本文選題：副溶血性弧菌　切入點(diǎn)：flaE基因　出處：《暨南大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：副溶血性弧菌(Vibrio parahaemolyticus, VP)是存在于近岸海洋環(huán)境的嗜鹽細(xì)菌,它常常因?yàn)槲廴竞Ｎ妒称范鹑祟惣毙晕改c炎,也可引起反應(yīng)性關(guān)節(jié)炎,是一種重要的食源性致病菌。副溶血性弧菌具有極性鞭毛和周身鞭毛兩個(gè)鞭毛系統(tǒng)以適應(yīng)不同環(huán)境,其鞭毛蛋白有良好的免疫原性,可作為抗原制備抗體。本實(shí)驗(yàn)通過(guò)構(gòu)建原核表達(dá)極性鞭毛FlaE蛋白的重組大腸桿菌,經(jīng)IPTG誘導(dǎo)表達(dá)并通過(guò)親和層析純化獲得目的蛋白,以其作為抗原制備多克隆抗體,為制備免疫磁珠,建立VP的快速富集和檢測(cè)奠定基礎(chǔ)。本實(shí)驗(yàn)主要得研究?jī)?nèi)容和結(jié)果如下： 經(jīng)Genebank中查得Vibrio parahaemolyticus,BB22菌株極性鞭毛基因FlaE基因序列(登錄號(hào)為U12817.2),設(shè)計(jì)引物,利用PCR方法采用高保真酶從標(biāo)準(zhǔn)株ATCC17802基因組中擴(kuò)增出flaE基因片段,回收所得的擴(kuò)增產(chǎn)物與pMD 19-Tvector連接,轉(zhuǎn)化大腸桿菌DH5a,利用氨芐青霉素、藍(lán)白斑篩選和菌落PCR初步確認(rèn)陽(yáng)性重組子,隨后進(jìn)行測(cè)序鑒定。測(cè)序結(jié)果表明目的基因全長(zhǎng)1122bp,與Genebank上VP. BB22 flaE基因片段同源性達(dá)98%。雙酶切pMD 19-T重組子,再與經(jīng)相同雙酶切處理的表達(dá)載體pET22b(+)連接,得到重組表達(dá)載體pET22b-flaE,轉(zhuǎn)化大腸桿菌BL21(DE3)。0.8 mM IPTG,37℃,3h誘導(dǎo)重組蛋白表達(dá)。經(jīng)SDS-PAGE分析表明蛋白分子量約為40kDa(與DNASTAR計(jì)算結(jié)果一致),以包涵體形式表達(dá)。對(duì)目的蛋白的多聚組氨酸標(biāo)簽進(jìn)行親和層析純化目的蛋白,SDS-PAGE電泳分析純化結(jié)果,同時(shí)利用BCA法測(cè)定融合蛋白含量。純化蛋白經(jīng)佐劑乳化制備抗原,免疫新西蘭兔子,收獲抗血清,用ELISA方法測(cè)得抗體效價(jià)為1：204800.
[Abstract]:Vibrio parahaemolyticus (VPP) is a halophilic bacterium found in the coastal marine environment. It often causes acute gastroenteritis and reactive arthritis in humans by contaminating seafood. Vibrio parahaemolyticus has two flagellum systems, polar flagellum and peri-flagella to adapt to different environments, and its flagellin has good immunogenicity. The recombinant Escherichia coli expressing polar flagellum FlaE protein was constructed and expressed by IPTG. The target protein was purified by affinity chromatography and used as antigen to prepare polyclonal antibody. In order to prepare immunomagnetic beads and establish the rapid enrichment and detection of VP, the main contents and results of this experiment are as follows:. The FlaE gene sequence of polarity flagella gene of Vibrio parahaemolyticus bb22 strain (accession number U12817.2) was identified by Genebank. Primers were designed to amplify flaE gene fragment from the standard ATCC17802 genome by PCR method. The recovered amplified product was linked to pMD 19-T vector. E. coli DH 5a was transformed into E. coli, positive recombinant was identified by ampicillin, blue and white spot screening and colony PCR. The result of sequencing showed that the target gene had a total length of 1122 BP, and the homology with the VP. BB22 flaE gene fragment on Genebank was 98%. The recombinant pMD 19-T was digested by double enzyme, and then ligated with the expression vector pET22b (), which was treated with the same double enzyme digestion. The recombinant expression vector pET22b-flae was obtained and transformed into E. coli BL21(DE3).0.8 mM IPT GG for 3 h to induce the expression of recombinant protein. SDS-PAGE analysis showed that the molecular weight of the recombinant protein was about 40 kDa (consistent with the result of DNASTAR calculation and expressed as inclusion body). The polymorphic of the target protein was expressed in the form of inclusion body. The purification result of the target protein was analyzed by SDS-PAGE electrophoresis. At the same time, the fusion protein was determined by BCA method. The purified protein was emulsified with adjuvant to prepare antigens, immunize New Zealand rabbits and harvest antiserum. The titer of the purified protein was 1: 204800 by ELISA method.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R378

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