西安EV71分离株的VP1基因克隆测序及感染特性分析

发布时间：2018-05-30 04:04

本文选题：EV71 + VP1基因　；参考：《第四军医大学》2010年硕士论文

【摘要】： 肠道病毒71型,与脊髓灰质炎病毒、卡萨奇病毒及埃克病毒同属肠道病毒属,因其首次从1969年加利福尼亚患有中枢神经系统疾病的患儿粪便标本中分离发现之时,按照肠道病毒先后排列顺序,排名71位,故被命名为肠道病毒71型,简称EV71。EV71是一种单股正链RNA病毒,它同CoxA16和CoxA10一起被公认为是引起HFMD的主要病原。与柯萨奇病毒的致病性不同,EV71能引起包括中枢神经系统疾病在内的多种并发症。目前众所周知的,感染EV71会导致脑炎、无菌性脑膜炎和急性弛缓性麻痹等多种与神经系统相关的疾病。自上个世纪七十年代首次报道手足口病以来,至今已在世界范围内引起多次爆发与流行;近十年来,我国大陆及台湾等周边地区EV71疫情日趋严重,儿童感染后易引起严重并发症,病死率高,死亡速度快,造成了社会的不安定。人是EV71唯一的宿主,EV71的流行一年四季均可出现,常见于夏秋季,主要侵犯6岁以下婴幼儿,幼儿园等人群密集处是EV71流行和暴发的主要场所,它的主要传播方式是粪口传播。但近年来亚太地区的流行情况显示,EV71病毒也可通过呼吸道传播;而目前呼吸道的飞沫传播已被认为是引起疾病流行的重要传播方式。自1969年在加利福利亚的脑炎婴儿的粪便中首次发现EV71病毒后,随后福利亚和纽约于1972年在EV71引起神经系统感染的病例中真正分离出该病毒。EV71感染在澳大利亚、日本、匈牙利、马来西亚、新加坡、台湾等地区也多次暴发;1999年以来,我国大陆地区也相继报告了EV71感染的局部流行,但与1998年台湾地区有所不同,大陆所流行的由EV71引起的HFMD和神经系统症状都较轻。由于不同时期、不同地区发生的EV71型感染往往呈现不同的流行特点,临床特征等都有着不同程度的差异,因此进一步的流行病学研究十分必要。 EV71结构蛋白主要是由VP1、VP2、VP3和VP4四种蛋白构成,其中,VPl基因最具研究意义。首先,VP1蛋白暴露于病毒表面,成为主要的病毒中和决定因子, EV71病毒的抗原性就是由VP1蛋白决定的;其次,VP1基因具有与病毒血清型完全对应遗传多样性。本实验通过收集2009年西安市西京医院儿科HFMD的患儿的疱液、咽拭子,进行病毒分离及RT-PCR技术,从而获取EV71的VP1目的基因,将其与PQE30载体连接,构建出质粒,并对EV71西安地方株VP1基因进行遗传学研究,为EV71西安地方株特性的研究以及EV71地区性的预防和控制工作的开展打下坚实的基础。研究目的本实验首先将收集到的EV71阳性标本进行病毒分离和RT-PCR检测,通过RT-PCR扩增EV71外壳蛋白VP1基因,构建重组表达质粒PQE30/VP1 ,转化大肠杆菌BL21中,进行测序,对VP1基因分析,并与标准株阜阳株(序列号为EU913471)比较,进行核苷酸同源性分析;其次,选用6周龄BALB/c小鼠,经鼻腔接种,对其用有明显细胞病变的病毒液进行感染,通过分子生物学方法检测小鼠的各组织的感染情况,了解肠道病毒71型的感染特点。通过2008年4月至2009年3月449例西京医院门诊及住院患儿的疱液及咽拭子标本行EV71初步筛查,对其结果从季节、年龄、性别进行统计学分析,并结合阳性患儿临床特征,对西安地区EV71感染的流行病学特征进行初步研究。实验方法 (1)采集西安地区手足口病患儿的疱液、咽部分泌物进行病毒分离和逆转录聚合酶链式反应(RT-PCR)检测;通过RT-PCR扩增肠道病毒71型( EV71)外壳蛋白VP1基因,构建重组表达质粒PQE30/ VP1 ,转化大肠杆菌BL21中,对VP1基因进行初步分析。 (2)通过滴鼻EV71感染BALB/c小鼠,观察经呼吸道感染后小鼠的感染状况,并采集各器官组织进行病理切片光镜检测、病毒RNA检测、病毒分离及鉴定,了解经呼吸道感染EV71的临床特点和各器官病毒分布情况。 (3)根据西安地区EV71感染流行期间数据和实验室检测结果进行流行病学回顾性研究, 实验结果 (1)对肠道病毒71型( EV71)西安地方株VP1基因测序,并将其与阜阳株(序列号为EU913471)相比较,表明西安地区EV71分离株其核苷酸有1处一个碱基的插入(10)和1处三个碱基的插入(170一172)。 (2)实验组动物感染后5天,与空白对照组相比,出现了消瘦(每只小鼠体重减少约2-3g不等),精神萎靡,活动减少,但无严重症状。病毒RNA PCR检测结果,肺、血液、肠均为阳性,而脑和心未见阳性条带。肺、肠病理切片光镜显示有病变,脑、心脏显示无病变;组织PCR阳性组织RT-PCR方法鉴定,结果均阳性。 (3)人群分布:在36例阳性病例中,①性别比:男性23例,女性13例,男女性别比1.77:1。②年龄分布:6岁以下阳性例数占总数的94.4%。时间分布西安地区2009年EV71感染最早发病时间为3月底4月初,其高峰期在春季4～5月,春季阳性率13.5%,明显高于其余各季节(P0.05,有统计学意义)。结论 (1)通过EV71的VP1基因测序结果表明,西安地方株VP1基因与阜阳株相比,其核苷酸存在一定差异。与EV71标准株BrCr、深圳株相比,在核苷酸同源性也都有不同程度的差异,通过使用原核表达系统初步构建西安地区EV71外壳蛋白VP1,为西安地区EV71抗体检测以及EV71的病毒预防和控制等工作的开展,打下一定的基础。 (2)在实验中发现,感染了EV71的小鼠,临床表现轻微,并没有出现典型的竖毛、弓背等临床症状,更没有出现四肢麻痹及死亡等严重并发症;EV71直接感染了肺、小肠及血液,但并未到达中枢神经系统及心脏。这一实验结果从另一个侧面反映了西安地区EV71感染的基本特征,为西安地区EV71感染的研究奠定基础。 (3) EV71感染不同地点和不同时间,其流行病学特征有着明显的不同。西安地区EV71感染的临床表现与1998年的台湾、2004年的深圳、2007年的山东以及2008年的安徽阜阳的严重病例相比,主要引起上呼吸道感染,无严重并发症及死亡病例。感染高峰期,以春季为主,这一结果与2008年阜阳EV71的流行极为相似。
