山羊痘病毒毒力因子KLP2的表达及多克隆抗体的制备

Expression of Goatpox Virus Virulence Factor KLP2 and Preparation of Polyclonal Antibody

为了研究羊痘病毒(capripox virus),制备其毒力因子KLP2的多克隆抗体,试验采用基因同源重组的方法分别构建了羊痘病毒两个结构域(KLP2-1和KLP2-2)的原核表达载体并进行测序鉴定。提取质粒后转化大肠杆菌BL21感受态细胞并用IPTG诱导表达,目的蛋白采用SDS-PAGE和Western blotting检测,并用KCl染色切胶纯化,纯化蛋白加弗氏佐剂免疫家兔,制备的抗体用Western blotting和ELISA检测评价。结果表明,试验成功构建了表达载体p ET42b-KLP2-1和p ET42b-KLP2-2,测序结果显示KLP2-1和KLP2-2基因大小分别为657和984 bp,分别表达了约27和38 ku的蛋白,与理论值相符。切胶纯化的蛋白浓度在1~2 mg/m L之间,制备的抗体用Western blotting检测发现,蛋白有明显的特异条带,ELISA检测抗体的效价均为1∶512。说明试验成功表达了KLP2-1、KLP2-2蛋白,制备了相应的多克隆抗体。

In order to study capripox virus,and prepare polyclonal antibody of its virulence factor KLP2,the prokaryotic expression vector of two structure domains（ KLP2-1 and KLP2-2） were built with the method of homologous recombination genes and identified by sequencing. The plasmid were transformed into E. coli BL21 and inducing expressed by IPTG,objective protein were tested by SDS-PAGE and Western blotting,and purified by KCl dyeing rubber cutting.The rabbits were immuned with freund＇s adjuvant,the prepared antibody were evaluated by Western blotting and ELISA test. The results showed that the expression vector of p ET42b-KLP2-1 and p ET42b-KLP2-2 were built successfully,and KLP2-1 and KLP2-2 genes were 657 and 984 bp,and expressed about 27 and 38 ku protein,respectively,it was consistent with theories value. The concentrations of rubber cutting purified protein was 1 to 2 mg/m L. The antibody showed obvious specific bands detecting by Western blotting,and its titer were 1 ∶ 512 detecting by ELISA. This result indicated that KLP2-1 and KLP2-2 proteins were expressed successfully,and the corresponding polyclonal antibody were prepared.