【摘要】：目的 構建幽門螺桿菌vacA-hpaA融合基因；構建大腸桿菌-雙歧桿菌穿梭表達質粒pGEX-vacA-hpaA;分析重組質粒pGEX-vacA-hpaA在大腸桿菌BL21中的表達;電穿孔轉化兩歧雙歧桿菌(Bb),構建幽門螺桿菌重組Bb-vacA-hpaA候選疫苗。 方法 以質粒pQE-vacA、pET-hpaA為模板,PCR擴增獲得vacA和hpaA編碼基因序列；依次構建重組質粒pQE-hpaA, pQE-vacA-hpaA,以重組質粒pQE-vacA-hpaA為模板,PCR擴增構建融合基因vacA-hpaA。將融合基因vacA-hpaA定向連接至大腸桿菌-雙歧桿菌穿梭表達載體pGEX-1λT,構建重組質粒pGEX-vacA-hpaA。電穿孔將pGEX-vacA-hpaA導入BL21, SDS-PAGE分析pGEX-vacA-hpaA在大腸桿菌BL21中的表達；Western blot鑒定表達蛋白的抗原性。最后將pGEX-vacA-hpaA電轉化導入Bb,構建幽門螺桿菌重組Bb-vacA-hpaA候選疫苗。 結果 瓊脂糖凝膠電泳證實vacA-hpaA融合基因全長1500bp左右,測序結果顯示其與預期結果一致。雙酶切證實vacA-hpaA融合基因成功插入pGEX-1λT質粒,成功構建重組質粒pGEX-vacA-hpaA并成功轉入大腸桿菌BL21。SDS-PAGE結果顯示重組質粒在大腸桿菌BL21中經IPTG誘導4h表達了約85KDa的目的蛋白,Western blot分析顯示該蛋白能分別與兔抗VacA和兔抗HpaA免疫血清發(fā)生特異性結合。最后PCR及雙酶切鑒定證實重組質粒pGEX-vacA-hpaA成功轉入兩歧雙歧桿菌Bb,成功獲得rBb-vacA-hpaA候選疫苗。 結論 1.成功構建vacA-hpaA融合基因。 2.成功構建幽門螺桿菌穿梭表達載體pGEX-vacA-hpaA。 3.幽門螺桿菌穿梭表達質粒pGEX-vacA-hpaA能在大腸桿菌BL21中經IPTG誘導表達,而且所表達的重組蛋白同時具有VacA和HpaA蛋白單獨的抗原性。 4.成功構建幽門螺桿菌rBb-vacA-hpaA候選疫苗。
[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.
【學位授予單位】：大理學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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