發(fā)布時(shí)間：2019-06-28 12:01

【摘要】：目的:用構(gòu)建含沙眼衣原體(Chlamydia trachomatis, Ct)血清型D型外膜蛋白2(outer membrane protein 2, Omp2)167～434位氨基酸編碼基因(omp2’)的重組表達(dá)載體,在大腸桿菌E. coli BL21(DE3)中表達(dá)Omp2重組蛋白;免疫BALB/c雌鼠,研制并鑒定其單克隆抗體,為Ct感染診斷試劑盒研制與其致病機(jī)制的研究提供實(shí)驗(yàn)依據(jù)。 方法:將含重組質(zhì)粒pET28b(+)/omp2’在原核細(xì)胞中用IPTG誘導(dǎo)表達(dá),SDS-PAGE、Western blot分析和鑒定表達(dá)的蛋白;采用強(qiáng)變性尿素溶解包涵體,經(jīng)過(guò)親和層析法對(duì)重組蛋白進(jìn)行純化;Bradford法測(cè)定純化的重組蛋白的濃度。采用分步稀釋,逐漸降低變性劑的濃度,并在復(fù)性緩沖溶液中加入一定量氧化、還原型谷胱苷肽和甘氨酸等,進(jìn)行復(fù)性;用純化的Omp2重組蛋白(rOmp2’)皮下多點(diǎn)免疫BALB/c雌鼠;無(wú)菌操作取其脾細(xì)胞,以聚乙二醇作為融合劑,與骨髓瘤細(xì)胞融合;有限稀釋法進(jìn)行單克隆化;間接ELISE法篩選陽(yáng)性克隆;以瓊脂糖免疫雙擴(kuò)散法鑒定單抗亞類;間接ELISE法測(cè)定腹水效價(jià);細(xì)胞涂片染色鏡檢雜交瘤細(xì)胞株染色體數(shù)目;流式細(xì)胞儀分析雜交瘤細(xì)胞DNA含量;Western blot分析其特異性。 結(jié)果:SDS-PAGE分析顯示,在IPTG誘導(dǎo)下,基因工程菌表達(dá)一相對(duì)分子質(zhì)量(Mr)約為35KDa的目的蛋白,且主要以包涵體形式存在;經(jīng)親和層析后可獲得純度在96%左右的重組蛋白。純化的rOmp2’免疫BALB/c小鼠,經(jīng)細(xì)胞融
[Abstract]:Aim: to construct a recombinant expression vector containing Chlamydia trachomatis (Chlamydia trachomatis, Ct) serotype D outer membrane protein 2 (outer membrane protein 2, Omp2) 167 / 434 amino acid coding gene (omp2') and express Omp2 recombinant protein in E. coli BL21 (DE3), and to develop and identify its monoclonal antibody against BALB/c female mice, so as to provide experimental basis for the development of Ct infection diagnostic kit and the study of its pathogenic mechanism. Methods: the recombinant plasmid pET28b () / omp2' was induced and expressed in prokaryotic cells by IPTG, the expressed protein was analyzed and identified by SDS-PAGE,Western blot, the inclusion body was dissolved by strong denatured urea, and the recombinant protein was purified by affinity chromatography, and the concentration of the purified recombinant protein was determined by Bradford method. The concentration of denaturant was gradually decreased by step dilution, and a certain amount of oxidation, reduced glutathione and glycine were added to the renaturation buffer solution for renaturation. BALB/c female mice were immunized with purified Omp2 recombinant protein (rOmp2') subcutaneally. the spleen cells were obtained by aseptic operation and fused with myeloma cells with polyethylene glycol as fusion agent. The positive clones were screened by indirect ELISE. The monoclonal antibody subclasses were identified by agarose immunodouble diffusion method, the ascitic titer was determined by indirect ELISE method, the chromosome number of hybridoma cell line was detected by cell smear staining microscope, and the specificity of hybridoma cell DNA content; Western blot was analyzed by flow cytometry. Results: SDS-PAGE analysis showed that under the induction of IPTG, the relative molecular weight (Mr) was about the target protein of 35KDa, and mainly in the form of inclusion body, and the recombinant protein with purity of about 96% could be obtained by affinity chromatography. BALB/c mice were immunized with purified rOmp2' and melted by cells.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R374

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