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目的构建融合了幽门螺杆菌尿素膜通道蛋白(Ure I)、尿素酶B亚单位(Ure B)的表位基因及霍乱毒素B亚单位(CTB)的原核表达质粒pET28a(+)/ct B/ure I-B(简称BIB)及其原核表达工程菌,并研究其生物学特性。方法参考基因库中ure I、ure B、ct B的基因序列,合成不含信号肽的ct B基因及ure I、ure B优势表位基因,串联融合,形成多靶点重组ct B/ure I-B基因(简称BIB基因),并构建pET28a(+)/BIB原核表达质粒,经限制性内切酶Nco I、Xho I酶切以及DNA测序鉴定正确后,将重组质粒导入大肠杆菌BL21(DE3)中,构建含BIB基因的原核表达工程菌。经乳糖诱导后,SDS-PAGE电泳检测重组蛋白(rBIB),Western blot及肌注BALB/c小鼠实验检测rBIB的免疫反应性和免疫原型。结果构建的原核表达质粒pET28a(+)/BIB,经双酶切和测序分析显示,构建的BIB基因与设计序列100%一致。乳糖诱导后,SDS-PAGE电泳显示在33kD左右出现一条明显蛋白条带,Western blot检测在33kD左右出现特异反应条带,肌注免疫BALB/c小鼠产生了较高的抗体水平。结论成功构建了pET28a(+)/BIB原核表达质粒及BIB原核表达工程菌,该工程菌可表达重组蛋白rBIB,且该重组蛋白具有良好的免疫反应性及免疫原性。
Objective To construct a prokaryotic expression plasmid pET28a (+) / ct B / C which integrates the epitopes of Ure I, Ure B and CTB of Helicobacter pylori, ure IB (referred to as BIB) and its prokaryotic expression engineering bacteria, and study its biological characteristics. Methods With reference to the gene sequences of ure I, ure B and ct B in the gene bank, the ct B gene without signal peptide and the predominant urease and ure B gene were synthesized and fused in series to form a multi-target recombinant ct B / ure IB (BIB gene). The prokaryotic expression vector pET28a (+) / BIB was constructed and verified by restriction endonucleases Nco I, Xho I and DNA sequencing. The recombinant plasmid was then introduced into E. coli BL21 (DE3) , Construction of prokaryotic expression engineering bacteria containing BIB gene. After induced by lactose, the recombinant protein (rBIB) was detected by SDS-PAGE and the immunoreactivity and immunophenotype of rBIB were detected by Western blot and intramuscular injection of BALB / c mice. Results The constructed prokaryotic expression plasmid pET28a (+) / BIB was digested and sequenced. The constructed BIB gene was 100% identical to the designed sequence. After induction with lactose, SDS-PAGE electrophoresis showed a significant protein band around 33kD, Western blot detection of specific bands appeared around 33kD, intramuscular immunization of BALB / c mice produced higher antibody levels. Conclusion The prokaryotic expression vector pET28a (+) / BIB and the prokaryotic expression vector BIB of BIB were successfully constructed. The recombinant protein rBIB was expressed in E. coli BL21 (DE3). The constructed recombinant protein has good immunoreactivity and immunogenicity.