重组人ω-干扰素的制备及活性分析

Preparation of recombination human ω-interferon and its active analysis

目的:建立人ω-干扰素(hIFN-ω)的大肠杆菌表达系统,为人ω-干扰素的生产及临床应用奠定基础。方法:自抗凝血中提取人基因组DNA,PCR扩增人ω-干扰素成熟肽编码序列克隆入pGEM-T-easy载体,进一步构建表达型重组质粒pBV-IFN-ω,温度诱导表达,表达产物行SDS-PAGE及Western blot鉴定。溶解并复性包涵体,细胞病变抑制法测定表达产物的抗病毒活性。结果:DNA序列分析证实,重组质粒pBV-IFN-ω含有开放读码框架正确的人ω-干扰素成熟肽编码序列。SDS-PAGE显示,诱导表达碎菌后的沉淀中有大小约为20kD的外源蛋白,Western blot证明此外源蛋白为重组人ω-干扰素,并经体外活性测定证实有良好的抗病毒活性。结论:成功地构建了人ω-干扰素(IFN-ω)的大肠杆菌表达系统,表达出具有抗病毒活性的重组人ω-干扰素。

Objective： To construct an E. coli expressing system of human interferon-ω（IFN-ω）. Methods：Extracted DNA from human blood and PCR human IFN-ω, cloned the human IFN-ω gene into plasmid T-easy and pBV-220. Expressed human IFN-ω in E. coli DHSct, the expressing product was analysed by SDS-PAGE, Western blot and anti-virus capacity test. Results：DNA sequence analysis showed the recombinant plasmid pBV- IFN-ω contained human IFN-ω. SDS-PAGE and Western blot proved that there were hIFN-ω in E. coli DHSct after temperature inducing and the expressing product has anti-virus activity. Conclusion：A human IFN-ω E. coli expressing system was constructed successfully, and the recombination human IFN-ω has anti-virus activity.