摘要
提取经植物血凝素诱导培养的梅花鹿外周血淋巴细胞总RNA,应用RT-PCR方法扩增出梅花鹿γ干扰素成熟蛋白基因并将其克隆到pMD18-T载体上,测序结果表明,扩增片段为梅花鹿γ干扰素成熟蛋白序列,与GenBank上发表的干扰素序列同源性为100%。将其重组到原核表达载体pET32a(+)上,并在大肠埃希菌BL21中实现了高效表达。表达产物以His-Tag融合蛋白的形式存在,表达量约占细菌总蛋白的32.6%。用镍亲和层析法对蛋白进行纯化,并利用VSV-MDBK/IBRV细胞系统分析其生物活性,重组梅花鹿γ干扰素抗病毒活性分别约为7.25×104U/mL和4.61×104U/mL。结果表明,重组梅花鹿γ干扰素特异性好,而且抗病毒活性比较稳定。
Total RNA was isolated from Cervus nippon peripheral blood lymphocytes,which were stimulated with PHA.Then the IFN-γ cDNA was amplified by RT-PCR.The amplified fragment was cloned into vector pMD18-T and the accuracy was confirmed by sequencing.The homology is 100%,compared with the CerIFN-γ gene published in the Genebank.The purified CerIFN-γ gene was Subcloned into the expression vector pET32a（＋） and this recombinant vector was transformed into the competence E.coli BL21,then induced by IPTG.The expressed production is His-Tag fusion protein which makes up of 32.6% proteins expressed by the bacteria.After the protein was purified by nickel affinity chromatography,its biological activity was analyzed using VSV-MDBK/IBRV cell systems.Results showed that recombinant CerIFN-γ anti-virus activity was about 7.25 ×10^4 U/mL,4.61× 10^4 U/mL.The result indicated that the recombination CerIFN-γ has a good specificity and a stable anti-viral activity.
出处
《动物医学进展》
CSCD
北大核心
2009年第11期1-5,共5页
Progress In Veterinary Medicine
基金
国家973项目(2006CB504401)
湖北省自然基金重大项目(2008CDA073)
艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2009ZX10602-14
2008ZX10301)
关键词
梅花鹿
Γ干扰素
抗病毒活性
Cervus nippon
interferon-gamma
antiviral activity
