發(fā)布時間：2019-03-19 14:09

【摘要】：目的:構(gòu)建結(jié)核分枝桿菌重組卡介苗,研究其免疫原性及抗結(jié)核作用,以期獲得結(jié)核病預(yù)防性和治療性疫苗。 方法:通過基因工程重組技術(shù)將結(jié)核分枝桿菌保護性抗原ESAT6、MPT64、Ag85A、Ag85B的編碼基因分別與穿梭質(zhì)粒載體PMD31、pYUB295重組,通過電穿孔技術(shù)導(dǎo)入卡介苗中,應(yīng)用PCR擴增、PAGE電泳鑒定重組卡介苗。將重組卡介苗免疫小鼠后,通過ELISA法檢測其血清中特異性抗體,用MTS法分析其脾淋巴細胞增殖指數(shù);結(jié)核分枝桿菌攻擊后,通過小鼠半數(shù)死亡時間、一定時間內(nèi)的死亡率、肺、肝、脾細菌生長最低稀釋度等指標評價各重組卡介苗對結(jié)核分枝桿菌感染的預(yù)防作用和治療效果。 結(jié)果:通過PCR擴增獲得esat6、mpt64、ag85a、ag85b基因,與穿梭表達載體PMD31、pYUB295重組后,通過PCR擴增、限制性內(nèi)切酶酶切、DNA測序鑒定表明成功地構(gòu)建了esat6、mpt64、ag85a、ag85b基因PMD31、pYUB295重組質(zhì)粒。 將各重組質(zhì)粒通過電穿孔導(dǎo)入卡介苗。質(zhì)粒PMD31重組卡介苗在抗性培養(yǎng)上不能很好生長,質(zhì)粒pYUB295重組卡介苗在抗性培養(yǎng)基上生長良好。pYUB295-目的基因重組卡介苗基因組DNA的PCR擴增以
[Abstract]:Aim: to construct recombinant BCG vaccine of Mycobacterium tuberculosis and study its immunogenicity and anti-tuberculosis effect in order to obtain preventive and therapeutic vaccine against tuberculosis. Methods: the gene encoding the protective antigen ESAT6,MPT64,Ag85A,Ag85B of Mycobacterium tuberculosis was recombined with shuttle plasmid vector PMD31,pYUB295 by gene engineering technique. The gene was introduced into BCG by electroporation technique and amplified by PCR. Recombinant BCG was identified by PAGE electrophoresis. After the mice were immunized with recombinant BCG vaccine, the specific antibodies in serum were detected by ELISA method, and the proliferation index of spleen lymphocytes was analyzed by MTT method. After Mycobacterium tuberculosis infection, the preventive and therapeutic effects of recombinant BCG vaccine on Mycobacterium tuberculosis infection were evaluated by the indexes of half death time, mortality within a certain time, minimum dilution of lung, liver and spleen bacteria growth and so on. Results: esat6,mpt64,ag85a,ag85b gene was amplified by PCR. After recombination with shuttle expression vector PMD31,pYUB295, esat6,mpt64,ag85a,ag85b gene PMD31, was successfully constructed by PCR amplification, restriction endonuclease digestion and DNA sequencing. PYUB295 recombinant plasmid. The recombinant plasmids were introduced into BCG vaccine by electroporation. Plasmid PMD31 recombinant BCG did not grow well in resistant culture and plasmid pYUB295 recombinant BCG grew well on the resistant medium. PCR amplification of genomic DNA of pYUB295-target recombinant BCG
【學位授予單位】：北京市結(jié)核病胸部腫瘤研究所
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R392

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