猕猴川西亚种肿瘤坏死因子α基因的克隆及表达

发布时间：2018-04-15 00:24

本文选题：猕猴川西亚种 + 肿瘤坏死因子α　；参考：《四川农业大学》2016年硕士论文

【摘要】：肿瘤坏死因子α(tumor necrosis factor-alpha,TNF-α)是一种强效的细胞因子,在参与免疫反应、炎症、损伤反应、控制细胞增殖、细胞分化以及细胞凋亡均能发挥作用。TNF-α及其抑制剂对于多种疾病的早期诊断、预后判断和治疗方面都有重要作用。猕猴川西亚种已用于多种人类重大疾病非人灵长类模型的构建,是研究TNF-α生物制剂对多种疾病影响的高价值实验动物。到目前为止,TNF-α在人和其他动物方面研究较多,但对猕猴TNF-α的研究较少,国内还未见猕猴川西亚种(Macaca mulatta lasiotis，Mm) TNF-α克隆表达的相关报道。本研究通过基因克隆技术获得MmTNF-α,并构建MmTNF-α基因的原核和真核表达菌株,并进行表达,为深入研究MmTNF-α蛋白活性功能、结构功能、制备单克隆抗体、开发检测试剂盒奠定物质基础,也为保护猕猴川西亚种提供资料。1、猕猴川西亚种TNF-α基因的克隆与原核表达参照NCBI/GenBank上登录的猕猴TNF-α mRNA序列设计一对引物,利用基因克隆技术获得MmTNF-α全长转录本为模板,PCR扩增目的基因,将其定向插入pMD-19T上,构建重组菌DH5a/pMD-19T-MmTNF-α,随机挑取阳性重组子进行PCR鉴定及序列测定分析。扩增获得的MmTNF-α基因为702bp,测序结果和GenBank上发布的猕猴TNF-α序列(NCBI Reference Sequence:NM_001047149.1)比对,同源性为99%。根据测序成功的MmTNF-α基因序列设计合成一对表达引物,经亚克隆构建原核表达载体pGEX-4T-1-MmTNF-α,进行PCR、双酶切鉴定,并在E.coliBL21 (DE3)中进行表达。在37℃下,0.1mmol/L IPTG诱导表达原核表达菌株E.coli BL21(DE3)/pGEX-4T-1-MmTNF-α4 h,获得大小约为52 kDa且主要以包涵体形式存在的融合蛋白,与预期大小一致经过WB分析证实重组蛋白具有良好免疫反应性。2、猕猴川西亚种TNF-α真核表达载体的构建与表达将实验室克隆的MmTNF-α基因插入pPIC9K多克隆位点, Sal I线性化重组质粒pPIC9K-MmTNF-α,经电击转化GS115。PCR鉴定阳性克隆,用PCR、MM和MD平板进行转化子表型鉴定,G418梯度平板筛选高拷贝重组菌。重组菌pPIC9K-MmTNF-α/GS115利用甲醇诱导,SDS-PAGE和Western-blot分析表达结果。真核表达菌株GS115/pPIC9k-MmTNF-α表型鉴定为甲醇诱导缓慢型Muts,在30℃下,1%甲醇连续诱导72 h后在上清中检测到大小约为26 kDa的目的蛋白,WB分析表达产物具有免疫反应性。
[Abstract]:Tumor necrosis factor alpha (tumor alpha necrosis factor-alpha, TNF-) is a potent cytokine, involved in immune response, inflammation, injury, control of cell proliferation, cell differentiation and cell apoptosis could play a role in.TNF- alpha and its inhibitor for the early diagnosis of various diseases, prognosis judgement and treatment have an important role. Macaca mulatta lasiotis has been used to construct a variety of major human diseases nonhuman primate model, is of high value in experimental animal study on effect of TNF- alpha biological agents for a variety of diseases. So far, TNF- alpha in human and other animal study more, but the research on rhesus monkey TNF- alpha less, there is no domestic monkeys in Western Sichuan species (Macaca mulatta lasiotis, Mm) reported a cloning and expression of TNF-. This study was MmTNF- by gene cloning, MmTNF- gene and construct the prokaryotic and eukaryotic expression strain, And for the further study of MmTNF- expression, protein activity, structure and function, preparation of monoclonal antibody, lay the material foundation development kit,.1 also provides information for the protection of Macaca mulatta lasiotis, Macaca mulatta lasiotis cloning TNF- gene and prokaryotic expression of monkey TNF- alpha mRNA sequence NCBI/ reference GenBank login a pair of primers were designed to obtain full-length transcripts, alpha MmTNF- as template by gene cloning technique, PCR gene, inserted into pMD-19T, construction of recombinant DH5a/pMD-19T-MmTNF- alpha, randomly selected positive recombinant was analyzed by PCR identification and sequence amplification. The MmTNF- gene was 702bp, rhesus TNF- alpha sequence released sequencing the results on the GenBank (NCBI Reference Sequence:NM_001047149.1) on 99%. homology according to the sequencing of successful MmTNF- alpha gene sequence pairs of primers were designed and synthesized, by PCR was subcloned to construct the prokaryotic expression vector of pGEX-4T-1-MmTNF- alpha, and by double digestion, and E.coliBL21 (DE3) for expression. At 37 DEG C, 0.1mmol/L IPTG induced expression of prokaryotic expression strain E.coli BL21 (DE3) /pGEX-4T-1-MmTNF- alpha 4 h, was about the size of the fusion protein of 52 kDa and mainly in the form of inclusion in the body, consistent with the expected size after WB analysis confirmed that the recombinant protein had a good immune response of.2, Macaca mulatta lasiotis construction and expression of MmTNF- alpha gene cloned into pPIC9K multiple cloning sites of eukaryotic expression vector of TNF- alpha, Sal I linearized recombinant plasmid pPIC9K-MmTNF- alpha, GS115.PCR was identified by electroporation cloning, PCR, MM and MD tablet of transformant phenotype, G418 gradient plate screening high copy recombinant bacteria. Recombinant pPIC9K-MmTNF- alpha /GS115 induced by methanol, SDS-PAGE and Western-blot expression analysis Results the eukaryotic expression strain GS115/pPIC9k-MmTNF- alpha phenotype was identified as methanol induced slow Muts. At 30 C, 1% methanol was continuously induced for 72 h, and the target protein of 26 kDa was detected in the supernatant. The expression product of WB analysis showed immunoreactivity.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78

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