本文選題：EV71 + VP1基因��； 參考：《第四軍醫(yī)大學》2010年碩士論文

【摘要】： 腸道病毒71型,與脊髓灰質炎病毒、卡薩奇病毒及埃克病毒同屬腸道病毒屬,因其首次從1969年加利福尼亞患有中樞神經系統(tǒng)疾病的患兒糞便標本中分離發(fā)現(xiàn)之時,按照腸道病毒先后排列順序,排名71位,故被命名為腸道病毒71型,簡稱EV71。EV71是一種單股正鏈RNA病毒,它同CoxA16和CoxA10一起被公認為是引起HFMD的主要病原。與柯薩奇病毒的致病性不同,EV71能引起包括中樞神經系統(tǒng)疾病在內的多種并發(fā)癥。目前眾所周知的,感染EV71會導致腦炎、無菌性腦膜炎和急性弛緩性麻痹等多種與神經系統(tǒng)相關的疾病。自上個世紀七十年代首次報道手足口病以來,至今已在世界范圍內引起多次爆發(fā)與流行;近十年來,我國大陸及臺灣等周邊地區(qū)EV71疫情日趨嚴重,兒童感染后易引起嚴重并發(fā)癥,病死率高,死亡速度快,造成了社會的不安定。 人是EV71唯一的宿主,EV71的流行一年四季均可出現(xiàn),常見于夏秋季,主要侵犯6歲以下嬰幼兒,幼兒園等人群密集處是EV71流行和暴發(fā)的主要場所,它的主要傳播方式是糞口傳播。但近年來亞太地區(qū)的流行情況顯示,EV71病毒也可通過呼吸道傳播;而目前呼吸道的飛沫傳播已被認為是引起疾病流行的重要傳播方式。自1969年在加利福利亞的腦炎嬰兒的糞便中首次發(fā)現(xiàn)EV71病毒后,隨后福利亞和紐約于1972年在EV71引起神經系統(tǒng)感染的病例中真正分離出該病毒。EV71感染在澳大利亞、日本、匈牙利、馬來西亞、新加坡、臺灣等地區(qū)也多次暴發(fā);1999年以來,我國大陸地區(qū)也相繼報告了EV71感染的局部流行,但與1998年臺灣地區(qū)有所不同,大陸所流行的由EV71引起的HFMD和神經系統(tǒng)癥狀都較輕。由于不同時期、不同地區(qū)發(fā)生的EV71型感染往往呈現(xiàn)不同的流行特點,臨床特征等都有著不同程度的差異,因此進一步的流行病學研究十分必要。 EV71結構蛋白主要是由VP1、VP2、VP3和VP4四種蛋白構成,其中,VPl基因最具研究意義。首先,VP1蛋白暴露于病毒表面,成為主要的病毒中和決定因子, EV71病毒的抗原性就是由VP1蛋白決定的;其次,VP1基因具有與病毒血清型完全對應遺傳多樣性。本實驗通過收集2009年西安市西京醫(yī)院兒科HFMD的患兒的皰液、咽拭子,進行病毒分離及RT-PCR技術,從而獲取EV71的VP1目的基因,將其與PQE30載體連接,構建出質粒,并對EV71西安地方株VP1基因進行遺傳學研究,為EV71西安地方株特性的研究以及EV71地區(qū)性的預防和控制工作的開展打下堅實的基礎。 研究目的 本實驗首先將收集到的EV71陽性標本進行病毒分離和RT-PCR檢測,通過RT-PCR擴增EV71外殼蛋白VP1基因,構建重組表達質粒PQE30/VP1 ,轉化大腸桿菌BL21中,進行測序,對VP1基因分析,并與標準株阜陽株(序列號為EU913471)比較,進行核苷酸同源性分析;其次,選用6周齡BALB/c小鼠,經鼻腔接種,對其用有明顯細胞病變的病毒液進行感染,通過分子生物學方法檢測小鼠的各組織的感染情況,了解腸道病毒71型的感染特點。通過2008年4月至2009年3月449例西京醫(yī)院門診及住院患兒的皰液及咽拭子標本行EV71初步篩查,對其結果從季節(jié)、年齡、性別進行統(tǒng)計學分析,并結合陽性患兒臨床特征,對西安地區(qū)EV71感染的流行病學特征進行初步研究。 實驗方法 (1)采集西安地區(qū)手足口病患兒的皰液、咽部分泌物進行病毒分離和逆轉錄聚合酶鏈式反應(RT-PCR)檢測;通過RT-PCR擴增腸道病毒71型( EV71)外殼蛋白VP1基因,構建重組表達質粒PQE30/ VP1 ,轉化大腸桿菌BL21中,對VP1基因進行初步分析。 (2)通過滴鼻EV71感染BALB/c小鼠,觀察經呼吸道感染后小鼠的感染狀況,并采集各器官組織進行病理切片光鏡檢測、病毒RNA檢測、病毒分離及鑒定,了解經呼吸道感染EV71的臨床特點和各器官病毒分布情況。 (3)根據西安地區(qū)EV71感染流行期間數(shù)據和實驗室檢測結果進行流行病學回顧性研究, 實驗結果 (1)對腸道病毒71型( EV71)西安地方株VP1基因測序,并將其與阜陽株(序列號為EU913471)相比較,表明西安地區(qū)EV71分離株其核苷酸有1處一個堿基的插入(10)和1處三個堿基的插入(170一172)。 (2)實驗組動物感染后5天,與空白對照組相比,出現(xiàn)了消瘦(每只小鼠體重減少約2-3g不等),精神萎靡,活動減少,但無嚴重癥狀。病毒RNA PCR檢測結果,肺、血液、腸均為陽性,而腦和心未見陽性條帶。肺、腸病理切片光鏡顯示有病變,腦、心臟顯示無病變;組織PCR陽性組織RT-PCR方法鑒定,結果均陽性。 (3)人群分布:在36例陽性病例中,①性別比:男性23例,女性13例,男女性別比1.77:1。②年齡分布:6歲以下陽性例數(shù)占總數(shù)的94.4%。時間分布西安地區(qū)2009年EV71感染最早發(fā)病時間為3月底4月初,其高峰期在春季4～5月,春季陽性率13.5%,明顯高于其余各季節(jié)(P0.05,有統(tǒng)計學意義)。 結論 (1)通過EV71的VP1基因測序結果表明,西安地方株VP1基因與阜陽株相比,其核苷酸存在一定差異。與EV71標準株BrCr、深圳株相比,在核苷酸同源性也都有不同程度的差異,通過使用原核表達系統(tǒng)初步構建西安地區(qū)EV71外殼蛋白VP1,為西安地區(qū)EV71抗體檢測以及EV71的病毒預防和控制等工作的開展,打下一定的基礎。 (2)在實驗中發(fā)現(xiàn),感染了EV71的小鼠,臨床表現(xiàn)輕微,并沒有出現(xiàn)典型的豎毛、弓背等臨床癥狀,更沒有出現(xiàn)四肢麻痹及死亡等嚴重并發(fā)癥;EV71直接感染了肺、小腸及血液,但并未到達中樞神經系統(tǒng)及心臟。這一實驗結果從另一個側面反映了西安地區(qū)EV71感染的基本特征,為西安地區(qū)EV71感染的研究奠定基礎。 (3) EV71感染不同地點和不同時間,其流行病學特征有著明顯的不同。西安地區(qū)EV71感染的臨床表現(xiàn)與1998年的臺灣、2004年的深圳、2007年的山東以及2008年的安徽阜陽的嚴重病例相比,主要引起上呼吸道感染,無嚴重并發(fā)癥及死亡病例。感染高峰期,以春季為主,這一結果與2008年阜陽EV71的流行極為相似。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R373;R725.1

