罗非鱼无乳链球菌LrrG-Sip融合基因原核表达载体的构建及表达

LrrG和表面免疫原性蛋白（Sip）是无乳链球菌（Streptococcus agalactiae）的2种表面蛋白,具有良好的免疫原性。为获得罗非鱼无乳链球菌表面蛋白LrrG和Sip蛋白的融合蛋白,该试验采用基因拼接技术中的双酶切法分2步逐个将Sip和LrrG基因插入pColdⅡ载体中,构建原核表达载体pColdⅡ-LrrG-Sip。将成功构建的融合基因原核表达载体转化感受态细胞BL21（DE3）,进行诱导表达条件的优化。结果显示,15℃、IPTG 0.5 mmol·L-1诱导9 h,目的蛋白呈可溶状态的表达量最高。Western Blot检测结果显示LrrG-Sip融合蛋白大小与预测一致（162kDa）,说明成功构建了融合基因,为罗非鱼源无乳链球菌亚单位疫苗的研制奠定了基础。

Construction and expression of prokaryotic expression vector for LrrG-Sip fusion gene of Streptococcus agalactiae in tilapia

LrrG （leucine-rich repeat protein from GBS） and Sip （surface immunogenic protein）, which are two kinds of surface anti- gen proteins from Streptococcus agalactiae in tilapia, have good immunogenicity. To obtain the LrrG-Sip fusion protein via coalescing surface antigen protein LrrG and Sip of S. agalctiae in tilapia, we cloned Sip and LrrG genes into vector pCold 11 one by one using double enzyme method of gene splicing technology, and constructed a prokaryotic expression vector pCold II -LrrG-Sip. The recombinant plasmid was transformed into E. coli BL21 （DE3） , and the result indicated that 9 h, 15 ℃ , 0. 5 mmol·L^-1 IPTG were the optimum inducing conditions under which fusion protein was most soluble and abundant. Western blotting test showed that the LrrG-Sip fusion protein was about 160 kDa, consistent with the prediction （162 kDa） , which suggested the prokaryotic expression vector pCold II -LrrG-Sip was constructed successfully and laid the foundation for developing subunit vaccines for S. agalctiae in tilapia.