嗜水气单胞菌孔蛋白OmpF重组表达及其免疫原性分析  

Recombinant Expression and Immunogenicity Analysis of the Porin Protein OmpF of Aeromonas hydrophila

作　　者：高云山 刘丹丹 徐俊林 桑雨浓 梁夏夏 刘建欣 王文彬 GAO Yun-shan;LIU Dan-dan;XU Jun-lin;SANG Yu-nong;LIANG Xia-xia;LIU Jian-xin;WANG Wen-bin(College of Marine Life and Fishers,Jiangsu Ocean University,Lianyungang 222005;Jiangsu Key Laboratory of Marine Biotechnology,Jiangsu Ocean University,Lianyungang 222005)

机构地区：[1]江苏海洋大学海洋生命与水产学院,连云港222005 [2]江苏海洋大学江苏省海洋生物技术重点实验室,连云港222005

出　　处：《生物技术通报》2019年第9期234-243,共10页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31701680);江苏省自然科学基金项目(SBK2017041308);江苏省优势学科建设工程资助项目(PAPD)

摘　　要：气单胞菌外膜蛋白 OmpF 是β-桶状结构的孔蛋白,参与渗透压环境适应调控,在人鱼共患病原气单胞菌属中保守性较高,作为免疫检测靶点具有较高的研究价值。通过人工合成得到了 GenBank 已发表的嗜水气单胞菌(Aeromonas hydrophila)OmpF 基因片段,将其插入质粒 pET-28a(+)后转入大肠杆菌 BL21(DE3),经异丙基硫代半乳糖苷(IPTG)诱导后表达出分子量为 40 kD 左右的重组蛋白。经过优化表达温度和 IPTG 诱导浓度等后,重组蛋白主要以包涵体的形式表达。采用纯化的包涵体免疫BALB / c 小鼠,ELISA 结果表明小鼠血清与煮沸灭活的 11 株气单胞菌中的 10 株(10/11)均有特异性交叉反应,效价在 1∶81 000,与副溶血性弧菌、创伤弧菌、霍乱弧菌、鳗弧菌、哈维氏弧菌及地衣芽孢杆菌无交叉反应。上述结果表明重组蛋白 OmpF 具有较好的免疫原性和气单胞菌保守性,可以用于制备气单胞菌交叉型抗体。Outer membrane protein(Omp)F in Aeromonas spp. is β-barrel like porin,and involves in the adaption of osmotic pressure and is highly conservative in this common pathogen for human and fish,thus poses a promising research value as immune-detection target. The A. hydrophila OmpF fragment published in GenBank was synthesized,and inserted into plasmid pET-28a(+). The recombinant plasmid was transformed into Escherichia coli BL21(DE3)and induced by isopropy-β-D-thiogalactoside(IPTG). The molecular size of the expressed protein was about 40 kD,which mainly expressed as inclusion bodies after optimization of inducing temperatures and IPTG concentrations. BALB/c mice were immunized with purified OmpF inclusion bodies. The results of ELISA showed that the immunized mice serum cross-reacted with 10 of 11 boiled and deactivated strains of Aeromonas spp.,the serum titers were around 1∶81 000. However,the mice serum did not react with Vibrio cholerae,Bacillus licheniformis,V. parahaemolyticus,V. vulnificus,and V. anguillarum. The above results indicate that the protein OmpF is immunogenic,conserved and was promising in developing Aeromonas cross-reactive antibodies.

关 键 词：嗜水气单胞菌 外膜蛋白OmpF 重组表达 免疫原性 

分 类 号：S941.42[农业科学—水产养殖]