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目的构建含幽门螺杆菌尿素酶保守基因序列ureI和ureB的双基因(简称IB)多表位原核表达质粒以及原核表达工程菌,并研究其微生物学特性。方法通过生物信息学方法从ureI和ureB基因中筛选T细胞和B细胞优势表位基因序列,人工合成所选择的表位基因序列并构建原核重组表达质粒pET28a(+)/IB。经限制性内切酶(EcoR I,Xho I)鉴定及DNA测序鉴定后,将重组质粒导入大肠杆菌BL21(DE3)。该工程菌经IPTG诱导后,用SDS-PAGE检测重组蛋白(rIB)的表达情况,用Western blot检测rIB的免疫反应性。结果构建的双基因多表位基因序列与所设计的一致;工程菌经IPTG诱导后做SDS-PAGE电泳显示,在28kD左右有一条明显蛋白条带,Western blot结果显示在28kD左右出现特异反应条带。结论成功构建含ureI和ureB双基因序列的原核表达质粒pET28a(+)/IB及工程菌,该工程菌表达的重组蛋白rIB能被抗幽门螺杆菌悉尼株(H.pylori SS1)的特异抗体所识别,表明该新重组蛋白与幽门螺杆菌有高度相关性。
OBJECTIVE: To construct a prokaryotic expression vector of double gene (referred to as IB) containing ureI and ureB gene of Helicobacter pylori urease and prokaryotic expression engineering bacteria, and to study its microbiological characteristics. Methods The sequences of dominant epitopes of T cells and B cells were screened from ureI and ureB genes by bioinformatics methods. The selected epitopes were synthesized and the prokaryotic recombinant plasmid pET28a (+) / IB was constructed. After identification by restriction enzyme (EcoR I, Xho I) and DNA sequencing, the recombinant plasmid was introduced into E. coli BL21 (DE3). After induced by IPTG, the recombinant protein (rIB) was detected by SDS-PAGE and the immunoreactivity of rIB was detected by Western blot. Results The constructed double-gene multi-epitope gene sequence was consistent with that of the designed one. After induced by IPTG, SDS-PAGE electrophoresis showed that there was a distinct protein band around 28kD. Western blot showed that the specific reaction band appeared at about 28kD band. Conclusion The prokaryotic expression plasmid pET28a (+) / IB and the engineered bacteria containing ureI and ureB double gene sequences were successfully constructed. The recombinant protein rIB expressed in this engineering bacterium can be detected by the specific antibody against H. pylori SS1 Identification, indicating that the new recombinant protein and Helicobacter pylori have a high degree of correlation.