肠道病毒71型的检测与分型及人类巨细胞病毒包膜蛋白UL115原核表达与分离纯化

发布时间：2018-04-28 00:02

本文选题：手足口病 + 沙窝镇　；参考：《暨南大学》2017年硕士论文

【摘要】：1.2012年沙窝镇手足口病疫情中肠道病毒71型的检测与分型肠道病毒71型(EV71)感染人后通常会伴随手足口病的发生。在中国,每年有成千上万的儿童感染肠道病毒71型,该病毒严重影响公众的健康。研究肠道病毒71型的病毒学特性对预防与控制手足口病的爆发是十分关键的。本实验研究报道了2012年9月至10月期间湖北沙窝镇感染手足口病的105个患儿的情况。实验通过反转录PCR技术,荧光定量PCR技术,分离及培养病毒技术来检测病毒。对肠道病毒71型的VP1基因进行测序分析,同时构建系统进化树对肠道病毒71型进行基因分型。病例表明,多于90%的患儿小于9周岁,3-6周岁大的患儿比例占50%左右。患儿普遍感染肠道病毒71型,表明在沙窝镇爆发的手中口病大多数是肠道病毒71型的感染并引起相关的并发症。这些手足口病的病例中,有66位确诊是肠道病毒感染引起的,48位患儿被肠道病毒71型感染,另外,柯萨奇病毒A16型感染患儿数目达到16位。测序与进化树分析结果表明,沙窝镇出现的肠道病毒71型病毒株属于C4基因亚型。值得注意的是,沙窝与南昌的地理距离较远,而与武汉的地理距离较近,但是,系统进化分析显示沙窝出现的肠道病毒71型病毒株,与曾经爆发过手足口病的南昌的肠道病毒71型病毒株的遗传距离较为接近,但与同样曾经爆发过手足口病的武汉的肠道病毒71型病毒株的遗传距离较远。沙窝镇感染手足口病的患儿中,女患儿感染肠道病毒71型的比例较高。然而,曾经出现肠道病毒感染的武汉和南昌,男患儿感染肠道病毒71型的比例较高。通过该实验研究,加深了对肠道病毒的认识,特别是在中国引起流行性手足口病的肠道病毒。本实验对预防与控制肠道病毒的的爆发有很大的研究意义。2.人类巨细胞病毒包膜蛋白UL115原核表达与分离纯化人巨细胞病毒(HCMV),是感染人类的一种疱疹病毒。对于免疫系统发育不全和免疫抑制患者可引起相关病症,甚至致死。HCMV拥有20个以上的包膜蛋白,它们位于病毒的最外层,与病毒趋向性,吸附,融合,子代病毒包装,成熟,传播过程密切相关。了解清楚HCMV包膜蛋白的结构,生物学功能,可为病毒的生物学机制提供新的认识,加深病毒与宿主间关系的理解。同时提供新的思路去设计,研发抗人巨细胞病毒的相关药物,并且对病毒疫苗的研制,具有重要的研究基础和临床意义。UL115(g L)蛋白是包膜蛋白的其中一员,UL115包膜蛋白作为外来抗原,除了具有良好的免疫反应性外,还有免疫原性,可为制备相应的抗原诊断单克隆抗体和开发HCMV快速诊断试剂盒提供可选原料。因此,研究HCMV-UL115生物学功能对阐明HCMV致病机制及疫苗的研制是有重要价值的。本实验首先通过常规PCR技术克隆UL115基因,接着构建UL115克隆重组菌株以及重组表达菌株,成功表达带有组氨酸标签的UL115重组蛋白。经实验鉴定,该重组蛋白在原核细胞内,以包涵体形式表达。在此基础上,浅层摸索优化了表达菌株的表达条件。本实验的摸索的较好诱导表达重组蛋白的条件为:31℃,0.03 m M/L IPTG,诱导时间9 h。经过镍柱分离纯化,获得该重组蛋白。使用BCA法对初步纯化的UL115重组蛋白进行定量,该重组蛋白浓度可达到0.105μg/μl。本实验成功构建UL115重组蛋白原核表达体系,并且探索了表达体系表达条件,分离纯化后获得该重组蛋白。这为研究UL115蛋白单克隆抗体相关生物学功能打下基础,对诊断检测试剂盒和疫苗的研制有重要意义,同时也为其他包膜蛋白的原核表达提供了相关实验经验。
[Abstract]:In 1.2012 years, the detection of enterovirus 71 in the epidemic situation of hand foot and mouth disease in Sha Wo town and the type of enterovirus 71 (EV71) infected people usually accompanied by hand foot and mouth disease. In China, thousands of children infected with enterovirus 71 each year. The virus seriously affects the public health. The virological characteristics of enterovirus 71 are studied to prevent the disease. The outbreak of hand foot and mouth disease (HFMD) is critical. This study reported 105 children infected with hand foot and mouth disease in Sha Wo Town, Hubei, from September 2012 to October. The experiment was carried out by reverse transcriptional PCR, fluorescence quantitative PCR technology, isolation and culture of virus technology to detect the virus. The VP1 gene of enterovirus 71 was sequenced. At the same time, the phylogenetic tree was constructed to genotyping of enterovirus 71. Cases showed that more than 90% of the children were less than 9 years old, and the proportion of children aged 3-6 years old accounted for about 50%. The children were generally infected with enterovirus 71, which showed that most of the hand mouth diseases in Sha Wo town were infected by enterovirus 71 and caused related complications. Of the cases of hand foot and foot disease, 66 were diagnosed with enterovirus infection, 48 were infected with enterovirus 71, and the number of coxsackievirus type A16 infected children reached 16. The results of sequencing and phylogenetic tree analysis showed that the enterovirus 71 disease of Sha Wo town belonged to the C4 gene subtype. The geographical distance of Nanchang is far away, and the geographical distance from Wuhan is close. However, the phylogenetic analysis shows that the enterovirus 71 virus strain of the sand nest is close to the genetic distance of the enterovirus 71 virus strain of Nanchang, which had once had hand foot and foot disease, but it