【參考文獻】

相關期刊論文 前10條

1 王中林;腸道病毒71感染的研究進展[J];國外醫(yī)學(兒科學分冊);2001年06期

2 陸一涵;姜慶五;;人腸道病毒71型與手足口病[J];疾病控制雜志;2008年03期

3 李文先;葉冬青;;EV71病毒感染并發(fā)神經源性肺水腫研究進展[J];疾病控制雜志;2008年03期

4 周世力;李琳琳;何雅青;;深圳市腸道病毒71型血清流行病學初步調查[J];熱帶醫(yī)學雜志;2007年01期

5 常宏偉;王明麗;;人腸道病毒71型的研究進展[J];中國生物制品學雜志;2009年06期

6 劉素琴;;更昔洛韋治療嬰幼兒手足口病的療效[J];實用兒科臨床雜志;2006年24期

7 田波,段海生,榮一兵,周世力;腸道病毒71型分子流行病學研究進展[J];中國病毒學;2004年04期

8 楊智宏,朱啟昒,李秀珠,王曉紅,王建設,胡家瑜,唐偉,崔愛利;2002年上海兒童手足口病病例中腸道病毒71型和柯薩奇病毒A組16型的調查[J];中華兒科雜志;2005年09期

9 朱汝南;錢淵;鄧潔;邢江峰;趙林清;王芳;廖斌;任曉旭;李穎;張琪;李杰;;北京市兒童手足口病與腸道病毒71型和柯薩奇病毒A組16型感染有關[J];中華流行病學雜志;2007年10期

10 張敏濤;;清熱瀉脾散治療小兒手足口病50例[J];中醫(yī)兒科雜志;2007年05期

，

本文編號：1953827