本文關(guān)鍵詞： 分支桿菌 結(jié)核 熱休克蛋白質(zhì)70 基因表達(dá) 原核細(xì)胞 蛋白質(zhì)類/分離與提純　出處：《吉林大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:構(gòu)建結(jié)核分支桿菌HSP70原核表達(dá)載體pQE30-Mtb HSP70并誘導(dǎo)其表達(dá)、純化及鑒定目的蛋白。 方法:利用PCR技術(shù)從牛型結(jié)核分支桿菌基因組中擴(kuò)增出Mtb HSP70 DNA序列,構(gòu)建重組質(zhì)粒pGEM T-Mtb HSP70,經(jīng)酶切、PCR和測(cè)序鑒定后,將Mtb HSP70基因亞克隆到原核表達(dá)質(zhì)粒pQE30,構(gòu)建重組表達(dá)質(zhì)粒pQE30-Mtb HSP70,在大腸桿菌M15中誘導(dǎo)表達(dá)。用鎳凝膠親和層析的方法純化目的蛋白,Westren Blot雜交鑒定純化蛋白。 結(jié)果:經(jīng)測(cè)序證實(shí),獲得的目的基因與GeneBank公布的結(jié)核桿菌Mtb HSP70基因序列一致。構(gòu)建的原核表達(dá)載體pQE30-Mtb HSP70在大腸桿菌M15中經(jīng)1mmol·L-1 IPTG誘導(dǎo)后表達(dá)出相對(duì)分子量約為70 400的蛋白,鎳柱純化后經(jīng)抗組氨酸單克隆抗體進(jìn)行Westren blotting,在相對(duì)分子量約70400處可見特異性著色帶。 結(jié)論:成功構(gòu)建了原核表達(dá)載體pQE30-Mtb HSP70,并成功誘導(dǎo)了Mtb HSP70蛋白的表達(dá),通過鎳凝膠親和層析法獲得純度較高的Mtb HSP70蛋白,為Mtb HSP70蛋白作為免疫佐劑和免疫原在抗腫瘤、抗結(jié)核等方面的進(jìn)一步研究提供條件。
[Abstract]:Aim: to construct the prokaryotic expression vector pQE30-Mtb HSP70 of Mycobacterium tuberculosis (HSP70) and induce it to express, purify and identify the target protein. Methods: the Mtb HSP70 DNA sequence was amplified from the genome of Mycobacterium bovis by PCR technique, and the recombinant plasmid pGEM T-Mtb HSP70 was constructed and digested by enzyme. After PCR and sequencing, Mtb HSP70 gene was subcloned into prokaryotic expression plasmid pQE30 and the recombinant expression plasmid pQE30-Mtb HSP70 was constructed. Expression was induced in E. coli M15 and purified by nickel gel affinity chromatography. The purified protein was identified by Westren Blot hybridization. Results: it was confirmed by sequencing. The obtained target gene was identical to the sequence of Mtb HSP70 gene of Mycobacterium tuberculosis published by GeneBank. The prokaryotic expression vector pQE30-Mtb was constructed. HSP70 was induced by 1 mmol 路L -1 IPTG in Escherichia coli M15 and expressed a relative molecular weight of 70,400. After purification by nickel column, Westren blotting was carried out by anti histidine monoclonal antibody. The specific band was observed at the relative molecular weight of about 70400. Conclusion: the prokaryotic expression vector pQE30-Mtb HSP70 was successfully constructed and the expression of Mtb HSP70 protein was induced successfully. High purity Mtb HSP70 protein was obtained by nickel gel affinity chromatography. Mtb HSP70 protein was used as immune adjuvant and immunogen in anti-tumor. Further research on anti-tuberculosis is necessary.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 黃波,馮作化,張桂梅,李東,王洪濤;腫瘤細(xì)胞混合肽誘導(dǎo)特異性抗腫瘤免疫應(yīng)答[J];中國(guó)科學(xué)(C輯:生命科學(xué));2002年02期

2 傅慶國(guó),孟凡東,郭仁宣;熱休克蛋白70誘導(dǎo)抗腫瘤免疫的機(jī)制研究[J];細(xì)胞與分子免疫學(xué)雜志;2002年04期

3 鐘文英,普雄明;熱休克蛋白的分子生物學(xué)研究進(jìn)展[J];醫(yī)學(xué)綜述;2005年02期

4 張友會(huì);惡性腫瘤基因治療研究的現(xiàn)狀與展望[J];中國(guó)腫瘤生物治療雜志;2000年04期

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