| 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。 |
| 关键词: 秦岭大熊猫 IFN-γ基因 基因表达 生物活性 |
| DOI： |
| 分类号: |
| 基金项目:国家林业局大熊猫国际合作项目（WH0632） |
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| Expression of Ailuropoda melanoleuca qinlingensis interferon-gamma and bioactivity analysis of the expressed product |
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ZHU Hui1 MA Qing yi2 QIAO Qi1 ZHU Xiao fu3 CHEN De kun1
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| Abstract: |
| 【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 detected by SDS PAGE and Western blot,and it was further purified by Nickel column 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. |
| Key words: Giant Panda in Qinling interferon-γ gene gene expression bioactivity |
