猪源Mx1基因的原核表达及其抗血清制备

Prokaryotic expression and identification of porcine Mx1 gene and preparation of the mouse antiserum

利用1对特异性引物,从pT-poMx1中扩增出Mx1基因开放式阅读框(1 992 bp),并将其编码区插入到携带GST标签的原核表达载体pGEX-6p-1中,经酶切和序列测定后,重组质粒pGEX-poMx1转化入大肠杆菌BL21(DE3),利用IPTG诱导表达融合蛋白并分析表达效果。结果表明:经终浓度为0.8 mmol.L-1 IPTG诱导后4 h,猪源Mx1基因(poMx1)在BL21(DE3)中获得高效表达,表达量占菌体蛋白的32.6%,SDS-PAGE显示融合蛋白相对分子质量为99×103。将目的蛋白切胶后免疫Balb/c小鼠,制备了特异性多抗血清,Western blot结果显示该抗血清与融合蛋白孵育反应后有明显的特异性条带。结论:poMx1基因表达成功。

Based on a pair of specific primers,the open reading frame of porcine Mx1（1 992 bp）gene（poMx1）was amplified from pT-poMx1 by PCR.The poMx1 gene was cloned into the prokaryotic expression vector of pGEX-6p-1 and verified by enzyme digestion and DNA sequencing.Then this recombinant plasmid was transformed into Escherichia coli BL21（DE3）for overexpression under opitimization conditions.The expression product had a molecular mass about 99×103 as expected.The poMx1 protein was separated in gel slices and used to immunize Balb/c mice.Antiserum was prepared and specificity analyzed by Western blot.High titer of antiserum against porcine Mx1 protein was obtained.The successful expression of porcine Mx1 protein in E.coli BL21 and the preparation of poMx1 specific mouse antiserum laid foundation for the further study on the antiviral mechanism.