扬子鳄α型干扰素蛋白的原核表达和纯化及其多克隆抗体的制备

Prokaryotic expression,purfication and preparation of polyclonal antibody of Chinese alligator IFN-α

为了进一步研究扬子鳄IFN-α,构建原核表达载体pET32a-IFN,导入大肠杆菌表达系统后诱导表达,并纯化靶蛋白。使用纯化重组蛋白作为抗原,加入佐剂乳化后免疫3只雌性的6周龄BALB/c小鼠,免疫后第35天对小鼠采血分离血清并测定效价。结果显示,成功构建原核表达载体pET32a-IFN,Western-blot鉴定显示,扬子鳄IFN-α重组蛋白成功表达,大小与预期相符,约为43 ku;免疫后的1号和2号小鼠所产生的多克隆抗体效价均为1∶51200,3号小鼠所产生的多克隆抗体效价为1∶25600,具有良好的特异性,这为后续扬子鳄IFN-α抗病毒的研究奠定了基础。

In order to further study the Chinese alligator IFN-α,the prokaryotic expression vector p ET32 a-IFN was constructed and introduced into the Escherichia coli expression system,target recombinant protein was induced and purified,and was used as the antigen to immunize 3 female 6-week-old BALB/c mice after emulsification with adjuvant for antibody.On the 35 th day,blood was collected from the mice to separate the serum and its titer was determined,and the response between antigen and antibody was done by Western-blot.The results showed that the prokaryotic expression vector p ET32 a-IFN was successfully constructed,and Western-blot identification showed that the Chinese alligator IFN-αrecombinant protein was successfully expressed,and the protein size was about 43 ku as expected.The titer of polyclonal antibodies produced by mice No.1 and No.2 is 1∶51200,and the polyclonal antibody produced by mice No.3 is 1∶25600,all of which have good specificity.The experiment laid the foundation for the follow-up research on the antiviral effect of Chinese alligator IFN-α.