吉林白鹅α干扰素基因的原核表达及抗血清的制备

摘要

The interferon a gene of Jilin white goose was amplified from genome DNA, the segment coding mature peptide of the GolFN -α gene was cloned into prokaryotic expression vector pGEX - 6p - 1 to construct recombinant plasmid pGEX - IFN -α. pGEX - IFN -α was transformed into E. coli BL - 21 ( DE3 ) , which can express fused protein GST - IFN -α in E. coli BL - 21 ( DE3 ) by IPTG inducing. The expressed product was identified by SDS - PAGE and Western blot. The results showed that fusion protein GST - IFN -α was about 45 ku and it could react with interferon antibody. After purificating with Glutathione Sepharose -4B affinity column, and then refolded by dialysis against water, antisera against goose IFN -a was generated by immunizing rabbis with the purified protein. The expression and purification of recombinant goose IFN - ct fusion protein and preparation of the antisera against goose IFN -α will lay a foundation for the future application of goose IFN -α.%采用PCR方法扩增了吉林白鹅α干扰素（JL-GolFN—α）成熟肽编码序列，将IFN—α片段定向插入原核表达载体pGEX-6p-1中，构建重组质粒pGEX—IFN—α，将重组质粒转入大肠杆菌BL21（DE3）的感受态细胞里，在IPTG诱导下表达可溶性的融合蛋白（GST—IFN—α）。SDS—PAGE、Western—blot检测结果表明，重组吉林白鹅IFN—α融合蛋白（rJL—GoIFN-α）的分子量大小约为43ku，表达量占菌体总蛋白的25％。重组吉林自鹅仅干扰素，经谷胱甘肽Sepbarose-4B亲和柱层析纯化后，经过透析复性，得到纯化的目的蛋白，含量可达0．25mg／mL。将复性蛋白免疫新西兰白兔3次，制备高滴度的鹅IFN—α抗血清。本实验表达和纯化了鹅的IFN—α，并制备了兔抗鹅的IFN—α抗血清，为下一阶段鹅α干扰素重组蛋白的应用奠定了基础。