新加坡石斑鱼虹彩病毒ORF162蛋白的表达、纯化及抗体制备

Expression,Purification and Antibody Preparation of Singapore Grouper Iridovirus ORF162

将新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)的ORF162的开放式阅读框插入pET-32a表达载体T7启动子控制下的6-His·Tag编码基因上游,构建SGIVORF162原核表达质粒pET-ORF162。表达质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导,成功表达SGIV ORF162融合蛋白。对IPTG浓度、诱导温度、诱导时间等诱导表达条件进行优化后,确定在0.7mmol/LIPTG、16℃条件下诱导14h时可溶性SGIV ORF162重组蛋白占重组蛋白总量的95%。经镍琼脂糖凝胶纯化,获得纯度为90%以上的SGIV ORF162蛋白。用纯化的SGIV ORF162蛋白免疫小鼠,获得高效特异的SGIV ORF162多克隆抗体。

Full length of SGIV ORF162 was inserted into the prokaryotic vector pET-32a fused with the down stream 6-His-Tag. The expression vector was transformed into the E. coli BL21 （DE3） strain and SGIV ORF162 fusion protein was successfully expressed. The optimum condition for the expression of soluble SGIV ORF162 fusion protein was 0.7 mmol/L IPTG 16 ℃, 14h. The fusion protein were purified by Ni-IDA His.Bind affinity column and the purity of recombinant SGIV ORF 162 protein was above 90%. The polyclonal antibody is prepared by immunizing mice with purified SGIV ORF162 protein and proved to be specific against SGIV ORF162 protein. The purified SGIV ORF162 fusion protein and the polyclonal antibody can be used for further functional and structural studies.