plasmid pQE-hpaA, pQE-vacA-hpaA, recombinant plasmid pQE-vacA-hpaA as the template, and PCR amplification and construction of the fusion gene vacA-hpaA. to connect the fusion gene vacA-hpaA directed to the Escherichia coli shuttle expression vector pGEX-1 lambda, were constructed by PCR amplification. To construct recombinant plasmid pGEX-vacA-hpaA. electroporation, pGEX-vacA-hpaA was introduced into BL21, SDS-PAGE was used to analyze the expression of pGEX-vacA-hpaA in Escherichia coli BL21; Western blot was used to identify the antigenicity of the expressed protein. Finally, pGEX-vacA-hpaA electrical conversion was transferred into Bb to construct a recombinant Bb-vacA-hpaA candidate vaccine for Helicobacter pylori.
Result
The agarose gel electrophoresis confirmed that the vacA-hpaA fusion gene was about 1500bp in length. The sequencing results showed that it was in agreement with the expected results. The double enzyme cut confirmed that the vacA-hpaA fusion gene was successfully inserted into the pGEX-1 lambda T plasmid, successfully constructed the recombinant plasmid pGEX-vacA-hpaA and successfully transferred to the Escherichia coli BL21.SDS-PAGE results to show that the recombinant plasmid was in Escherichia coli BL21. The target protein of about 85KDa was expressed by 4H by IPTG, and Western blot analysis showed that the protein could be specifically combined with Rabbit anti VacA and Rabbit anti HpaA immune sera. Finally, the identification of the recombinant plasmid pGEX-vacA-hpaA successfully transferred to Bifidobacterium Bifidobacterium Bb, and the rBb-vacA-hpaA candidate vaccine was successfully obtained by the identification of the Rabbit anti VacA and the Rabbit anti HpaA immune sera.
conclusion
1. the vacA-hpaA fusion gene was successfully constructed.
2. The shuttle expression vector pGEX-vacA-hpaA was successfully constructed.
3. Helicobacter pylori shuttle expression plasmid pGEX-vacA-hpaA can be induced by IPTG in Escherichia coli BL21, and the recombinant protein expressed at the same time has the antigenicity of VacA and HpaA protein alone.
4. Successful construction of candidate vaccine for Helicobacter pylori rBb-vacA-hpaA.
【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
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