[Abstract]:Enterovirus 71, which is the same enterovirus with poliovirus, CAD and EK virus, is named as enterovirus 71 for the first time when it was isolated from the stool specimens of children with central nervous system disease in 1969 in California, and was named as enterovirus 71, called EV71.E. V71 is a single strand of positive chain RNA virus, which is recognized together with CoxA16 and CoxA10 as the main cause of HFMD. Unlike Coxsackie virus, EV71 can cause a variety of complications, including central nervous system disease. It is known that infection with EV71 can lead to encephalitis, aseptic meningitis, and acute flaccid paralysis. Many kinds of diseases related to the nervous system. Since the first report of hand foot and mouth disease in the 70s of last century, it has caused many outbreak and epidemic in the world. In the past ten years, the epidemic situation of EV71 in the surrounding areas of China and Taiwan is becoming more and more serious. It has become a social unrest.
People are the only host of EV71, and the epidemic of EV71 can occur throughout the year. It is common in summer and autumn, mainly invades infants under 6 years of age, and the concentrated place in the kindergarten is the main place for the epidemic and outbreak of EV71. The main mode of transmission is the spread of fecal mouth. However, the prevalence of EV71 in the Asia Pacific region shows that the EV71 virus can also pass through the respiratory tract in recent years. The current respiratory droplet transmission has been considered an important way of spreading the disease. EV71 virus was first discovered in the feces of infantile encephalitis in California in 1969, and the virus.EV71 infection in Australia was subsequently separated from Australia and New York in the cases of EV71 infection in 1972. Leah, Japan, Hungary, Malaysia, Singapore, Taiwan and other regions have also occurred many times. Since 1999, the local epidemic of EV71 infection has been reported in mainland China, but it is different from that in Taiwan in 1998. The symptoms of HFMD and the divine system, which are caused by EV71 in the mainland, are lighter. EV71 infection often presents different epidemic characteristics, and clinical characteristics have different degrees of difference. Therefore, further epidemiological research is very necessary.
