摘要
病毒性肝炎是由多种肝炎病毒引起的常见传染病,具有传染性强、传播途径复杂、流行面广泛,发病率较高等特点。临床上主要表现为乏力、食欲减退、恶心、呕吐、肝肿大及肝功能损害,部分病人可有黄疸和发热。有些患者出现荨麻疹、关节痛或上呼吸道症状。病毒性肝炎呈世界性分布,我国是病毒性肝炎的高发区域,其中全国各地人群中乙型肝炎的发病率最高约为50%—70%。乙型肝炎是由乙型肝炎病毒(hepatitis B virus,HBV)感染所至。HBV属嗜肝DNA病毒科,为环状,双股DNA病毒,外层是脂蛋白包膜,含乙肝表面抗原(HBsAg);内部是核心,含有核酸及乙肝核心抗原(HBcAg)。对人类血清中HBV的电镜检查,显示三种颗粒:即Dane颗粒、球型颗粒和丝状颗粒。HBV感染人体肝细胞后,对肝细胞并无直接致病作用,而是通过细胞免疫和体液免疫造成肝细胞的病毒性免疫损害,并从而引起各种临床表现。大部分患者呈急性发病过程,通常于6个月内痊愈;10%-20%患者可演变成慢性肝炎,继而发展成肝硬化,甚至肝细胞癌;90%以上新生儿感染者成为慢性携带者或慢性肝炎患者。但值得庆幸的是目前已有乙肝疫苗用于预防HBV感染,而且事实证明HB疫苗预防接种已降低了全球流行区人群中HBV的携带率及急性死亡率,且HB疫苗有极好的安全性。
然而随着乙肝疫苗普遍推行,对可逃逸HBV中和抗体的表面抗原基因(S基因)变异株感染逐步有所认识。有研究报道,目前在接受乙肝疫苗免疫和肝移植后接受HB免疫球蛋白治疗的患者中,不断发现HBsAg“α”决定簇突变的病毒,同时研究表明常规预防接种所产生的中和抗体对这些病毒无效。对该变异株进行进一步的测序发现,编码“α”决定簇的核苷酸有点突变(587位G→A),导致HBV S基因区145位氨基酸由甘氨酸(GLy)→精氨酸(Arg);突变发生的区域是能与疫苗诱导的抗体相结合的重要的抗原决定簇区,因此突变株不能被中和抗体识别,且五年后机体内依旧可以检测到此突变株,提示这种突变是有复制能力且遗传稳定的。HBsAg“α”决定簇145位变异株不仅流行广泛,而且同野生病毒株一样,具有致病性和传播能力,而且与肝炎的不良预后有关。研究表明,145位点的突变会导致HBsAg抗原性改变,致使临床上广泛使用的HBsAg诊断试剂对其无法检出,造成漏检。更为严重的是,用现行乙肝疫苗接种产生的抗体不能中和该变异株。在大范围人群接种乙肝疫苗后,由HBsAg“α”决定簇145位变异株引起的感染,很可能会成为新的公共卫生问题。因此,及时快速准确地检测出“α”决定簇突变变异株的感染很有意义。
目前,HBV的变异可通过PCR和DNA测序等技术来检测,但这些方法费用较高、技术难度大,尚不适于大量临床标本的分析。HBV S区145位点变异为HBsAg“α”决定基发生率较高的变异,因此研究该位点变异的快速检测方法具有非常重要的意义。在本文中,克隆145位点变异的HBV S基因,制备HBV变异株的诊断抗原,以此制备单克隆抗体,利用单克隆抗体及其多克隆抗体建立了145位突变HBsAg的快速检测方法,该方法具有较高的检测特异性及敏感性,有望解决隐性HBV持续性感染的漏检问题,从而使更多的人免受HBV的侵袭。建立乙肝变异株的快速诊断方法,为开展流行病学调查和相关的遗传进化关系分析奠定基础。
As a serious infection disease, viral hepatitis, is caused by various kinds of hepatitis virus and featured as dangerous infection, complicated route of transmission, broad epidemicity, and high morbidity. Acute infection with hepatitis B virus (HBV) is associated with acute viral hepatitis and begins with general ill-health, loss of appetite, nausea, vomiting, body aches, mild fever, dark urine, and then progresses to development of jaundice. Viral hepatitis is prevalent in the world, especially in China, where there are around 50-70% of the population exposed to the infection of HBV. Hepatitis B (HB) is caused by hepatitis B virus, a kind of hepadnavirus. The genome of Hepatitis B virion is a small circular, partially double stranded DNA, encoding an outer protein shell (envelop, contain HBsAg) and an inner body (core, contain HBcAg, HBeAg, HBV-DNA and DNAP). Viewed with electron microscope, three particles could be identified in human serum containing HBV. They are Dane, spherical and filiform particles. After invading in human liver cells, HBV not only has direct pathopoiesis effect on liver cells but also destroys the viral immune system in those cells through both cell mediated and humoral mediated immunity. Meanwhile, these processes lead to many clinical manifestations. Most patients with acute viral hepatitis could recovery in 6 months. About 10~20% patients are infected with chronic Hepatitis B virus, and eventually suffer hepatic cirrhosis and hepatocellular carcinoma. More than 90% of infected newborns are chronic HBsAg carriers or infected with acute Hepatitis B virus. Hepatitis B vaccines has been used to prevent HBV infection, and the incidence and mortality have been decreased efficiently after inoculation in the epidemic areas over the world.
Some HBV variants emerge together with the increasing inoculation of Hepatitis B vaccines and we should be noted to the S gene variants, which might escape from the HBV neutralization antibody. The variants with HBsAg mutation were reported in inoculated subjects and patients grafted with HBV liver from donor who had been received the HB immune globulin(IG) therapy. Some reports indicated that the neutralization antibody induced by common vaccine was not sufficient to recognize these variants. Sequence analysis revealed a single nucleotide mutation in the DNA region coding for the HBsAg“α”epitope, which results in the alteration of amino acid 145 GLy of HBV S gene. The HBsAg“α”epitope might be very important for binding the antibody, and the recognition might be defected due to the mutation in the S variants, so that the variants can still be detected in patients in 5 years. Furthermore, it has been shown that this mutation is replicable and could be stably inherited. Like the wild hepatitis virus, HBsAg“α”145 variants has the ability to infect and transmit, and is associated with the bad prognosis of viral hepatitis. It has been shown that the 145 mutation may attenuate the antigenicity of HBsAg, and subsequently leads to failure of detection by the HBsAg diagnostic reagent. The antibody induced by Hepatitis B vaccines couldn’t neutralize the variant. Although most people were inoculated by Hepatitis B vaccines, infection by HBsAg“α”145 variants may become a new public health problem. Therefore, it’s urgent to promptly identify whether a subject is infected by such variants with HBsAg“α”epitope mutation.
So far, HBV variant could be detected by polymerase chain reaction (PCR) and DNA sequencing technology. But these methods are expensive, time-consuming and difficult in technology when analyzing mass clinical specimens. The 145 nucleotide in HBV S gene mutated with high rate, so it is significant to establish a method to detect such point mutation promptly. In this study, we first cloned HBV S gene, and then prepared HBV variant antigen and its antibodies. Further, the monoclonal and polyclonal antibodies against HBV variant antigen were applied to establish the ELISA-based diagnosis method for HBV. This method was highly specific and sensitive, which was able to resolve the mentioned problems that recessive HBV of persistent infection could not be detected. This method could protect many people from the attack of HBV. The rapid diagnosis assay for HBV mutant strain would be helpful for epidemiological investigation and relationship analysis in term of its heredity and evolution.
引文
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