本文選題：結(jié)核分枝桿菌 + MPT64��； 參考：《寧夏醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:構(gòu)建結(jié)核分枝桿菌MPT64原核表達(dá)載體pET32a-MPT64并誘導(dǎo)其表達(dá)、純化及鑒定目的蛋白。 方法:采用PCR從MTB H37Rv株DNA基因組中擴(kuò)增MPT64基因,將目的片段克隆至pET32a載體中進(jìn)行測(cè)序。鑒定陽性重組質(zhì)粒pET32a-MPT64,經(jīng)測(cè)序證實(shí)與Genebank公布的基因序列基本一致。將MPT64基因亞克隆入pET32a原核表達(dá)載體,酶切鑒定陽性重組質(zhì)粒pET32a-MPT64,并用IPTG進(jìn)行誘導(dǎo)表達(dá)。SDS-PAGE檢測(cè)和Western-blot鑒定表明,在相對(duì)分子量為24kDa的位置有特異性目的蛋白表達(dá)。采用MagExtractor-His-tag蛋白純化試劑盒對(duì)目的蛋白進(jìn)行純化 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MPT64基因序列基本一致。構(gòu)建的原核表達(dá)載體pET32a-MPT64在大腸桿菌BL21中經(jīng)IPTG誘導(dǎo)后表達(dá)出相對(duì)分子量約為24000的蛋白,MagExtractor-His-tag蛋白純化試劑盒純化后經(jīng)抗組氨酸單克隆抗體進(jìn)行Westren blotting,在相對(duì)分子量約24000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pET32a-MPT64,并成功誘導(dǎo)了MPT64蛋白的表達(dá),通過MagExtractor-His-tag蛋白純化試劑盒純化獲得純度較高的MPT64蛋白,為MPT64蛋白作為免疫原在下一步研究中提供條件。
[Abstract]:Aim: to construct the prokaryotic expression vector pET32a-MPT64 of Mycobacterium tuberculosis MPT64 and induce its expression, purify and identify the target protein. Methods: the MPT64 gene was amplified by PCR from the DNA genome of MTB H37Rv strain and cloned into pET32a vector for sequencing. The positive recombinant plasmid pET32a-MPT64 was identified by sequencing, and the sequence was basically the same as that published by Genebank. The MPT64 gene was subcloned into the prokaryotic expression vector of pET32a. The recombinant plasmid pET32a-MPT64 was identified by restriction endonuclease digestion, and the expression was induced by IPTG. SDS-PAGE detection and Western-blot identification showed that there was a specific target protein expression in the position where the relative molecular weight was 24kDa. Purification of Target protein by MagExtractor-His-tag protein purification Kit Results: it was confirmed by sequencing that the obtained target gene was consistent with the sequence of MPT64 gene of Mycobacterium tuberculosis published by GeneBank. The constructed prokaryotic expression vector pET32a-MPT64 was induced by IPTG in Escherichia coli BL21 to express a relative molecular weight of about 24000 protein, MagExtractor-His-tag protein purification kit was purified by anti-histidine monoclonal antibody to Westren blotting.The relative molecular weight was about 24000. A specific ribbon can be seen. Conclusion: the prokaryotic expression vector pET32a-MPT64 was successfully constructed, and the expression of MPT64 protein was induced successfully. The purified MPT64 protein was purified by MagExtractor-His-tag protein purification kit, which provided the condition for MPT64 protein to be used as immunogen in the further study.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378

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