The structure protein of EV71 is mainly composed of four proteins, VP1, VP2, VP3 and VP4. Among them, the VPl gene has the most significance. First, the VP1 protein is exposed to the surface of the virus and becomes the major virus neutralization factor. The antigenicity of EV71 virus is determined by the VP1 protein; secondly, the VP1 gene has a complete genetic diversity with the serotype of the virus. By collecting the blister fluid, pharynx swab, virus isolation and RT-PCR technology, the VP1 target gene of EV71 was obtained by collecting the blisters and swabs of the children HFMD of the pediatric HFMD of Xijing Hospital in 2009. The plasmids were connected with the PQE30 vector, and the VP1 gene of the local strain of EV71 in Xi'an was studied in order to study the local strain of EV71 in Xi'an and EV7. 1 the development of regional prevention and control has laid a solid foundation.
research objective
In this experiment, the EV71 positive specimens were isolated and detected by RT-PCR, and the EV71 coat protein VP1 gene was amplified by RT-PCR, and the recombinant expression plasmid PQE30/VP1 was constructed and transformed into the Escherichia coli BL21 to be sequenced. The VP1 gene was analyzed and the nucleotide homology was compared with the standard strain Fuyang strain (sequence number is EU913471). Secondly, 6 weeks old BALB/c mice were selected to infect the virus liquid with obvious cytopathic disease through the nasal cavity inoculation. The infection of all the tissues of the mice was detected by molecular biology methods and the infection characteristics of the enterovirus 71 were understood. From April 2008 to March 2009, 449 cases of the outpatient and inpatient blister fluid and pharynx in Xijing Hospital The swab specimens were screened by EV71, and the results were statistically analyzed from season, age and sex, and the epidemiological characteristics of EV71 infection in Xi'an were preliminarily studied in combination with the clinical characteristics of positive children.
Experimental method
(1) collect the blister fluid of children with hand foot and mouth disease in Xi'an area, detect the pharyngeal secretion by virus isolation and reverse transcriptase polymerase chain reaction (RT-PCR), and amplify the VP1 gene of the enterovirus 71 (EV71) shell protein (EV71) by RT-PCR, and construct the recombinant expression plasmid PQE30/ VP1 and transform the Escherichia coli BL21 to the preliminary analysis of the VP1 gene.
(2) to infect BALB/c mice with nasal drops EV71, the infection status of mice after respiratory tract infection was observed, and the pathological sections of various organs were collected for pathological examination, virus RNA detection, virus isolation and identification, to understand the clinical characteristics of respiratory tract infection EV71 and the distribution of various organs.
(3) epidemiological retrospective study was carried out based on the data of EV71 infection and laboratory test results in Xi'an area.
experimental result
(1) the VP1 gene of the enterovirus 71 (EV71) Xi'an local strain was sequenced and compared with the Fuyang strain (sequence number EU913471). It showed that the nucleotides of the EV71 isolates in Xi'an region had 1 bases insertion (10) and 1 insertion of three bases (170 172).
(2) on the 5 day after the infection in the experimental group, compared with the blank control group, there was a loss of weight (the weight loss of each mouse was about 2-3g), the mental malaise, the decrease of activity, but no serious symptoms. The results of RNA PCR detection of the virus, lung, blood, and intestines were positive, while the brain and heart did not have positive bands. Lung, and intestinal pathological sections showed lesions, brain, and heart display. No pathological changes. PCR positive tissues of RT-PCR were identified and the results were all positive.
(3) population distribution: among 36 positive cases, the sex ratio: male 23 cases, female 13 cases, sex ratio of male and female, 1.77:1. age distribution: the 94.4%. time distribution of positive cases under 6 years old, the earliest onset time of EV71 infection in Xi'an area in 2009 is the end of March at the end of April, the peak period is 4~5 months in spring, and the positive rate of spring is 13.5%, obviously higher than that of it The remaining seasons (P0.05, statistically significant).
conclusion
(1) the VP1 gene sequencing results of EV71 showed that the VP1 gene of Xi'an local strain was different from that of Fuyang strain. Compared with the EV71 standard strain BrCr and the Shenzhen strain, the nucleotide homology of the local strain also had different degrees of difference. The EV71 shell protein VP1 in Xi'an region was constructed by using the primary nucleoexpression system, and it was the EV7 of Xi'an region. 1 the development of antibody detection and EV71 virus prevention and control has laid a solid foundation.
(2) in the experiment, it was found that the mice infected with EV71 had mild clinical manifestations and no typical symptoms such as the vertical hair, the back of the bow, and other serious complications such as limbs paralysis and death; EV71 was directly infected with the lungs, small intestine and blood, but did not reach the central divine system and the heart. The experimental results were reflected from the other side. The basic characteristics of EV71 infection in Xi'an area lay the foundation for the study of EV71 infection in Xi'an area.
(3) the epidemiological characteristics of EV71 infection at different locations and different times are distinct. The clinical manifestations of EV71 infection in Xi'an region are mainly caused by the infection of the upper respiratory tract in Taiwan, Shenzhen in 2004, Shandong in 2007, and Fuyang in Anhui in 2008. There are no serious complications and death cases. The peak period is mainly spring, and this result is very similar to the popularity of EV71 in Fuyang in 2008.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R373;R725.1

【参考文献】