摘要
从健康人外周血分离单核细胞,并提取总RNA,利用RT-PCR扩增出hIFN-γ基因。构建了重组表达菌株BL21(DE3)(pET32a(+)-hIFN-γ)。经酶切鉴定和序列测定证实,构建的重组质粒pET32a(+)-hIFN-γ含有hIFN-γ基因,且基因序列和阅读框架正确。经W estern blot鉴定证实为hIFN-γ,并实现了在大肠杆菌中的高效表达。表达产物占菌体总蛋白的52%。同时对其抗病毒活性进行了测定,从而为hIFN-γ的生物活性和生产工艺研究提供了可靠的试验数据。
Total RNA was prepared from monocytes which were obtained from healthy volunteers.Human interferon-γ（hIFN-γ） gene was amplified with RT-PCR.The recombinant plasmid pET32a（＋）-hIFN-γ containing hIFN-γ gene was obtained and transformed into Escherichia coli BL21（DE3）.The recombinant plasmid pET32a（＋）-hIFN-γ contained the gene hIFN-γ which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis.The result of Western blot showed that the expression protein was correct and expressed high in Escherichia coli.The expression level of the hIFN-γ protein were about 52% of total cellular protein.The antiviral activity of hIFN-γ was tested.This provided experimental data to research the biology activity and productive technique of hIFN-γ.
出处
《中国兽药杂志》
2011年第1期16-19,共4页
Chinese Journal of Veterinary Drug
基金
国家自然科学基金项目资助(30972188)
