發(fā)布時間：2018-05-17 16:31

  本文選題：卡介苗 + ERP基因　； 參考：《石河子大學》2006年碩士論文

【摘要】： 結(jié)核病是人類主要的傳染性疾病之一,全球每年新發(fā)結(jié)核病人數(shù)超過800萬,死亡人數(shù)約300萬,全世界結(jié)核桿菌感染人數(shù)超過1/3。近年來HIV的廣泛流行及多重耐藥結(jié)核菌株的出現(xiàn)使結(jié)核病的發(fā)病率和死亡率呈上升趨勢。世界衛(wèi)生組織于1993年宣布“全球結(jié)核病緊急狀態(tài)”,并指出應優(yōu)先發(fā)展有效的結(jié)核病疫苗。 卡介苗是目前全球唯一一種用于預防結(jié)核病的疫苗,它是牛分支桿菌的減毒活菌苗株。卡介苗能夠有效預防嚴重的兒童結(jié)核病,但對成人結(jié)核病的保護效力在不同的地區(qū)人群中有顯著差異(0-80%),因而亟需一種更為有效的結(jié)核病疫苗。目前正在研究中的新型疫苗包括亞單位疫苗、DNA疫苗、減毒活疫苗、重組卡介苗等。 由于卡介苗具有廣泛使用的安全性(全球已有超過30億人接種了卡介苗),對卡介苗進行改造是開發(fā)新疫苗的一種策略。ERP基因是結(jié)核桿菌中的一個重要的毒力基因,研究表明,它不僅存在于有毒力的結(jié)核桿菌中,也存在于卡介苗中,免疫功能缺陷者(如HIV患者)接種卡介苗可能引起嚴重的播散性結(jié)核病。 目的本研究利用基因敲除技術(shù)和電穿孔技術(shù)構(gòu)建卡介苗ERP基因缺失突變株,為初步探討卡介苗突變株的功能奠定了基礎(chǔ),并為研制新型結(jié)核病疫苗進行了有益的探索。 方法對卡介苗菌進行體外培養(yǎng),用改進的方法手抽卡介苗菌基因組DNA,PCR擴增ERP基因兩側(cè)的兩個800bp左右的片段,經(jīng)純化后連接T載體,通過藍白斑篩選及菌落PCR鑒定后挑選陽性克隆送測序,測序正確的兩個片段分別連接到PKO載體預定位點,構(gòu)建卡介苗ERP基因置換型打靶載體,經(jīng)抗生素,菌落PCR、質(zhì)粒PCR及酶切鑒定后挑選陽性克隆再次測序,驗證載體構(gòu)建成功與否。將構(gòu)建好的打靶載體電穿孔卡介苗,通過抗生素及PCR鑒定出卡介苗ERP基因缺失突變株。 結(jié)果1.提取卡介苗菌基因組DNA。2.擴增出兩個目的片段。3.將兩個目的片段插入到PKO載體的預定位點,構(gòu)建卡介苗ERP基因置換型打靶載體。4.將打靶載體電穿孔入卡介苗,篩選并鑒定卡介苗ERP基因缺失突變株。 結(jié)論構(gòu)建了卡介苗ERP基因置換型打靶載體,并構(gòu)建篩選卡介苗ERP基因缺失突變株。
[Abstract]:Tuberculosis is one of the major infectious diseases in human beings. Worldwide, the number of new TB cases is more than 8 million every year, the death toll is about 3 million, and the number of tuberculosis bacilli infection in the world is more than one third. In recent years, the prevalence of HIV and the emergence of multidrug resistant tuberculous bacilli have increased the incidence and mortality of tuberculosis. The World Health Organization (WHO) declared a global TB emergency in 1993 and stated that priority should be given to the development of effective TB vaccines. BCG is the only vaccine used to prevent tuberculosis in the world. It is a live attenuated strain of Mycobacterium bovis. BCG vaccine can effectively prevent severe tuberculosis in children, but the protective effect of BCG vaccine on adult tuberculosis is significantly different among different populations in different regions. Therefore, a more effective TB vaccine is urgently needed. New vaccines are currently under study, including subunit DNA vaccine, live attenuated vaccine, recombinant BCG vaccine and so on. Because the BCG vaccine has been widely used safely (more than 3 billion people have been vaccinated with BCG vaccine in the world, the modification of BCG vaccine is a strategy for developing new vaccine. ERP gene is an important virulence gene in Mycobacterium tuberculosis. It exists not only in noxious Mycobacterium tuberculosis, but also in BCG vaccine. Vaccination of BCG in patients with immune deficiency (such as HIV patients) may cause serious disseminated tuberculosis. Objective to construct BCG ERP mutant by gene knockout technique and electroporation technique, and to establish a foundation for the function of BCG mutant strain, and to explore the development of new TB vaccine. Methods BCG was cultured in vitro. Two 800bp fragments were amplified from the genomic DNA of BCG by hand extraction, and then ligated into T vector after purification. The positive clones were selected and sequenced by blue-white spot screening and colony PCR identification. The two correct fragments were sequenced and linked to the predetermined site of the PKO vector respectively. The BCG ERP gene replacement targeting vector was constructed, and antibiotics were used to construct the BCG ERP gene replacement target vector. The positive clones were selected and sequenced after colony PCR, plasmid PCR and restriction endonuclease digestion to verify the success of the construction of the vector. The target vector electroporation BCG was constructed and the ERP gene deletion mutant of BCG vaccine was identified by antibiotics and PCR. Result 1. The genomic DNA of Bacillus Calmette-Guerin (BCG) was extracted. Two target fragments. Two target fragments were inserted into the predetermined site of PKO vector to construct BCG ERP gene replacement targeting vector. 4. The target vector was electroporated into the BCG vaccine to screen and identify the BCG ERP gene deletion mutant. Conclusion BCG ERP gene replacement targeting vector was constructed and BCG ERP gene deletion mutant was constructed.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

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本文編號：1902064