| 摘要: |
| [目的]构建幽门螺杆菌空泡毒素(VacA)P58亚单位酵母双杂交诱饵表达载体,为应用酵母双杂交系统筛选与空泡毒素P58亚单位相互作用的蛋白奠定基础.[方法]采用PCR方法,从幽门螺杆菌s1/m1型标准毒株NCT11637基因组中扩增空泡毒素P58亚单位,经EcoRⅠ和BamHⅠ双酶切后克隆入经同样酶切处理的酵母双杂交诱饵载体pGBKT7中,构建pGBKT7-VacA P58重组质粒,并将其转化E.coli DH5α,提取质粒经BamHⅠ、EcoRⅠ双酶切、测序鉴定正确后,转化入宿主酵母菌AH109,色氨酸(Trp)缺陷压力筛选,对阳性转化酵母菌扩大培养,提取总蛋白进行Western-blot鉴定,同时检测重组蛋白有无毒性、渗漏和自激活活性.[结果]成功扩增长度为1 202 bp的空泡毒素P58片段,重组质粒pGBKT7-VacA P58经BamHⅠ、EcoRⅠ双酶切可得到1 202 bp大小的片段,测序结果显示扩增产物序列与参考序列完全匹配.重组质粒pGBKT7-VacA P58成功地转化至酵母细胞AH109中.表达的诱饵蛋白无毒性、无渗漏及无自激活活性,Western-blot分析证实酵母菌AH109细胞正确表达了重组蛋白.[结论]成功构建了幽门螺杆菌空泡毒素P58亚单位酵母诱饵表达载体,其可在酵母菌系统中成功表达,且表达产物正确. |
| 关键词: 幽门螺杆菌 酵母双杂交 空泡毒素 P58亚单位 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金 |
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| Construction and identification of the Yeast Two-Hybrid System bait expression vector of Helicobacter pylori Vacuolating cytotoxin A P58 subunit |
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| Abstract: |
| 【Objective】 The study was to construct the bait expression plasmid pGBKT7-VacA P58 of Helicobacter pylori Vacuolationg cytotoxin A P58 subunit in order to lay a foundation for screening unknown protein interacting with P58 subunit from the Yeast Two Hybrid Systen.【Method】 The P58 gene fragment was amplified from Helicobacter pylori type strain NCTC11637 genomic DNA and in frame into vector pGBKT7.Verified by sequencing,the bait vector was transfected into AH109 yeast cells.And the expression of the bait protein was analyzed by Western blot.Meanwhile,the toxicity and self activation of the bait protein were detected.【Result】 Helicobacter pylori Vacuolating cototoxin A P58 subunit was amplified and cloned into the vector pGBKT7 successfully.The bait vector was then transferred into AH109 yeast cells successfully,and no toxicity and self activation were found.The expression of the bait protein was confirmed by Western blot.【Conclusion】 The bait expression vector pGBKT7 VacA P58 was constructed successfully,thus providing a foundation for further studies on the proteins interacting with Vac A in order to reveal the molecule mechanisms of invasion and toxic effect and signal transduction pathway ultimately. |
| Key words: Helicobacter pylori Yeast Two Hybrid System VacA P58 subunit |
