發(fā)布時(shí)間：2018-01-19 05:12

  本文關(guān)鍵詞： 肺炎衣原體 主要外膜蛋白 基因表達(dá) 蛋白純化 免疫活性　出處：《南華大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：構(gòu)建含肺炎衣原體(Chlamydia pneumoniae，Cpn)主要外膜蛋白(MOMP)優(yōu)勢(shì)表位(147～297aa)基因的重組表達(dá)體，在大腸桿菌BL21中進(jìn)行誘導(dǎo)表達(dá)，純化表達(dá)產(chǎn)物并進(jìn)行免疫原性和抗原性分析，為探索重組蛋白在肺炎衣原體血清學(xué)診斷中的應(yīng)用奠定前期實(shí)驗(yàn)基礎(chǔ)。 方法：通過(guò)生物信息學(xué)分析，篩選OmpA(MOMP的編碼基因)優(yōu)勢(shì)抗原表位的編碼基因，以Cpn AR-39標(biāo)準(zhǔn)株DNA基因組為模板，Taq聚合酶鏈反應(yīng)擴(kuò)增目的片段，將此片段克隆于pUCm-T載體，，經(jīng)酶切和PCR鑒定后，目的片段亞克隆于原核表達(dá)載體pET30a中，構(gòu)建重組體pET30a-MOMP_(VD2-VD3)，然后轉(zhuǎn)化至表達(dá)宿主菌E．coli BL21(DE3)，經(jīng)酶切、PCR和測(cè)序篩選陽(yáng)性克隆，挑取單個(gè)含重組質(zhì)粒pET30a-MOMP_(VD2-VD3)的工程菌陽(yáng)性克隆進(jìn)行培養(yǎng)和IPTG誘導(dǎo)表達(dá)，利用SDS-PAGE和Western-Blot進(jìn)行分析和鑒定表達(dá)產(chǎn)物；Ni-NTA親和層析柱純化重組蛋白，并進(jìn)行稀釋透析復(fù)性，BCA法測(cè)定純化蛋白濃度。用純化復(fù)性的MOMP_(VD2-VD3)重組蛋白免疫BALB／c小鼠，間接ELISA方法檢測(cè)免疫鼠血清中MOMP_(VD2-VD3)多克隆抗體的效價(jià)，對(duì)MOMP_(VD2-VD3)重組蛋白的免疫原性進(jìn)行分析；并以純化的MOMP_(VD2-VD3)重組蛋白包被微孔板，建立間接ELISA方法，檢測(cè)Cpn參比血清和臨床冠心病患者血清，同時(shí)與晶美公司Cpn IgG ELISA診斷試劑盒檢測(cè)結(jié)果進(jìn)行比較，根據(jù)間接ELISA檢測(cè)效果，評(píng)價(jià)重組抗原在Cpn血清學(xué)診斷中的應(yīng)用價(jià)值。 結(jié)果：軟件分析OmpA的抗原表位編碼基因并選擇了OmpA的442～873bp位堿基序列為目的基因(片段長(zhǎng)度為432bp，編碼144個(gè)氨基酸)，PCR擴(kuò)增
[Abstract]:Objective: to construct Chlamydia pneumoniae containing Chlamydia pneumoniae. The recombinant expression of the dominant epitope of CpN, the main outer membrane protein (MOMP), was induced and expressed in Escherichia coli (E. coli) BL21. The expression products were purified and immunogenicity and antigenicity analysis were carried out, which laid the experimental foundation for exploring the application of recombinant protein in the serological diagnosis of Chlamydia pneumoniae (Chlamydia pneumoniae). Methods: by bioinformatics analysis, the coding genes of dominant antigen epitopes of OmpA(MOMP were screened, and the DNA genome of Cpn AR-39 standard strain was used as template. The target fragment was amplified by Taq polymerase chain reaction and cloned into pUCm-T vector. After restriction endonuclease digestion and PCR identification, the target fragment was subcloned into prokaryotic expression vector pET30a. The recombinant pET30a-MOMPStace VD2-VD3 was constructed and then transformed into E. coli 21 (DE3), which was digested by enzyme. PCR and sequencing were used to screen the positive clones, and a single engineering strain containing recombinant plasmid pET30a-MOMPStace VD2-VD3 was selected for culture and IPTG induced expression. SDS-PAGE and Western-Blot were used to analyze and identify the expressed products. The recombinant protein was purified by Ni-NTA affinity chromatography and refolded by dilution and dialysis. BCA method was used to determine the concentration of purified protein. BALB/c mice were immunized with purified recombinant protein of MOMPS VD2-VD3. The titers of polyclonal antibodies against VD2-VD3 in the serum of immunized mice were detected by indirect ELISA method, and the immunogenicity of the recombinant protein was analyzed. The recombinant protein of MOMPD _ 2-VD3 was coated with micropore plate and indirect ELISA method was established to detect the reference serum of Cpn and the serum of clinical patients with coronary heart disease. At the same time, the results were compared with Cpn IgG ELISA diagnostic kit of Jingmei Company, according to indirect ELISA detection effect. To evaluate the value of recombinant antigen in the serological diagnosis of Cpn. Results: the antigenic epitope encoding gene of OmpA was analyzed by software and the target gene was selected as the target gene (the length of the fragment was 432bp). PCR Amplification of Encoding 144 Amino acids
【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】：2006
【分類號(hào)】：R374

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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2 劉鋼,胡翼云,趙德環(huán),李紅莉,王樹(shù)欣,江載芳,楊永弘;巢式聚合酶鏈反應(yīng)診斷肺炎衣原體感染的臨床應(yīng)用探討[J];中華微生物學(xué)和免疫學(xué)雜志;2001年04期

本文編號(hào)：1442772