秦岭大熊猫IFN-γ基因的表达及其生物活性分析  

Expression of Ailuropoda melanoleuca qinlingensis interferon-gamma and bioactivity analysis of the expressed product

作　　者：朱辉[1] 马清义 乔琪[1] 朱小甫[3] 陈德坤[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]陕西省珍稀野生动物抢救饲养研究中心,陕西周至710402 [3]咸阳职业技术学院,陕西咸阳712000

出　　处：《西北农林科技大学学报（自然科学版）》2009年第12期22-28,33,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家林业局大熊猫国际合作项目(WH0632)

摘　　要：【目的】探讨大熊猫秦岭种群和四川种群IFN-γ基因的差异,获得具有生物活性的秦岭大熊猫重组γ干扰素蛋白。【方法】采集秦岭大熊猫血样,采用RT-nested PCR技术扩增秦岭大熊猫IFN-γ基因,将其克隆到表达载体pET32a中,转入DH-5α,进行菌液PCR和双酶切鉴定,经序列测定与分析,构建表达质粒pET32a-IFN-γ,将其转入BL21(DE3)受体菌后,用IPTG诱导表达秦岭大熊猫IFN-γ蛋白。对重组IFN-γ蛋白进行SDS-PAGE电泳、Western-blot分析后,用镍柱亲和层析纯化可溶性蛋白。采用淋巴细胞增殖试验检测重组IFN-γ的活性。【结果】大熊猫秦岭种群和四川种群的IFN-γ基因序列在434位核苷酸处存在差异,秦岭大熊猫的为G,而四川大熊猫的为C;氨基酸序列相同。重组IFN-γ主要以可溶性形式存在,其分子质量为35.3 ku,约占菌体总蛋白的20%;纯化的可溶性蛋白经SDS-PAGE分析,纯度可达90%以上;重组IFN-γ具有促进淋巴细胞转化增殖的生物学活性。【结论】获得的重组IFN-γ蛋白表达量高且具有生物学活性。[Objective] The research was done to ascertain the difference of IFN-γ gene between Giant Pandas subspecies in Qinling and Sichuan subspecies,and obtain the recombinant IFN-γ protein with biological activity. [Method] The IFN-γ gene of Giant Panda was amplified by RT-nested PCR and PCR products were cloned into expression vector pET32a, then transferred to E. coli DH-5α. The vector pET32a-IFN-γ was obtained by PCR identication,dual enzyme digestion and sequence analysis, pET32a IFN -γ was transferred into E. coli BL21（DE3）and introduced by IPTG to express the recombinant IFN-γ. The recombinant IFN-γ protein was detecled by SDS PAGE and Western-blot,and it was further purified by Nickelcolumn affinity chromatography,the biological activity of the purified protein was detected by lymphocyte proliferation assay. [Result] Sequence analysis revealed the difference of IFN-γ gene was located in 434 nucleotide,compared with Giant Pandas subspecies in Qinling,434 nucleotide were mutated （G→C）. The IFN-γ gene of Giant Panda subspecies in Qinling was expressed effectively. The molecular weight of recombinant protein was about 35.3 ku,accounting for about 20% of total protein of bacterium. The recombinant IFN-γ existed in soluble form. And SDS-PAGE, the purification of the soluble recombinant protein could arrive to more than 90%. The anti-recombinant panda IFN-γ had the ability to stimulate transformation and proliferation of Lymphocyte. [Conclusion] The soluble recombinant IFN-γ of Giant Panda subspecies in Qinling was expressed effectively and had high biological activity.

关 键 词：秦岭大熊猫 IFN-Γ基因 基因表达 生物活性 

分 类 号：S852.4[农业科学—基础兽医学] Q786[农业科学—兽医学]