山羊痘病毒毒力因子KLP2的表达及多克隆抗体的制备

发布时间：2018-08-18 14:19

【摘要】：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。
[Abstract]:In order to study the preparation of polyclonal antibody to the virulence factor KLP2 of sheep pox virus (capripox virus), the prokaryotic expression vectors of two domains of sheep pox virus (KLP2-1 and KLP2-2) were constructed by homologous recombination and sequenced. The plasmid was extracted and transformed into Escherichia coli BL21 competent cells and expressed by IPTG. The target protein was detected by SDS-PAGE and Western blotting, purified by KCl staining, purified with Freund's adjuvant and immunized with Freund's adjuvant. The prepared antibody was evaluated by Western blotting and ELISA. The results showed that the expression vectors p ET42b-KLP2-1 and p ET42b-KLP2-2 were successfully constructed. The sequencing results showed that the size of KLP2-1 and KLP2-2 genes were 657 and 984bprespectively, and the proteins of about 27 ku and 38 ku were expressed respectively, which were consistent with the theoretical values. The concentration of purified protein was between 1 mg/m / L and 2 mg/m / L, and the titers of the prepared antibodies were 1: 512 by Western blotting detection, and the titers of Elisa were 1: 512. The results showed that KLP2-1 and KLP2-2 protein were successfully expressed and the corresponding polyclonal antibody was prepared.
【作者单位】：塔里木大学动物科学学院;新疆生产建设兵团塔里木畜牧科技重点实验室;
【基金】：国家自然基金(31360616) 塔里木畜牧科技兵团重点实验室开放课题(HS201307)
【分类号】：S852.654

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