扬子鳄Mx基因的原核表达和纯化及其多克隆抗体的制备

扬子鳄Mx以蛋白质的形式发挥其抗病毒功能,本试验通过构建pET28a-Mx质粒,将其转化入大肠杆菌感受态细胞中,用IPTG诱导其进行蛋白大量表达,并纯化蛋白。将纯化好的蛋白作为抗原,与弗氏佐剂充分乳化,免疫6周龄的BALB/c雌性小鼠,4次免疫后提取其血清,测定效价。应用Western-blot检测制备的鼠抗扬子鳄Mx多克隆抗体的特异性。结果显示,扬子鳄Mx蛋白原核表达载体构建成功,并且应用切胶纯化的方式成功纯化了该蛋白。制备的鼠抗扬子鳄Mx多克隆抗体的效价达1∶51 200以上。本试验研究结果为进一步研究扬子鳄Mx蛋白的抗病毒功能提供了物质条件。

Prokaryotic expression,purfication and preparation of polyclonal antibody of Chinese alligator Mx gene

Chinese alligator Mx protein plays an antiviral function.In this test,p ET28a-Mx plasmid was constructed and was transformed into Escherichia coli competent cells.IPTG induced lots of protein expression,then purified those protein. The purified protein was used as an antigen,emulsified with Freund’s adjuvant sufficiently,and 6-week-old BALB/c female mice were immunized. After the fourth immunization,the serum was extracted and the titer was determined. The specificity of the mouse anti-Chinese alligator Mx polyclonal antibody was detected by Western-blot.The results showed that the prokaryotic expression vector of Chinese alligator Mx protein was successfully constructed in this experiment,and the protein was successfully purified by gelatin purification. Mouse anti-Chinese alligator Mx polyclonal antibody was prepared by mixing the purified protein with Freund’s adjuvant,the valence of antibody was above 1 ∶ 51 200.The results of this experiment provide a material basis for further research on the antiviral function of Chinese alligator Mx protein.