【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家族,在熱應激下它們大量表達,因此稱為熱休克蛋白。熱休克蛋白表達量的提高與許多應激條件有關,如低氧、營養(yǎng)缺乏、病毒感染、吞噬和轉化作用。結核分枝桿菌是結核病病原菌。結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)。當機體感染結核分枝桿菌時,結核桿菌熱休克蛋白60顯示較強的疫原性,可誘導較強的B細胞和T細胞免疫應答。因此我們克隆結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,為進一步純化和研究該蛋白的免疫學特性奠定基礎。 二、方法 利用PCR技術從結核桿菌H37Rv株中擴增hsp60基因,并將其與pZero-T載體連接,進行DNA測序。用EcoRI+XbaI雙酶切,從TBhsp60-pZero-T質粒上切下hsp60基因,然后將該片段插入同樣經(jīng)過雙酶切的E.coli表達載體pProEXHTb中。構建重組原核表達質粒pProEXHTb-TBhsp60。并將pProEXHTb-TBhsp60重組原核表達質粒轉化入不同的E.coli菌株(BL21, DH5α, JM109),用不同摩爾濃度的IPTG誘導表達。 三、結果 成功地克隆了結核分枝桿菌hsp60基因。DNA測序證實,與GenBank公布的序列一致。含pProEXHTb-TBhsp60基因表達質粒的大腸桿菌經(jīng)IPTG誘導后,能夠高效表達相對分子質量(KD)約為60KD的融合蛋白。 四、結論 獲得了結核分枝桿菌hsp60基因,成功地構建了原核表達質粒pProEXHTb-TBhsp60,并在大腸桿菌得到表達,結果顯示重組質粒在E.coliBL21中表達時表達量最大,并隨時間的增加表達量也增加,但蛋白的表達不受IPTG濃度的影響。為大量獲得HSP60蛋白和研究其免疫學特性奠定了基礎。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R378

【參考文獻】

相關期刊論文 前2條

1 范雄林,徐志凱,白光春,李元,李別虎,薛瑩;結核分枝桿菌Ag85B基因的克隆及真核表達載體的構建[J];細胞與分子免疫學雜志;2000年04期

2 汪雁鶴,龔幼龍;我國結核病的疫情、原因及其影響因素[J];醫(yī)學與社會;2003年03期

，

本文編號：2506064