鸡IFN-γ基因的克隆及原核表达
摘要
γ-干扰素(Interferon-γ，IFN-γ)是一类主要由活化的T淋巴细胞和NK细胞产生的细胞因子，其作用包括广泛的抗微生物感染(病毒、细胞内寄生菌和寄生虫)、抗肿瘤和调节机体免疫功能。尤其在抗病毒感染和调节机体免疫功能方面作用显著，是动物机体内主要的免疫调节因子之一，因此可望研发成为新型天然生物佐剂。为此，本研究进行了鸡IFN-γ基因的克隆和原核表达，为进一步研究和应用奠定基础。研究内容：1．鸡IFN-γ基因的克隆。用刀豆素(ConA)刺激分离的脾淋巴细胞后，提取总RNA，反转录PCR(RT-PCR)扩增IFN-γ基因，然后将扩增产物与pGEM-T-easy载体连接，获得pGEM-T-easy／CHIFN-γ重组质粒，对其进行酶切、PCR及基因测序鉴定，结果表明，本研究克隆得到了完整的鸡IFN-γ基因，与GeneBank中登录的鸡IFN-γ基因序列完全一致，而且还发现ConA刺激诱导脾淋巴细胞6h、8h效果较好。2．鸡IFN-γ基因的原核表达。在鸡IFN-γ基因开放阅读框(ORF)两侧设计表达引物，以pGEM-T-easy／CHIFN-γ质粒为模板进行PCR扩增，将扩增产物和质粒pGEX-4T-1分别用EcoR Ⅰ和Xho Ⅰ双酶切，纯化回收后连接获得pGEX-4T-1／CHIFN-γ重组质粒；进一步将其转化大肠杆菌BL21，提取质粒，酶切、PCR及基因测序鉴定正确的重组菌用ITPG诱导表达，SDS-PAGE分析表达产物。结果表明，鸡IFN-γ基因插入方向正确，并在大肠杆菌中被成功表达，融合蛋白的分子量大小约为43KD。
IFN-γ is a cytokine predominantly secreted by activated T cells and NK cells, plays important part in anti-infections caused by various microorganisms (viruses, intracellular bacteria and parasites), anti-tumor proliferation and modulate immune function. It is one of main immune regulatory factors in animal body, especially is resistance to virus infections.It is predict great potential as a new type of romising natural adjuvants. So we cloned and expressed CHIFN-γ gene in prokaryoticly cells, which established a foundation for further research and development. Research content: 1. Cloning of CHIFN-γ gene. Total RNA was abstracted from chicken's spleen lymphocytes after being activated by concanavalin A (Con A), CHIFN-γ gene was amplified by Reverse Transcription—Polymerase Chain Reaction (RT—PCR) and ligated into pGEM-T-easy vector.Recombinant plasmid pGEM-T-easy/CHIFN-γ was indentified by endonuclease restriction digestion, PCR and DNA sequencing. The sequence is same to the sequence of CHIFN-γ gene submitted in GenBank. We also found that the result that spleen lymphocytes is activated 6h、 8h by ConA is good. 2. Prokaryotic expression of CHIFN-γ gene .The CHIFN-γ gene was amplified with the primers re-designed beside the ORF of CHIFN-γ gene.The amplified production and pGEX-4T-1 was digested with EcoR I and Xho I .The sequence was inserted into pGEX-4T-1 and the recombinant expression vetor was constructed,then transformed into Escherichia coli BL21.After being induced by IPTG to expess CHIFN-γ.The recombinant fusion protein ananlyzed by SDS—PAGE.The result showed that chicken's CHIFN-γ gene was successfully expressed in prokaryotic cells as a fusion protein with a molecular weight about 43, 000 Dalton.
IFN-γ is a cytokine predominantly secreted by activated T cells and NK cells, plays important part in anti-infections caused by various microorganisms (viruses, intracellular bacteria and parasites), anti-tumor proliferation and modulate immune function. It is one of main immune regulatory factors in animal body, especially is resistance to virus infections.It is predict great potential as a new type of romising natural adjuvants. So we cloned and expressed CHIFN-γ gene in prokaryoticly cells, which established a foundation for further research and development. Research content: 1. Cloning of CHIFN-γ gene. Total RNA was abstracted from chicken's spleen lymphocytes after being activated by concanavalin A (Con A), CHIFN-γ gene was amplified by Reverse Transcription—Polymerase Chain Reaction (RT—PCR) and ligated into pGEM-T-easy vector.Recombinant plasmid pGEM-T-easy/CHIFN-γ was indentified by endonuclease restriction digestion, PCR and DNA sequencing. The sequence is same to the sequence of CHIFN-γ gene submitted in GenBank. We also found that the result that spleen lymphocytes is activated 6h、 8h by ConA is good. 2. Prokaryotic expression of CHIFN-γ gene .The CHIFN-γ gene was amplified with the primers re-designed beside the ORF of CHIFN-γ gene.The amplified production and pGEX-4T-1 was digested with EcoR I and Xho I .The sequence was inserted into pGEX-4T-1 and the recombinant expression vetor was constructed,then transformed into Escherichia coli BL21.After being induced by IPTG to expess CHIFN-γ.The recombinant fusion protein ananlyzed by SDS—PAGE.The result showed that chicken's CHIFN-γ gene was successfully expressed in prokaryotic cells as a fusion protein with a molecular weight about 43, 000 Dalton.
引文
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