ltB-ureB融合基因原核表达系统的构建及其产物免疫性和佐剂活性的鉴定

构建ltB-ureB融合基因原核表达系统并对其表达产物的免疫性和佐剂活性进行鉴定.采用PCR和T-A克隆法从幽门螺杆菌(Helicobacter pylori,Hp)临床菌株Y06和大肠杆菌44851株DNA中获得了ureB和ltB全长基因扩增片段及其克隆,并构建了ltB-ureB融合基因及其原核表达系统pET32a-ltB-ureB-E.coli BL21DE3.在E.coli BL21DE3宿主菌中用不同浓度的IPTG诱导表达,并用Hp全菌抗体的Western blot、ELISA以及GM1ELISA分别证实了目的重组蛋白(rLTB-UreB)的免疫性和佐剂活性.与报道的相关序列比较,所克隆的ureB和ltB核苷酸序列同源性分别为96.88%～97.82%和99.12%～99.71%,氨基酸序列同源性为99.65%～99.82%和97.58%～99.19%.0.1～1.0 mmol/L的IPTG均能有效地诱导目的重组蛋白rLTB-UreB的表达,该蛋白主要以包涵体形式存在,其产量约为细菌总蛋白的35%.Western blot结果证实rLTB-UreB不仅能与商品化的Hp全菌抗体结合,免疫家兔后也能产生特异性抗体,表明rLTB-UreB有良好的免疫反应性及抗原性.GM1-ELISA结果显示rLTB-UreB能与牛GM1结合,表明rLTB-UreB有佐剂活性.以兔抗rLTB-UreB为一抗,发现所检测的109株Hp临床分离菌株均表达UreB;以rLTB-UreB为包被抗原,发现所检测的125例Hp感染者血清中均存在UreB抗体;表明UreB广泛存在于不同的Hp菌株中,并有很强的抗原性,也提示rLTB-UreB确有自然表达UreB的抗原特异性.本文成功地构建了LTB-UreB融合基因原核高效表达系统,所表达的LTB-UreB融合蛋白有良好的免疫性和佐剂活性,为Hp基因工程疫苗的产业化奠定了坚实的基础.

Construction of prokaryotic expression system of ltB-ureB fusion gene and identification of the recombinant protein immunity and adjuvanticity

To construct ltB-ureB fusion gene and its prokaryotic expression system and identify immunity and adjuvanticity of the expressed recombinant protein, the completed ureB gene from a clinical Helicobacter pylori strain Y06 and ltB gene from Escherichia coli strain 44851 and ltB-ureB fusion gene were amplified by PCR and cloned by T-A cloning method. A prokaryotic expression system of ltB-ureB fusion gene, named as pET32a-ltB-ureB-E.coli BL21DE3, was then constructed. The target recombinant protein (rLTB-UreB) was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot, ELISA assay, and GM-1-ELISA were applied to determine immunity and adjuvanticity of rLTB-UreB recombinant protein. In comparison with the reported corresponding sequences, homologies of the nucleotide sequences of the cloned ureB and ltB genes were 96.88%-97.82% and 99.12%-99.71%, while homologies of the putative amino acid sequences of the two cloned genes were 99.65%-99.82% and 97.58%-99.19%, respectively. IPTG with different dosages of 0.1-1.0 mmol/L could effectively induce pET32a-ltB-ureB-E.coli BL21DE3 to express the target recombinant protein rLTB-UreB mainly presented by the form of including body and the output was approximate 35% of the total bacterial proteins. Western blot results in this study demonstrated that rLTB-UreB could combine to a commercial antibody against whole cell of H.pylori and induce rabbit to produce specific antibody, which indicating the qualified immunoreactivity and antigenicity of rLTB-UreB. The combination of rLTB-UreB and bovine GM-1 confirmed by GM-1-ELISA revealed the existence of adjuvanticity. By using ELISA assays, all the 109 strains of H.pylori isolates were found to express UreB when rabbit anti-rLTB-UreB as the first antibody and all the serum samples from 125patients infected with H.pylori were positive for UreB antibody when rLTB-UreB as the coated antigen. Results of the two ELISA assays indicated that UreB with high antigenicity generally exists in different H.pylori isolates and rLTB-UreB really possessed antigen specificity of naturally expressed UreB. A recombinant prokaryotic expression system with high expression efficiency of the target fusion gene ltB-ureB was successfully established. The expressed rLTB-UreB showed that it has qualified immunogenicity, antigenicity and adjuvanticity. All the results mentioned above laid a firm foundation for further development of H.pylori genetic engineering vaccine.