重组产气荚膜梭菌plc毒素基因植物乳酸杆菌的构建  

Construction of recombinant Lactobacillus plantarum containing plc gene of Clostridium perfringens

作　　者：张艳[1,2] 周庆民[1] 冯万宇[1] 徐馨[1] 黄健[1] 王新[1,2] 李兰兰[2] 郑晓星 宁秀云 李广兴[2] 

机构地区：[1]黑龙江省兽医科学研究所,黑龙江齐齐哈尔161006 [2]东北农业大学动物医学学院,黑龙江哈尔滨150030 [3]黑龙江省讷河市动物防疫站,黑龙江齐齐哈尔161300

出　　处：《中国兽医杂志》2017年第10期31-34,37,共5页Chinese Journal of Veterinary Medicine

摘　　要：依据乳酸菌的肠道益生作用,选择产气荚膜梭菌关键致病因子α毒素/磷脂酶C为抗原,构建产气荚膜梭菌α毒素去除信号肽的plc基因片段重组植物乳杆菌,利用植物乳酸菌穿梭载体pSIP409构建重组质粒pSIP409-plc,经双酶切鉴定和序列测定正确后转化大肠杆菌BL21(DE3)感受态细胞并进行SppIP诱导表达。Western Blot证明重组蛋白表达成功,并且主要以包涵体形式存在,plc重组蛋白相对分子质量分别为40 kDa。重组质粒pSIP409-plc分别电转化植物乳杆菌NC8细胞,PCR和双酶切鉴定正确后进行SppIP诱导表达。Western Blot和间接免疫荧光鉴定表明,构建的重组植物乳酸杆菌具有诱导分泌plc蛋白的能力,可作为黏膜免疫的候选抗原。In this study, according to the intestinal efferveseent effect of lactic acid bacteria, the key pathogenic factor α-toxin / phospholipase C was selected as the antigen to construct the plc gene fragment of Clostridium perfringens alpha toxin by removing the signal peptide. The recombinant plasmid pSIP409 -plc was construeted by using pSIP409, which was transformed into Escherichia eoli BI21 （DE3） competent cells and identified by SppIP induction. Western Blot demonstrated that the recombinant protein was successfully expressed and contained mainly in inclusion bodies. The relative moleeular mass of pie recombinant protein was 40 kDa. The recombinant plasmid pSIP409 - plc was transformed into Lactohacillus plantarum NC8 cells by PCR and digested with double di- gestion. Western Blot and indirect immunofluoreseence showed that the constructed recombinant Lactobacillus plantar had the ability to induee pie protein secretion and could be used as candidate antigen for mucosal immunization.

关 键 词：产气荚膜梭菌 plc基因 植物乳酸杆菌 表达 

分 类 号：S858.31[农业科学—临床兽医学]