本文選題：結(jié)核分枝桿菌　切入點(diǎn)：ESAT-6　出處：《華中科技大學(xué)》2007年碩士論文

【摘要】： 目的構(gòu)建結(jié)核桿菌ESAT-6基因穿梭質(zhì)粒,在恥垢分枝桿菌中表達(dá)蛋白并對重組蛋白進(jìn)行鑒定,同時(shí)利用電轉(zhuǎn)化方法,將重組質(zhì)粒轉(zhuǎn)化到卡介苗中,為疫苗研究奠定實(shí)驗(yàn)基礎(chǔ);構(gòu)建能在畢赤酵母表達(dá)系統(tǒng)中表達(dá)結(jié)核桿菌ESAT-6蛋白的重組質(zhì)粒,并對其表達(dá)產(chǎn)物進(jìn)行基本生物學(xué)特性鑒定,為新型結(jié)核病疫苗的研究開發(fā)提供研究依據(jù)。 方法[1]從GenBank獲得結(jié)核分枝桿菌ESAT-6基因DNA序列,根據(jù)該序列及ps3000表達(dá)載體的要求,設(shè)計(jì)和合成一對引物,利用PCR技術(shù)擴(kuò)增ESAT-6基因,并插入pGEMT載體,進(jìn)行PCR,雙酶切及測序鑒定。[2]亞克隆法構(gòu)建ps3000—ESAT-6大腸桿菌--分枝桿菌穿梭質(zhì)粒,經(jīng)電轉(zhuǎn)化法導(dǎo)入到恥垢分枝桿菌和卡介苗中,Western blot鑒定其生物學(xué)活性。[3]設(shè)計(jì)和合成畢赤酵母表達(dá)引物并引入酶切位點(diǎn),擴(kuò)增ESAT-6基因,并插入pGEMT載體,進(jìn)行PCR,雙酶切及測序鑒定。[4]亞克隆法構(gòu)建pPICZaA-- ESAT-6穿梭質(zhì)粒,經(jīng)化學(xué)轉(zhuǎn)化法整合畢赤酵母GS115染色體中,PCR鑒定目的基因的整合,Zeocin抗性篩選多拷貝重組子并誘導(dǎo)表達(dá),表達(dá)產(chǎn)物用Western-blot鑒定其表達(dá)。 結(jié)果[1]成功擴(kuò)增了結(jié)核分枝桿菌ESAT-6基因;正確構(gòu)建穿梭質(zhì)粒ps3000-ESAT-6,用pET表達(dá)系統(tǒng)ESAT-6純化蛋白免疫小鼠的多抗血清通過Western blot證實(shí)了該重組恥垢桿菌中有表達(dá)并具有生物學(xué)活性。[2]從結(jié)核分枝桿菌H37Rv基因組中擴(kuò)增出ESAT-6基因片段,成功構(gòu)建重組表達(dá)質(zhì)粒pPICZaA-ESAT-6,SDS-PAGE顯示胞外并無表達(dá),酵母細(xì)胞經(jīng)裂解后ESAT-6基因翻譯的蛋白加上載體的信號肽序列和c-myc表位一共18kDa左右在蛋白膠上相應(yīng)位置有表達(dá),并且能被結(jié)核病人血清抗體所識別。 結(jié)論[1] ESAT-6基因重組恥垢分枝桿菌構(gòu)建成功,為下一步表達(dá)ESAT-6蛋白的重組BCG( Bacilli Calmette-Guérin)疫苗的研究奠定了基礎(chǔ)。[2]利用畢赤酵母表達(dá)系統(tǒng)獲得胞內(nèi)表達(dá)的結(jié)核桿菌ESAT-6重組蛋白,為結(jié)核病診斷抗原和疫苗研究奠定了基礎(chǔ)。
[Abstract]:Objective to construct the shuttle plasmid of ESAT-6 gene of Mycobacterium tuberculosis, express the protein in Mycobacterium smeareum and identify the recombinant protein. At the same time, the recombinant plasmid was transformed into BCG vaccine by the method of electrical transformation, which laid the experimental foundation for vaccine research. The recombinant plasmid which can express the ESAT-6 protein of Mycobacterium tuberculosis in Pichia pastoris expression system was constructed, and its expression product was identified by biological characteristics, which provides the basis for the research and development of new TB vaccine. Methods [1] the DNA sequence of ESAT-6 gene of Mycobacterium tuberculosis was obtained from GenBank. According to this sequence and the requirement of ps3000 expression vector, a pair of primers were designed and synthesized. ESAT-6 gene was amplified by PCR and inserted into pGEMT vector. Ps3000-ESAT-6 Escherichia coli-Mycobacterium shuttle plasmid was constructed by [2] subclone method. The biological activity of Pichia pastoris was identified by Western blot. [3] expression primers of Pichia pastoris were designed and synthesized, and restriction endonuclease sites were introduced to amplify the ESAT-6 gene and insert it into pGEMT vector. The pPICZaA- ESAT-6 shuttle plasmid was constructed by subcloning method. The recombinant plasmid was identified by chemical transformation method and identified by chemical transformation method. The integration of the target gene, Zeocin resistance, was screened for multiple copies of recombinant plasmids and induced expression, and the expression of pPICZaA- ESAT-6 shuttle plasmid was induced by chemical transformation method, which was used to identify the integration of the target gene in the GS115 chromosome of Pichia pastoris. The expressed product was identified by Western-blot. Results [1] Mycobacterium tuberculosis ESAT-6 gene was amplified successfully. The shuttle plasmid ps3000-ESAT-6 was constructed correctly. The polyclonal antibody of mouse immunized with ESAT-6 purified by pET expression system was confirmed to be expressed and bioactive by Western blot. [2] the ESAT-6 gene fragment was amplified from the H37Rv genome of Mycobacterium tuberculosis. The recombinant expression plasmid pPICZaA-ESAT-6 SDS-PAGE showed no extracellular expression. The protein translated by ESAT-6 gene and the signal peptide sequence and c-myc epitope of yeast cells were expressed in the corresponding position on the protein gel. And can be recognized by the tuberculosis patient serum antibody. Conclusion [1] the recombinant Mycobacterium ESAT-6 gene was successfully constructed, which laid a foundation for the further study of recombinant Bacilli Calmette-Gu 茅 rin vaccine expressing ESAT-6 protein. [2] Recombinant protein of Mycobacterium tuberculosis ESAT-6 was obtained by using Pichia pastoris expression system. It lays a foundation for the study of tuberculosis diagnosis antigen and vaccine.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346

【參考文獻(xiàn)】

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1 程繼忠,鄭波,肖紅梁,駒卿,皇甫永穆;結(jié)核桿菌HSP70在恥垢分枝桿菌中的表達(dá)及其免疫原性研究[J];中華微生物學(xué)和免疫學(xué)雜志;1998年05期

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本文編號：1697180