【摘要】：目的建立土撥鼠肝炎病毒(Woodchuck hepatitis virus, WHV)核酸的熒光定量PCR (Real-time PCR)檢測(cè)方法及土撥鼠肝炎病毒表面抗原(WHsAg)的改良雙抗加心ELISA檢測(cè)方法,應(yīng)用于土撥鼠肝炎病毒模型的創(chuàng)制研究。 方法核酸檢測(cè)：分別根據(jù)土撥鼠肝炎病毒核心抗原(WHcAg)和表面抗原(WHsAg)的DNA序列設(shè)計(jì)13對(duì)擴(kuò)增引物,分別以混合土撥鼠肝臟DNA的WHV核酸DNA為模板進(jìn)行PCR,從中篩選無(wú)非特異性擴(kuò)增及引物二聚體,且擴(kuò)增靈敏度高的引物,用于建立土撥鼠血清中WHV DNA的Real-time PCR檢測(cè)方法,并利用該方法對(duì)土撥鼠血清進(jìn)行了篩查。表面抗原檢測(cè)：原核表達(dá)WHsAg的N端區(qū)域,His·Tag鑷柱親和層析純化目的蛋白；用該蛋白分別免疫兔和蛋雞制備多克隆抗體并純化。建立用于檢測(cè)感染土撥鼠肝炎病毒的土撥鼠血清中WHsAg的雙抗夾心ELISA方法。 結(jié)果核酸檢測(cè)：根據(jù)WHsAg基因的5’端設(shè)計(jì)的一對(duì)引物WHVSF1與WHVSR1,檢測(cè)靈敏度可達(dá)1×10’拷貝／u L,病毒拷貝數(shù)與Real-time PCR Ct值的標(biāo)準(zhǔn)曲線的R2值為0.997,且電泳未見(jiàn)明顯非特異性條帶及引物二聚體。表面抗原檢測(cè)：純化后的WHsAg蛋白純度達(dá)到95%,免疫兔和蛋雞后分別制備相應(yīng)的抗WHsAg血清,純化得到相應(yīng)抗體,ELISA結(jié)果顯示兔抗WHsAg抗體的效價(jià)達(dá)1：1,024,000,雞抗WHsAg抗體的效價(jià)為1：64,000。本實(shí)驗(yàn)建立的雙抗加心ELISA法檢測(cè)WHsAg的檢測(cè)限為10ng/mL。 結(jié)論初步建立了土撥鼠血清中WHV DNA的Real-time PCR檢測(cè)方法及感染土撥鼠肝炎病毒的土撥鼠血清中WHsAg的改良雙抗加心ELISA檢測(cè)方法,上述方法為進(jìn)一步研究土撥鼠肝炎病毒模型奠定了基礎(chǔ)。
[Abstract]:Objective to establish a fluorescence quantitative PCR (Real-time PCR) method for the detection of groundhog hepatitis virus (Woodchuck hepatitis virus, WHV) nucleic acid and an improved double-antibody plus heart ELISA method for detection of groundhog hepatitis virus surface antigen (WHsAg). It was applied to the creation of groundhog hepatitis virus model. Methods nucleic acid detection: according to the DNA sequence of groundhog hepatitis virus core antigen (WHcAg) and surface antigen (WHsAg) (WHsAg), 13 pairs of amplified primers were designed. The WHV nucleic acid DNA of mixed groundhog liver DNA was used as template for PCR,. The nonspecific amplification and primer dimer were screened from the samples and the primers with high sensitivity were used to establish a method for the detection of WHV DNA in groundhog serum. The method was used to screen the groundhog serum. Surface antigen detection: the target protein was purified by affinity chromatography of N-terminal, His Tag tweezers which expressed WHsAg in prokaryotic cells, and the polyclonal antibody was prepared and purified by immunizing rabbits and laying hens respectively. A double antibody sandwich ELISA method was developed for the detection of WHsAg in the serum of groundhog infected with groundhog hepatitis virus. Results nucleic acid detection: the sensitivity of a pair of primers WHVSF1 and WHVSR1, designed according to the 5 'end of WHsAg gene could reach 1 脳 10' copy / u L, and the R2 value of the standard curve of virus copy number and Real-time PCR Ct value was 0.997. Moreover, there were no obvious nonspecific bands and primer dimer on electrophoresis. Surface antigen detection: the purity of purified WHsAg protein reached 95%. After immunizing rabbits and laying hens, the corresponding anti-WHsAg serum was prepared, and the corresponding antibody was purified. The ELISA results showed that the titer of rabbit anti-WHsAg antibody was 1: 1 024000. The titer of chicken anti WHsAg antibody was 1: 64000. The detection limit of double antibody plus center ELISA method for detection of WHsAg is 10 ng / mL. Conclusion the Real-time PCR detection method for WHV DNA in groundhog serum and the modified double antibody plus heart ELISA method for detection of WHsAg in groundhog serum infected with groundhog hepatitis virus were established. These methods lay a foundation for further study of groundhog hepatitis virus model.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332;R373

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