鳗弧菌毒力相关基因fata的克隆及其在大肠杆菌中的表达毕业论文-细胞生物学专业毕业论文.docxVIP

山东师范大学硕士学位论文后的该融合蛋白制备抗原，免疫动物得到的抗血清的效价为1：128。 山东师范大学硕士学位论文 后的该融合蛋白制备抗原，免疫动物得到的抗血清的效价为1：128。 通过本课题的实验，就为今后以外膜蛋白FatA为靶蛋白，将其编码基因连接 到乳酸菌特异的P170表达系统内，而后转化乳酸菌，制备重组乳酸菌活载体疫苗 奠定了基础。这就在一定意义上为预防与治疗鱼类弧菌病提供了新的思路与途径。 关键词：鳗弧菌；fatA基因：基因克隆：原核表达 中图分类号：Q7 2 山东师范大学硕士学位论文Abstract 山东师范大学硕士学位论文 Abstract Vibrio anguillarum，a severe pathogen of marirle fish，is the causative agent of vibriosis which is a highly fatal hemorrhagic septicemic disease．In this paper,fatA gene， the important virulence gene of Hbrio anguillarum，was cloned by the method of molecular cloning and recombinant technics．The recombinant expression vector pET-28a(+)／fatA was constructed and E．coli BL21(DE3)was transformed to express the FatA protein，which is the most important component of the iron transport system in I,Tbrio anguillarum，The expressed protein was purified and used as antigen to iln／ilune animals to gain the antisermn．Tlle titer of the antiserum was measured by the methods of double agar diffusion．nlis provided important data for the designing and application ofthe living vector vaccine for Vibrio anguillarum． The pEIB 1 plasmid was extracted from Vibrio anguillarum MVM425 by the method of SDS cracking and the gene offata Was amplified by the method ofPCR from this plasmid．Then the recombinant expression vector pET-28a(+)／fatA was constructed after double enzyme digestion and the host strain Dh5a was transformed，Ⅱle selected clones were verified by double enzyme digestion and DNA sequencing．DNA sequencing results showed that the sequence of DNA fragments inserted into the vector was identical to the one that had been reported before．111e predict protein coded by the recombinant vector is a fused protein．which has a His—tag at the carboxylic end． The recombinant expression vector pET-28a(+)／fatA was then transformed into E．coli BL21fDE3)and the FatA protein was expressed under the induction of IPTG SDS—PAGE results showed that a protein with a molocular weight