本文選題：馬爾尼菲青霉 + 基因克隆�。� 參考：《廣西醫(yī)科大學(xué)》2005年碩士論文

【摘要】：背景和目的:馬爾尼菲青霉(Penicillium marneffei, P.m)是一種雙相致病真菌,由其所致的馬爾尼菲青霉病(Penicilliosis marneffei)主要流行于東南亞和我國的南方地區(qū),主要見于免疫缺陷患者,現(xiàn)已成為該地區(qū)人類免疫缺陷病毒(human immunodeficiency virus,HIV)感染者的一種重要的繼發(fā)性感染。P.m 侵犯人體的單核巨噬系統(tǒng),沒有特異的臨床表現(xiàn),過去的確診常依賴于真菌培養(yǎng),但常使時(shí)間延誤。病理診斷有時(shí)易和其它巨噬細(xì)胞內(nèi)病原體混淆。香港大學(xué)發(fā)現(xiàn)了編碼P.m 細(xì)胞壁甘露糖蛋白的MP1 基因,并開發(fā)出的免疫學(xué)診斷試劑,取得較好的使用效果,但有時(shí)仍和曲霉(Aspergillus)出現(xiàn)交叉反應(yīng)造成誤診。因此,通過構(gòu)建P.m 的cDNA 文庫,尋找新的編碼P.m特異性抗原蛋白的基因,制備特異性抗體用于馬爾尼菲青霉病的診斷以提高診斷效率是很有必要的。 方法:(1)構(gòu)建P.m 28℃和37℃雙相的cDNA 文庫并對(duì)文庫進(jìn)行基因測序,通過生物信息學(xué)方法對(duì)基因序列進(jìn)行分析,以發(fā)現(xiàn)有價(jià)值的基因。(2)用生物信息學(xué)方法對(duì)新發(fā)現(xiàn)的P.m 的PYA_024_60(簡稱60)和PYA_012_92(簡稱92)兩條全長基因進(jìn)行功能和定位的預(yù)測。(3)用原核表達(dá)的方法,將這兩條基因的部分序列插入表達(dá)質(zhì)粒pGEX-5X-1,用大腸桿菌表達(dá)成融合谷胱甘肽-硫-轉(zhuǎn)移酶(glutathione S-transferase,GST)標(biāo)簽的蛋白質(zhì)(GST-60p, GST-92p),經(jīng)Glutathione Sepharose 4B 親合層析提純后以此蛋白質(zhì)為抗原對(duì)新西蘭兔進(jìn)行皮下免疫注射制備兔抗血清,再用免疫親合層析法用這兩種蛋白質(zhì)從血清中提純特異性的多克隆抗體。(4)用純化的抗體做Western 免疫印跡,驗(yàn)證60p 和92p 在P.m 細(xì)胞壁上的特異性。(5)用伴刀豆蛋白A(Concanavalin A,ConA)初步驗(yàn)證60p 和92p 是否為糖蛋白。(6)將這兩個(gè)抗體用于P.m 及各種真菌病的病理組織的免疫組化診斷,初步驗(yàn)證其特異性和靈敏性。
[Abstract]:Background & objective: Penicillium marneffeii (P.mm) is a biphasic pathogenic fungus. Penicilliosis marneffeieii, which is caused by Penicillium marneffeii, is mainly prevalent in Southeast Asia and southern China. It has become an important secondary infection. P. m invades the human mononuclear macrophage system in this area. The diagnosis in the past often depends on fungal culture, but it often delays the time. Pathological diagnosis is sometimes easily confused with other pathogens in macrophages. The MP1 gene encoding P.m cell wall mannose glycoprotein was found in the University of Hong Kong, and the immunological diagnostic reagent was developed. The results show that the MP1 gene can be used well, but sometimes it is misdiagnosed with Aspergillus aspergillus. Therefore, by constructing the cDNA library of P.m, a new gene encoding P.m specific antigen protein was found. It is necessary to prepare specific antibodies for the diagnosis of penicilliosis marneffei. Methods: a biphasic cDNA library of P.m28 鈩，

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