本文選題：EB病毒 + 病毒衣殼抗原　； 參考：《青島大學(xué)》2007年碩士論文

【摘要】： 目的原核表達(dá)EB病毒(Epstein-Barr virus,EBV)衣殼蛋白p23-p18融合蛋白,研制EBV特異性衣殼抗原(viralcapsid antigen,VCA)抗體ELISA檢測試劑。 方法①選擇p23編碼基因BLRF2(氨基酸1～162)和p18編碼基因BFRF3的C端(氨基酸105～176)基因序列,設(shè)計特異性引物,以EBV標(biāo)準(zhǔn)株B95-8為模板,采用重疊延伸PCR技術(shù),將兩段基因通過多肽接頭(Gly_4Ser)_3 DNA序列連接以獲得融合基因p23-p18。②融合基因經(jīng)測序證實(shí)序列準(zhǔn)確無誤后,導(dǎo)入表達(dá)載體pThioHis A構(gòu)建原核表達(dá)克隆,轉(zhuǎn)染E.coli BL21后IPTG誘導(dǎo)表達(dá),重組蛋白經(jīng)鎳-敖合物瓊脂糖樹脂柱親和層析純化獲得融合抗原,Western blot鑒定其特異性。③用標(biāo)準(zhǔn)陽性血清滴定抗原效價,選擇最適濃度包被ELISA反應(yīng)板,配套商品化的辣根過氧化酶標(biāo)羊抗人IgM、IgG及其它一系列試劑,反復(fù)優(yōu)化反應(yīng)條件,組裝成EBV特異性VCA IgM和IgG間接ELISA診斷試劑。④選擇血清標(biāo)本進(jìn)行檢測,并與“金”標(biāo)準(zhǔn)即間接免疫熒光(IFA)和NOVATEC公司生產(chǎn)的VCAIgM和IgG ELISA檢測試劑盒進(jìn)行比較,計算該試劑的敏感性、特異性、約登指數(shù)、符合率、陽性預(yù)測值和陰性預(yù)測值等指標(biāo),同時檢測診斷試劑的重復(fù)性和穩(wěn)定性。 結(jié)果p23和p18融合基因成功構(gòu)建并有效表達(dá),Western blot顯示融合抗原具有較高的特異性和敏感性。與IFA比較,采用融合衣殼抗原制備的EBVVCAIgG診斷試劑盒的敏感性、特異性、約登指數(shù)、符合率、陽性預(yù)測值和陰性預(yù)測值分別為97.3%、97.4%、0.947、97.3%、99.8%和77.6%;IgM ELISA診斷試劑盒的上述各項指標(biāo)分別為94.8%、99.5%、0.943、98.7%、97.3%和98.9%。融合抗原ELISA與NOVATEC公司同類產(chǎn)品有較高的符合率(IgG為97.7%,IgM為96.6%),且具有良好的重復(fù)性和穩(wěn)定性。 結(jié)論采用p23-p18融合蛋白制備的EBV VCA IgG和IgM ELISA檢測試劑敏感特異,簡便快速,重復(fù)性和穩(wěn)定性好,進(jìn)一步完善后可用于臨床EBV感染的診斷及流行病學(xué)調(diào)查。
[Abstract]:Objective to express the p23-p18 fusion protein of Epstein-Barr virus (EBV) capsid protein in prokaryotic expression and to prepare a ELISA assay for EBV specific capsid antigens. Methods (1) the sequence of p23 gene BLRF2 (amino acid 1: 162) and p18 gene BFRF3 (amino acid 105h176) were selected. Specific primers were designed. The standard EBV strain B95-8 was used as template, and the overlapping extension PCR technique was used. The fusion gene p23-p18.2 fusion gene was cloned into the expression vector pThioHis A to construct the prokaryotic expression clone after the sequence of the fusion gene p23-p18.2 fusion gene was confirmed by sequencing. The expression of IPTG was induced by transfection of E.coli BL21. The recombinant protein was purified by agarose resin affinity chromatography. The fusion antigen was identified by Western blot. The titrated antigen titrated with standard positive serum was used to determine the specificity of the recombinant protein. The optimal concentration was chosen to cover the ELISA reaction plate. The commercial horseradish peroxidase labeled goat anti-human IgMN IgG and a series of other reagents were repeatedly optimized and assembled into EBV specific VCA IgM and IgG indirect ELISA diagnostic reagent .4 to select serum samples for detection. The sensitivity, specificity, Yorden index, coincidence rate, positive predictive value and negative predictive value of the reagent were calculated by comparing with the "gold" standard (IFA) and the VCAIgM and IgG ELISA test kits produced by NOVATEC Company, and the sensitivity, specificity, Yorden index, coincidence rate, positive predictive value and negative predictive value of the reagent were calculated. The reproducibility and stability of diagnostic reagents were also detected. Results the fusion gene p23 and p18 were successfully constructed and expressed effectively. Western blot showed that the fusion antigen had high specificity and sensitivity. Compared with IFA, the sensitivity, specificity, Yorden index, coincidence rate, positive predictive value and negative predictive value of EBVVCAIgG diagnostic kit prepared by fusion capsid antigen were 99.8% and 99.8%, respectively, and 998.98%, respectively. The fusion antigen ELISA has a high coincidence rate with that of the similar products of NOVATEC company. It has a good reproducibility and stability. Conclusion EBV VCA IgG and IgM ELISA prepared by p23-p18 fusion protein are sensitive and specific, simple, rapid, reproducible and stable, and can be used in the diagnosis and epidemiological investigation of clinical EBV infection.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

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本文編號：1907967