嗜水气单胞菌外毒素和外膜蛋白双基因融合表达载体的构建和高效表达

CONSTRUCTION AND HIGH EXPRESSION OF AN act-OmpTS FUSION VECTOR OF AEROMONAS HYDROPHILA

用基因融合方式 ,以嗜水气单胞菌基因组DNA为模板 ,设计引物通过聚合酶链式反应 (PCR)把去除部分毒性活性编码区的细胞毒肠毒素基因 (act)与去除信号肽的外膜蛋白基因 (OmpTS)连接一起 ,两基因之间插入一个link er(Gly4Ser) 3 经BamHⅠ和HindⅢ双酶切 ,得到 2 .1kb的双基因融合片段 ,克隆于表达质粒pQE 30中 ,构建了双基因重组表达载体pQE30 /act GS ompTS ,转化大肠杆菌M15 (pREP4 ) ,经IPTG诱导 ,表达出预期大小 (81.0kD)的融合蛋白Act GS OmpTS ,此蛋白占菌体总蛋白的 4 2 %。Westernblot检测结果显示 ,该蛋白与抗Act兔血清和抗OmpTS兔血清都呈阳性反应 ,表明融合蛋白保留了外毒素和外膜蛋白的反应原性 。

Cytotoxic enterotoxin and outer membrane protein have been proved to be protective antigens against Aeromonas hydrophila. Primers P1,P2,P3 and P4 were designed based on the sequences of cytotoxic enterotoxin gene (act) and outer membrane gene (OmpTS) of Aeromonas hydrophila in Genbank (primers P1,P2 for act, primers P3,P4 for ompTS). The genomic DNA of a strain of Aeromonas hydrophila isolated in Guangdong Province was extracted and used as PCR template. Then the partial act fragmetnt and ompTS fragment without the signal sequence were amplified separately and purified by DNA agarose gel purification Kit. With these two fragments mixed together as the template, one target fragment about 2.1kb was amplified with primer P1 and P4 after the second step PCR amplification. A linker, (Gly 4Ser) 3, was inserted between these two genes. The down stream primer of act overlapped the upstream primer of OmpTS in 21bp and the linker (Gly 4Ser) 3 was encoded by this two primers together. The 2.1 kb fragment was digested by BamHⅠ and HindⅢ, ligated into BamHⅠ/HindⅢ linearized pQE-30 plasmid(Qiagen Co.). pQE30/act-GS-OmpTS,an expression vector with the fusion fragment was then constructed. After transformed into E. coli M15 (pREP4) and induced with IPTG, pQE30/act-GS-OmpTS was hyper-expressed. To optimize the expression of the recombinant fusion protein, expression conditions ranging in salinity, pH value, IPTG concentration, induced time, induced temperature and medium type were tested. The optimal condition was proved to be pH7, 0?0.2% NaCl,LB,30℃and 0.2mmol/L IPTG for 3-5h. The recombinant fusion protein (Act-GS-OmpTS) exhibited a molecular weight of about 81.0 kDa in 12% SDS-PAGE, which was identical to what had been anticipated. Scanned by CS-950 spot scanning densitometer (SHIMADZU TM),the band of the recombinant fusion protein showed about 42% of total E. coli proteins. Western blot analysis shown that rabbit polyantibody to Act and OmpTS all reacted with Act-GS-OmpTS, which indicated the fusion protein may have similar epitopes to those of the natural proteins.