文献类型：期刊文献

中文题名：嗜水气单胞菌外膜蛋白(OMP)的原核表达及其对BALB/c小鼠的免疫保护研究

英文题名：Prokaryotic Expression of Outer Membrane Protein(OMP) from Aeromonas hydrophila and the Immunoprotection in BALB/c Mice

作者：胡春霞[1,2];李梅[1];李卫芬[1];许梓荣[1];沈文英[3]

机构：[1]浙江大学教育部动物分子营养学重点实验室;[2]绍兴文理学院元培学院生命科学系;[3]绍兴文理学院生命科学学院

年份：2010

期号：2

起止页码：218

中文期刊名：农业生物技术学报

外文期刊名：Journal of Agricultural Biotechnology

收录：CSTPCD、、CSCD2011_2012、北大核心2008、北大核心、CSCD

基金：浙江省重大科技专项项目(2006C12086)资助

语种：中文

中文关键词：嗜水气单胞菌;外膜蛋白;原核表达;小鼠;免疫保护

外文关键词：Aeromonas hydrophila; Outer membrane protein; Prokaryotic expression; Mice; Immune protection

中文摘要：为确定嗜水气单胞菌(Aeromonas hydrophila,Ah)AS1.927株外膜蛋白(OMP)的原核表达产物是否具有抗原性及其对小鼠的免疫保护力。本研究根据GenBank上嗜水气单胞菌外膜蛋白ompA基因序列(GenBank登录号:AF146597)设计1对引物,克隆出ompA全长基因1020bp(GenBank登录号:EU309491),经SalⅠ和XholⅠ双酶切后连接pET-30a(+),转化大肠杆菌(Escherichia coli)BL21,IPTG诱导表达,并进行SDS-PAGE及Western blot分析鉴定。结果显示重组基因工程菌E.coli[pET-30a-ompA]表达的融合蛋白分子量约为40.7kD,可以被鼠抗嗜水气单胞菌外膜蛋白抗血清识别。将重组基因工程菌E.coli[pET-30a-om-pA]口服免疫BALB/c小鼠(Mus mussulus);末次免疫1周后用放射免疫分析小鼠肠粘膜分泌型免疫球蛋白A(sIgA)水平,检测结果揭示,该重组基因工程菌能提高sIgA的分泌(P<0.05);同时在小鼠血清中测出特异性抗体IgG(P<0.05)。末次免疫2周后用100LD50的AhAS1.927(3.3×105cfu/mL)腹腔注射攻击小鼠,口服重组菌对小鼠的相对免疫保护力(relative percent survival,RPS)为75%,表明重组基因工程菌可诱导小鼠对AhAS1.927产生一定的免疫保护作用。

外文摘要：Outer membrane protein（OMP） of Aeromonas hydrophila（Ah） AS1.927 strain can be ideal antigen candidates for vaccine design.To prove its antigenicity and immunoprotection of the expressed OMP, a 1 020 bp fragment encoding ompA was cloned from the Ah AS1.927 genome by a pair of primers designed according to the sequence of ompA in GenBank（AF146597） After digesting the fragment by Sal Ⅰ＋Xhol Ⅰ, the fragment was inserted into pET-30a（＋） vector to construct the recombinant plasmid pET-30a-ompA .After transformed into the expressing Escherichia coli strain BL21, the aimed protein was expressed induced by IPTG.The expressed OMP was analyzed by SDS-PAGE and Western blot.Results showed that the OMP fusion protein was about 40.7 kD and could react with anti-sera against OMP from mice.BALB/c mice（Mus musculus） were orally inoculated at 1st, 2nd,3rd, 20th, 21th, and 22th day with the engineering bacteria E.coli pET-30a-ompA.Serum IgG was detected by ELISA at 7th day post-vaccination.On the 14th day after the last immunization, the mice were challenged with 3.3×10^5 of live Ah AS1.927（100 LD50）.Results showed that mice vaccinated with E.coli pET-30a-ompA significantly increased antigen-specific IgG level in the serum（P0.05） and secretory immunoglobulin A（sIgA） level in the intestinal mucosa compared with the control.Besides, the immunized group had nearly 75% relative percent survivals（RPS）.This indicated that oral immunization of E.coli pET-30a-ompA can protective initiate an immune response upon challenge with live Ah AS1.927 in BALB/c mice.The present study will provide technical basis for development of efficient gene engineering vaccine against A.hydrophila.