發(fā)布時間：2018-08-06 21:44

【摘要】：隨著人民生活水平提高,甲型肝炎的發(fā)病率逐年下降,群體免疫力卻隨之減弱,極易造成大規(guī)模的爆發(fā)流行。國家因此通過人工主動免疫的方式提升甲型肝炎群體免疫力。但甲型肝炎疫苗的普種卻引發(fā)了一些負面問題�，F(xiàn)行的甲型肝炎診斷試劑,如被用于檢測甲型肝炎病毒感染的Abbott HAV Ab診斷試劑盒只能檢測病毒結(jié)構(gòu)蛋白誘發(fā)的抗體,自然感染和疫苗接種時都可以誘發(fā)機體產(chǎn)生此類抗體,因此試劑盒無法區(qū)分上述兩種情況。甲型肝炎病毒自然感染能誘發(fā)機體產(chǎn)生針對病毒結(jié)構(gòu)蛋白和非結(jié)構(gòu)蛋白的抗體,而滅活疫苗接種誘生的抗體只針對病毒結(jié)構(gòu)蛋白,減毒活疫苗接種誘發(fā)的抗非結(jié)構(gòu)蛋白的抗體水平低下。因此可以建立一種特異檢測甲肝病毒非結(jié)構(gòu)蛋白抗體的ELISA方法來表征病毒復(fù)制的發(fā)生。已有文獻證實,在實驗感染野生型病毒株的靈長類血清和自然感染的人血清中能夠檢測到anti-3Cpro抗體的存在(3Cpro為一種病毒非結(jié)構(gòu)蛋白),同時文獻還指出迄今為止,3Cpro是甲肝病毒中唯一的能夠通過原核表達系統(tǒng)產(chǎn)生功能蛋白的病毒非結(jié)構(gòu)蛋白。重組3Cpro蛋白通過原核表達系統(tǒng)以包涵體的形式大量表達,可以通過標準的生物化學(xué)方法進行純化,得到均質(zhì)的蛋白。 將3Cpro蛋白的基因插入到T7啟動子控制外源蛋白表達的、含有硫氧還蛋白編碼序列的表達質(zhì)粒M48中,構(gòu)建融合蛋白的基因工程表達質(zhì)粒。目的蛋白在Escherichia coli工程菌中進行表達,以包涵體的形式存在,可占菌體總蛋白量的40%以上。融合蛋白通過親和層析和陰離子交換后可達到高度均質(zhì)�？衫肧DS-PAGE來鑒定純化結(jié)果。 在這項研究中,我們檢測了建立的ELISA方法是否能夠鑒定出實驗室水平和臨床水平鑒定的急性期病人血清中的anti-3C IgM抗體,并與接種減毒活疫苗半年內(nèi)的兒童血清結(jié)果進行對比。結(jié)果顯示,利用HAV 3Cpro作為診斷抗原建立的ELISA方法能夠有效地檢測出針對非結(jié)構(gòu)蛋白的抗體,可以將自然感染和疫苗接種的情況區(qū)分開來。另外,這種ELISA的方法可以用來檢測體內(nèi)病毒沒有達到檢測水平、癥狀沒有出現(xiàn)時病毒的有限復(fù)制。這個檢測方法也利于我們更好地了解非結(jié)構(gòu)蛋白抗體在機體對HAV感染的免疫應(yīng)答中所起的作用。
[Abstract]:With the improvement of people's living standard, the incidence of hepatitis A is decreasing year by year, but the group immunity is weakened. Therefore, the country through artificial active immunization way to enhance hepatitis A group immunity. However, hepatitis A vaccine has caused some negative problems. The current diagnostic reagents for hepatitis A, such as the Abbott HAV Ab diagnostic kit used to detect hepatitis A virus infection, can only detect antibodies induced by structural proteins of the virus, and both natural infection and vaccination can induce the body to produce such antibodies. Therefore, the kit can not distinguish the above two cases. The natural infection of hepatitis A virus can induce the body to produce antibodies against virus structural proteins and non-structural proteins, while the antibodies induced by inactivated vaccine inoculation only target virus structural proteins. The level of antibody against non-structural proteins induced by live attenuated vaccine was low. Therefore, we can establish a specific ELISA method for the detection of hepatitis A virus nonstructural protein antibodies to characterize the occurrence of viral replication. It has been confirmed in the literature that The presence of anti-3Cpro antibodies (3Cpro is a viral nonstructural protein) can be detected in primate sera infected with wild-type virus strains and in human sera infected naturally. Virus non-structural proteins that produce functional proteins through prokaryotic expression systems. The recombinant 3Cpro protein was expressed in the form of inclusion body by prokaryotic expression system, and the homogenized protein could be purified by standard biochemical method. The gene of 3Cpro protein was inserted into the expression plasmid M48 containing thioredoxin coding sequence controlled by T7 promoter to construct the gene engineering expression plasmid of fusion protein. Objective protein was expressed in Escherichia coli engineering bacteria and existed as inclusion body, which accounted for more than 40% of the total bacterial protein. The fusion protein was highly homogenized by affinity chromatography and anion exchange. SDS-PAGE can be used to identify the purification results. In this study, we examined whether the established ELISA assay could identify anti-3C IgM antibodies in the serum of acute stage patients with laboratory and clinical levels, and compare the results with those of children who were vaccinated with live attenuated vaccine within half a year. The results showed that the ELISA method using HAV 3Cpro as diagnostic antigen could effectively detect antibodies against non-structural proteins and distinguish natural infection from vaccination. In addition, the ELISA method can be used to detect the limited replication of the virus when the virus does not reach the detection level and symptoms do not occur. This method also helps us to better understand the role of non-structural protein antibodies in the immune response to HAV infection.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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