LTB-hpaA融合基因原核表达产物的鉴定及其免疫保护作用

Identification and immunoprotective effect of prokaryotic expression product of ltB-hpaA fusion gene.

分别从幽门螺杆菌（Helicobacter pylori，Hp）临床菌株Y06和大肠杆菌44851株DNA中扩增1tB和hpaA基因，构建1tB-hpaA融合基因厦其原核表达系统，鉴定其表达产物的免疫原性、佐荆活性及其对Hp SS1株感染小鼠的保护作用．采用PCR分别从上述菌株DNA中扩增hpaA基因和1tB基因片段，构建1tB-hpaA融合基因，T—A克隆后测定核苷酸序列．采用质粒pOE32和宿主菌E．coliM15构建1tB-hpaA融合基因表达系统，并用不同浓度的IPTG诱导表达．采用western blot和GM1—ELISA鉴定表达产物的抗原性、免疫反应性和佐荆活性．建立HP SS1株感染BALB／e小鼠模型，检测rLTB-HpaA的免疫保护效果．采用ELISA检测125例胃、十二指肠粘膜活检标本尿素酶阳性患者血清中HpaA抗体和41株Hp临床菌株HpaA表达情况．与Gen Bank登录的相关序列比较，所构建的ltB-hpaA融合基因核苷酸序列同源性分别为94．25％～99．71％和95．38％～99．19％．所构建的表达系统pQE32-1tB-hpaA—M15的目的重组蛋白（rLTB-HpaA）表达量高达细菌总蛋白的1／3左右．rLTB-HpaA能与Hp全菌抗体发生结合反应，免疫家兔能产生特异性抗体．GM3-ELISA结果显示rLTB-HpaA能与牛GM1结合．rHpaA免瘦小鼠的保护率仅为66．7％，rLTB-HpaA免疫小鼠的保护率可增至83．3％.81．6％患者血清（102／125）HpaA抗体检测结果阳性，所有菌株均含有HpaA．本文成功地构建了ltB-hpaA融合基因高效原核表达系统，所表达的rLTB-HpaA有良好的免疫原性和佐剂活性，并对Hp感染小鼠有较强的免疫保护作用．

LtB gene and hpaA gene were amplified from DNAs of a clinical Helicobacter pylori strain Y06 and Escherichia coli strain 44851, respectively, hB-hpaA fusion gene and its prokaryotic expression system were construc ted, and to the immunogenicity, adjuvanticity and immunoprotection in H. pylori strain SS1 infected mice of the product were determined. By using PCR, hpaA and ltB genes from DNAs of the bacteria mentioned above were amplified and ltB-hpaA fusion gene was constructed. Amplification fragments of the target genes were sequenced after T-A cloning. Plasmid pQE32 and E. coli strain M15 were applied to construct expression system of the fusion gene. This prokaryotic expression system was expressed under inducement with different dosages of IPTG. Antigenieity, immunoreactivity and adjuvantieity of the expressed protein were identified by Western blot and GM, ELISA. The antibody against HpaA in sera of 125 patients with urease positive in gastric and duodenal mucosal biopsy samples and the HpaA expression of 41 of H. pylori isolates were examined by ELISA. H. pylori strain SSI infected BALB/c mouse modal was established to measure immunoprotective effect of rLTB HpaA. In comparison with the corresponding sequences registered in GenBank, similarities of the nucleotide and putative amino acid sequences of the constructed ltB-hpaA gene were 94.25% 99.71% and 95.38%-99.19%, respectively. Expression output of the target recombinant protein （rLTB-HpaA） by the constructed expression system pQE32-hB-hpaA-M15 was as high as approximate 1/3 of the total bacterial proteins, rLTB-HpaA was able to combine with antibody against whole cell of H. pylori and induce rabbit to produce specific antibody. Combination of rLTB-HpaA and bovine GM1 was confirmed by the result of GM1-ELISA. The protective rate in the mice immunized with rHpaA alone was as low as 66.7%. When immunized with rLTB-HpaA in the mice, the protective rate was increased to 83.3%. 81.6% of the patients serum samples （102/125） were HpaA antibody positive and all of the H. pylori isolates were HpaA contained. A prokaryotic expression systems with high efficiency of ltB-hpaA fusion gene was successfully established in this study. The expressed rLTB-HpaA showed well immunogenieity and adjuvanticity, and stronger immunoprotective effect in H. pylori infected mice.