甲型肝炎病毒3C蛋白的原核表达及其抗原性研究

发布时间：2018-08-06 21:44

【摘要】：随着人民生活水平提高,甲型肝炎的发病率逐年下降,群体免疫力却随之减弱,极易造成大规模的爆发流行。国家因此通过人工主动免疫的方式提升甲型肝炎群体免疫力。但甲型肝炎疫苗的普种却引发了一些负面问题。现行的甲型肝炎诊断试剂,如被用于检测甲型肝炎病毒感染的Abbott HAV Ab诊断试剂盒只能检测病毒结构蛋白诱发的抗体,自然感染和疫苗接种时都可以诱发机体产生此类抗体,因此试剂盒无法区分上述两种情况。甲型肝炎病毒自然感染能诱发机体产生针对病毒结构蛋白和非结构蛋白的抗体,而灭活疫苗接种诱生的抗体只针对病毒结构蛋白,减毒活疫苗接种诱发的抗非结构蛋白的抗体水平低下。因此可以建立一种特异检测甲肝病毒非结构蛋白抗体的ELISA方法来表征病毒复制的发生。已有文献证实,在实验感染野生型病毒株的灵长类血清和自然感染的人血清中能够检测到anti-3Cpro抗体的存在(3Cpro为一种病毒非结构蛋白),同时文献还指出迄今为止,3Cpro是甲肝病毒中唯一的能够通过原核表达系统产生功能蛋白的病毒非结构蛋白。重组3Cpro蛋白通过原核表达系统以包涵体的形式大量表达,可以通过标准的生物化学方法进行纯化,得到均质的蛋白。将3Cpro蛋白的基因插入到T7启动子控制外源蛋白表达的、含有硫氧还蛋白编码序列的表达质粒M48中,构建融合蛋白的基因工程表达质粒。目的蛋白在Escherichia coli工程菌中进行表达,以包涵体的形式存在,可占菌体总蛋白量的40%以上。融合蛋白通过亲和层析和阴离子交换后可达到高度均质。可利用SDS-PAGE来鉴定纯化结果。在这项研究中,我们检测了建立的ELISA方法是否能够鉴定出实验室水平和临床水平鉴定的急性期病人血清中的anti-3C IgM抗体,并与接种减毒活疫苗半年内的儿童血清结果进行对比。结果显示,利用HAV 3Cpro作为诊断抗原建立的ELISA方法能够有效地检测出针对非结构蛋白的抗体,可以将自然感染和疫苗接种的情况区分开来。另外,这种ELISA的方法可以用来检测体内病毒没有达到检测水平、症状没有出现时病毒的有限复制。这个检测方法也利于我们更好地了解非结构蛋白抗体在机体对HAV感染的免疫应答中所起的作用。
[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.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【相似文献】