产气荚膜梭菌NetB毒素基因重组植物乳酸杆菌的构建

Construction of Recombinant Lactobacillus plantaram Containing Netb Gene of Clostridium perfringens

选择产气荚膜梭菌坏死性肠炎毒素B(NetB)为抗原,构建产气荚膜梭菌NetB毒素基因重组植物乳酸杆菌,利用植物乳酸杆菌穿梭载体pSIP409构建重组质粒pSIP409-NetB,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,NetB重组蛋白相对分子质量为36 ku。重组质粒pSIP409-NetB电转化植物乳酸杆菌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NetB蛋白的能力,可作为黏膜免疫的候选抗原。

Clostridium perfringens necrosis enteritis toxin B （NetB）was selected as antigen to construct Clostridium perfringens NetB toxin gene recombinant Lactobacillus plantarum, and Lactobacillus plantarum shuttle vector pSIP409 was used to construct recombinant plasmid pSIP409-NetB, after double enzyme digestion, identification and sequencing, the cells were transformed into Escherichia coli BL21 （DE3）competent cells and induced by SppIP. Westeru blot of recombinant protein expression was proved successful, and which was mainly in the form of inclusion body, NetB recombinant protein with a relative molecular mass was 36 ku. pSIP409-NetB recombinant plasmids were electroporated into Lactobacillus NC8 cells, SpplP was induced to experss after PCR and double digestion. Western Blot and indirect immunoiluorescence assay showed that the recombinant LactobaciUus had the ability to induce secretion of NetB protein and could be used as candidate antigen for mucosal immunity.