【摘要】： 一、目的 熱休克蛋白(heat shock protein,Hsp)屬于廣泛存在于自然界中高度保守的蛋白質(zhì)家族,在熱應(yīng)激下它們大量表達(dá),因此稱為熱休克蛋白。熱休克蛋白表達(dá)量的提高與許多應(yīng)激條件有關(guān),如低氧、營(yíng)養(yǎng)缺乏、病毒感染、吞噬和轉(zhuǎn)化作用。結(jié)核分枝桿菌是結(jié)核病病原菌。結(jié)核分枝桿菌基因組攜帶有兩組GroEL基因,GroEL1和GroEL2。它們分別編碼熱休克蛋白60(cpn60.1)和熱休克蛋白65(cpn60.2)。當(dāng)機(jī)體感染結(jié)核分枝桿菌時(shí),結(jié)核桿菌熱休克蛋白60顯示較強(qiáng)的疫原性,可誘導(dǎo)較強(qiáng)的B細(xì)胞和T細(xì)胞免疫應(yīng)答。因此我們克隆結(jié)核分枝桿菌熱休克蛋白60(Hsp60)編碼基因,并在大腸桿菌中表達(dá),為進(jìn)一步純化和研究該蛋白的免疫學(xué)特性奠定基礎(chǔ)。 二、方法 利用PCR技術(shù)從結(jié)核桿菌H37Rv株中擴(kuò)增hsp60基因,并將其與pZero-T載體連接,進(jìn)行DNA測(cè)序。用EcoRI+XbaI雙酶切,從TBhsp60-pZero-T質(zhì)粒上切下hsp60基因,然后將該片段插入同樣經(jīng)過雙酶切的E.coli表達(dá)載體pProEXHTb中。構(gòu)建重組原核表達(dá)質(zhì)粒pProEXHTb-TBhsp60。并將pProEXHTb-TBhsp60重組原核表達(dá)質(zhì)粒轉(zhuǎn)化入不同的E.coli菌株(BL21, DH5α, JM109),用不同摩爾濃度的IPTG誘導(dǎo)表達(dá)。 三、結(jié)果 成功地克隆了結(jié)核分枝桿菌hsp60基因。DNA測(cè)序證實(shí),與GenBank公布的序列一致。含pProEXHTb-TBhsp60基因表達(dá)質(zhì)粒的大腸桿菌經(jīng)IPTG誘導(dǎo)后,能夠高效表達(dá)相對(duì)分子質(zhì)量(KD)約為60KD的融合蛋白。 四、結(jié)論 獲得了結(jié)核分枝桿菌hsp60基因,成功地構(gòu)建了原核表達(dá)質(zhì)粒pProEXHTb-TBhsp60,并在大腸桿菌得到表達(dá),結(jié)果顯示重組質(zhì)粒在E.coliBL21中表達(dá)時(shí)表達(dá)量最大,并隨時(shí)間的增加表達(dá)量也增加,但蛋白的表達(dá)不受IPTG濃度的影響。為大量獲得HSP60蛋白和研究其免疫學(xué)特性奠定了基礎(chǔ)。
[Abstract]:Objective heat shock protein (heat shock protein,Hsp) belongs to a highly conserved protein family which widely exists in nature and is highly expressed under heat stress, so it is called heat shock protein. The increase of heat shock protein expression is related to many stress conditions, such as hypoxia, nutritional deficiency, virus infection, phagocytosis and transformation. Mycobacterium tuberculosis is the pathogen of tuberculosis. There are two groups of GroEL genes, GroEL1 and GroEL2., carried in the genome of Mycobacterium tuberculosis. They encode heat shock protein 60 (cpn60.1) and heat shock protein 65 (cpn60.2), respectively. When the body was infected with Mycobacterium tuberculosis, Mycobacterium tuberculosis heat shock protein 60 showed strong epidemic genicity and could induce strong B cell and T cell immune response. Therefore, we cloned the heat shock protein 60 (Hsp60) coding gene of Mycobacterium tuberculosis and expressed it in E. coli, which laid a foundation for further purification and study of the immunological characteristics of the protein. Methods hsp60 gene was amplified from Mycobacterium tuberculosis H37Rv strain by PCR and ligated with pZero-T vector for DNA sequencing. The hsp60 gene was cut off from TBhsp60-pZero-T plasmid by EcoRI XbaI double enzyme digestion, and then the fragment was inserted into the E.coli expression vector pProEXHTb, which was also digested by double enzyme digestion. Construction of Recombinant prokaryotic expression plasmid pProEXHTb-TBhsp60. The recombinant prokaryotic expression plasmid of pProEXHTb-TBhsp60 was transformed into different E.coli strains (BL21, DH5 偽, JM109) and induced by IPTG with different molar concentrations. Results the hsp60 gene of Mycobacterium tuberculosis was successfully cloned and confirmed by DNA sequencing, which was consistent with the sequence published by GenBank. E. coli containing pProEXHTb-TBhsp60 gene expression plasmid was induced by IPTG to express fusion protein whose relative molecular weight (KD) was about 60KD. Conclusion the hsp60 gene of Mycobacterium tuberculosis was obtained, and the prokaryotic expression plasmid pProEXHTb-TBhsp60, was successfully constructed and expressed in E. coli. The results showed that the expression of recombinant plasmid in E.coliBL21 was the highest, and also increased with the increase of time, but the expression of protein was not affected by the concentration of IPTG. It laid a foundation for obtaining a large number of HSP60 proteins and studying their immunological characteristics.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 范雄林,徐志凱,白光春,李元,李別虎,薛瑩;結(jié)核分枝桿菌Ag85B基因的克隆及真核表達(dá)載體的構(gòu)建[J];細(xì)胞與分子免疫學(xué)雜志;2000年04期

2 汪雁鶴,龔幼龍;我國(guó)結(jié)核病的疫情、原因及其影響因素[J];醫(yī)學(xué)與社會(huì);2003年03期

，

本文編號(hào)：2506064