摘要
目的构建幽门螺杆菌(Hp)vacA基因片段原核表达载体并进行表达。方法采用PCR技术,扩增HpvacA基因的1个片段B,并克隆入pGEM-3Zf(-)质粒。测序正确后,再克隆入质粒pRSETA中,构建重组表达载体pRSE-TA-B。结果经过转化E.coliBL21DE3(plysS)后,诱导表达出相对分子质量(Mr)为33000的蛋白。SDS-PAGE分析显示,表达量占全菌总蛋白质的47.8%,上清中目的蛋白占全菌总蛋白的10.9%。ELISA和Westernblot分析表明,表达蛋白能与兔抗VacA多抗结合。结论成功地构建原核表达载体pRSETA-B,为进一步探讨该片段的生物学活性,以及临床上通过检测患者血清预测Hp的感染提供了实验依据。
Aim To construct prokaryotic expression vector carrying a fragment of Helicobacter pylori (Hp) vacuolating cytotoxin A(vac A) gene and to express it in Ecoli. Methods Using PCR and recombinant DNA techniques, the segment B of Hp vac A gene was amplified, cloned into clone vector pGEM 3Zf( ), sequenced and then cloned into expression vector pRSETA. Thus the prokaryotic expression vector of a segment related with vacuolating activity of Hp vac A gene was constructed. Results After induction with 5×10-3mmol/L IPTG for 4h, a fusion protein with relative molecular mass(Mr) 33 000 was expressed, representing 47.8% of total bacterial protein in E.coli. The fusion protein in lysate supernatant amounted to 10.9% of total bacterial protein. It was showed that the protein could be bound to rabbit anti Vac A antibody by ELISA and Western blot analysis. Conclusion The prokaryotic expression vector is constructed successfully, so that provide an experiment basis for analyzing the biological function of this segment and predicting Hp infection by virtue of detecting the patient's sera.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2001年第3期237-240,共4页
Chinese Journal of Cellular and Molecular Immunology