was similar to the enterovirus 71 of Wuhan, which had also had hand foot and mouth disease. The genetic distance of the virus is far away. Among the children infected with hand foot and mouth disease in Shawo Town, the proportion of the female children infected with enterovirus 71 is higher. However, in Wuhan and Nanchang, which had been infected by enterovirus, the proportion of the male children infected with enterovirus 71 was higher. Enteroviruses that cause the epidemic of hand foot and mouth disease. This experiment has great significance for the prevention and control of the outbreak of enterovirus,.2. human cytomegalovirus envelope protein UL115 prokaryotic expression and isolation and purification of human cytomegalovirus (HCMV), is a human herpes virus. For the immune system development and immunosuppressive patients It can cause related diseases and even death.HCMV has more than 20 enveloped proteins. They are located in the outermost of the virus. They are closely related to viral tendencies, adsorption, fusion, packaging, maturation and transmission of the progeny virus. Understanding the structure of HCMV envelope protein and biological power can provide new understanding for the biological mechanism of the virus and deepen the disease. The understanding of the relationship between poison and host. Meanwhile, it provides new ideas to design and develop anti human cytomegalovirus related drugs, and the development of the virus vaccine has important research basis and clinical significance.UL115 (g L) protein is one of the envelope proteins, UL115 coated egg white as an external antigen, in addition to a good immune response There are also immunogenicity, which can provide alternative raw materials for the preparation of the corresponding antigen diagnostic monoclonal antibody and the development of HCMV rapid diagnostic kit. Therefore, it is of great value to study the biological function of HCMV-UL115 to elucidate the pathogenesis of HCMV and the development of the vaccine. This experiment first cloned the UL115 gene by conventional PCR technology and then constructed the UL1. 15 the recombinant strain was cloned and the recombinant expression strain was successfully expressed. The recombinant protein with histidine label was expressed successfully. The recombinant protein was expressed in the prokaryotic cell and expressed in the form of inclusion body. On this basis, the expression condition of the expression strain was optimized in the shallow layer. The condition of the test was better to induce the expression of the recombinant protein. The recombinant protein was obtained by separation and purification by nickel column at 31 C, 0.03 m M/L IPTG and 9 h.. The recombinant protein was quantified by BCA method. The concentration of the recombinant protein could reach 0.105 mu g/ Mu L., and the recombinant protein prokaryotic expression system was successfully constructed, and the expression condition of the expression system was explored, and the purification of the recombinant protein was isolated and purified. The recombinant protein is obtained, which lays the foundation for the study of the biological functions of the monoclonal antibody of UL115 protein, and is of great significance for the development of diagnostic kit and vaccine. It also provides relevant experimental experience for the prokaryotic expression of other enveloped proteins.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.1

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