犬γ干扰素多克隆抗体的制备及生物活性检测  

Preparation of rabbit antisera against CaIFN-γ and evaluation of CaIFN-γ bioactivity

作　　者：李一经[1] 吴昱[1] 唐丽杰[1] 葛俊伟[1] 乔薪瑗[1] 崔文[1] 姜艳平[1] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030

出　　处：《东北农业大学学报》2012年第12期73-77,共5页Journal of Northeast Agricultural University

基　　金：黑龙江省高校科技创新基金(2011TD001)

摘　　要：将犬γ干扰素基因和表达载体pET30a双酶切后构建了重组质粒pET30a-CaIFN-γ,并转化到E.coliRosetta感受态中进行诱导表达。SDS-PAGE和Western blot分析显示,重组蛋白约23 ku并主要以包涵体形式存在。包涵体通过切胶纯化后肌注家兔,制备出CaIFN-γ的抗血清,测得血清效价为1:12 800。对复性后的包涵体通过细胞试验进行抗病毒活性检测,表明重组犬γ干扰素在终浓度为0.09 mg·mL-1时具有一定的抗病毒活性。本试验成功的原核表达、纯化了犬γ干扰素蛋白,制备CaIFN-γ的抗血清,为CaIFN-γ蛋白生物学特深入研究和生产应用奠定基础。The canine IFN-y gene was digested and was inserted into plasmid pET30a to construct recombinant plasmid pET30a-CalFN-γ, then the recombinant plasmid was transformed into E. coil Rosetta. The recombinant protein was expressed after inducing. SDS-PAGE and Western-blot results showed that the recombinant protein was about 23 ku and mainly in the form of inclusion bodies. CalFN-γ antiserum was generated by immunizing the rabbit with the purified recombinant CalFN-γ protein by gel isolation. ELISA result showed that the serum titer was 1 12 800. The biological activity of renaturated protein was checked by cell experiment. Cell experiment result showed that CalFN-γ with the terminal concentration of 0.09 mg · mL-1 possesses characteristic of antivirus. The experiment successfully expressed and purified the CalFN-γ protein and obtained the CalFN-γ antiserum, and provided a reliable basis for the further study on CaIFN-γ protein biological characteristics and its application in the productive practice.

关 键 词：犬IFN-γ 原核表达 抗病毒活性 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]