柔嫩艾美耳球虫AMA1蛋白多克隆抗体的制备及在乳酸乳球菌中的表达  

Preparation of polyclonal antibody against AMA1 protein of Eimeria tenellaand its expression in Lactococcus lactis

作　　者：王芬[1] 马春丽[2] 马德星[1] 张跃[1] 黄宇晨[1] 潘龙[1] 高铭扬[1] 洪琴[1] 杨贵君[1] 康健[3] 

机构地区：[1]东北农业大学动物医学学院,江苏南京210095 [2]东北农业大学食品学院,黑龙江哈尔滨150030 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《中国兽医科学》2015年第11期1130-1135,共6页Chinese Veterinary Science

基　　金：黑龙江省自然科学基金项目(C201422);黑龙江省博士后启动金项目(LBH-Q14019)

摘　　要：将柔嫩艾美耳球虫子孢子顶膜抗原AMA1胞外域基因片段(EtAMA1)克隆入原核表达载体pET-30a(+)中,构建重组质粒pET-30a(+)-EtAMA1。质粒经鉴定正确后转化入大肠杆菌中筛选阳性菌,阳性菌经IPTG诱导后采用Western-blot检测目的蛋白的表达情况。用表达的重组蛋白免疫新西兰白兔制备多克隆抗体。应用间接ELISA测定抗体效价,并采用Western-blot检测抗体特异性。将EtAMA1克隆入乳酸菌表达载体pTX8048中构建阳性质粒pTX8048-EtAMA1,质粒电转化入宿主菌乳酸乳球菌(Lactococcus lactis)NZ9000中筛选阳性菌pTX8048-EtAMA1-L.lactis NZ9000。阳性菌经Nisin诱导后采用Western-blot检测目的蛋白的表达情况。结果表明,EtAMA1在大肠杆菌中以包涵体形式表达。多克隆抗体的效价为218。Western-blot检测证实,制备的抗体可与子孢子反应。重组菌pTX8048-EtAMA1-L.lactis NZ9000经Nisin诱导后,采用Western-blot检测到约61ku的目的蛋白。上述研究结果为基于EtAMA1的鸡球虫病乳酸菌口服疫苗的研制提供了重要参考。The gene fragment encoding the extracellular domain of apical membrane antigen AMA1 fromEimeria tenellasporozoites（EtAMA1）was cloned into prokaryotic expression vector pET-30a（＋）to construct recombinant plasmid pET-30a（＋）-EtAMA1.The characterized plasmid was transformed into the Escherichiacolicompetent cells to screen positive strains.The positive strains were induced with IPTG,and then the expression of objective protein was detected by Western-blot.To produce polyclonal antibody,the expressed protein was immunized New Zealand white rabbits.The antibody titer was detected by indirect enzyme-linked immunosorbent assay,and the antibody specificity was confirmed by Western-blot.The EtAMA1 fragment was cloned into the vector pTX8048 to screen positive plasmid,and the positive plasmid pTX8048- EtAMA1 was electrotransformed into host strains Lactococcus lactis NZ9000 to select the positive bacteria pTX8048-EtAMA1-L.lactis NZ9000.The expression of the objective protein was detected by Western-blot after the positive bacteria were induced by 5ng/mL Nisin.The results showed that EtAMA1 was expressed in E.coli strain in form of inclusion bodies.The titer of the prepared polyclonal antibody from rabbit was 218.The prepared antibody was characterized to bind with sporozoite by using Western-blot.The molecular weight of expressed protein in the positive strains pTX8048-EtAMA1-L.lactis NZ9000 was 61ku in size by using Western-blot.This study provided important references for the preparation of oral vaccine against avian coccidiosis.

关 键 词：柔嫩艾美耳球虫 顶膜抗原 多克隆抗体 乳酸乳球菌 

分 类 号：S852.723[农业科学—基础兽医学]