本文關(guān)鍵詞：解脲脲原體MB抗原保守區(qū)段的表達(dá)與抗體制備及初步應(yīng)用　出處：《南華大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 解脲脲原體 重組蛋白 表達(dá)與純化 MB抗原保守區(qū)段 多克隆抗體

【摘要】：目的：構(gòu)建含解脲脲原體(Ureaplsama urealyticum，Uu)膜蛋白MB抗原保守區(qū)段(20～130aa)基因的重組表達(dá)載體，在大腸桿菌中進(jìn)行誘導(dǎo)表達(dá)，純化產(chǎn)物免疫新西蘭兔獲得特異性的多克隆抗體(PcAb)，檢測(cè)Uu感染的臨床標(biāo)本，分析其敏感性與特異性，為制備Uu臨床檢測(cè)試劑盒奠定實(shí)驗(yàn)基礎(chǔ)。 方法：通過(guò)生物信息學(xué)分析，篩選MB抗原保守區(qū)基因片段優(yōu)勢(shì)抗原表位，以Uu1型血清型標(biāo)準(zhǔn)株基因組DNA為模板，通過(guò)聚合酶鏈反應(yīng)(PCR)擴(kuò)增目的片段，將其克隆至原核表達(dá)載體pQE30，構(gòu)建MB抗原基因保守區(qū)段重組表達(dá)質(zhì)粒pQE30／partMBA，然后轉(zhuǎn)化至表達(dá)宿主菌M15中進(jìn)行誘導(dǎo)表達(dá)，利用SDS-PAGE和Western-Blot對(duì)表達(dá)產(chǎn)物進(jìn)行分析和鑒定；鎳瓊脂糖凝膠F．F．親和層析柱純化重組蛋白，BCA法測(cè)定純化蛋白濃度。用純化的含MB抗原保守區(qū)段的重組蛋白免疫新西蘭兔，間接ELISA方法檢測(cè)免疫兔血清中抗MB抗原保守區(qū)段PcAb的效價(jià)，對(duì)表達(dá)的MB抗原保守區(qū)段重組蛋白的免疫原性進(jìn)行分析。然后用制備的特異性PcAb為一抗，將收集的臨床尿液標(biāo)本處理后包被微孔板，，建立間接ELISA方法檢測(cè)臨床標(biāo)本中Uu，同時(shí)與培養(yǎng)法進(jìn)行比較。根據(jù)PcAb檢測(cè)臨床樣本中Uu的敏感性與特異性，評(píng)價(jià)重組蛋白制備的特異PcAb在臨床標(biāo)本檢測(cè)中的應(yīng)用價(jià)值。 結(jié)果：軟件分析MB抗原的抗原性、疏水性和跨膜區(qū)，選取了MB抗原保守區(qū)段基因的第208～540bp位堿基序列為目的基因(片段長(zhǎng)度為333bp，編碼111
[Abstract]:Objective : To construct a recombinant expression vector containing a conserved segment ( 20 - 130aa ) of Ureaplaria ( Ureapl ) membrane protein MB antigen , induce expression in E . coli , purify the product immunized New Zealand rabbits to obtain the specific polyclonal antibody , and analyze its sensitivity and specificity , and lay a foundation for the preparation of the clinical detection kit . Methods : By bioinformatics analysis , the dominant epitope of MB antigen conserved region gene fragment was screened . The target fragment was amplified by polymerase chain reaction ( PCR ) . The recombinant expression plasmid pQE30 / partMBA was constructed by polymerase chain reaction ( PCR ) , then transformed into prokaryotic expression vector pQE30 . The expression product was analyzed and identified by SDS - PAGE and Western - Blot . F . The purified recombinant protein was purified by affinity chromatography column . The purified protein concentration was determined by BCA method . The titer of the conserved segment of anti - MB antigen in serum of immunized rabbits was analyzed by using purified recombinant protein containing MB antigen conserved segment , and the immunogenicity of the expressed MB antigen conserved segment recombinant protein was analyzed . Results : The antigenicity , hydrophobicity and transmembrane domains of MB antigen were analyzed by software . The nucleotide sequence of 208 锝

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