本文選題：肺炎支原體 + 呼吸道合胞病毒　； 參考：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：【目的】深入了解廣州地區(qū)呼吸道合胞病毒(RSV)及肺炎支原體(MP),四種不同亞型人冠狀病毒的流行病學情況及臨床特征,提高臨床診斷的確診率,并嘗試對鼠源單克隆雜交瘤細胞株3D7的抗HAdv-3中和抗體基因進行人源化改造,為治療性抗體的研發(fā)奠定基礎�！痉椒ā恳弧⒉杉�2012年1月~2014年1月期間在廣州地區(qū)急性呼吸道疾病患兒(≤14歲)咽拭子標本,提取標本核酸,利用熒光定量PCR進行RSV與MP檢測;并對其流行特征進行分析。二、采集2009年7月1日~2016年6月30日廣州醫(yī)科大學附屬第一醫(yī)院及中山大學孫逸仙紀念醫(yī)院患有急性呼吸道感染的兒科住院患兒的咽拭子標本,共11399例。采用了熒光定量PCR方法對上述標本進行4種人類冠狀病毒(Coronavirus,HCoV)檢測,包括HCoV-229E,HCoV-NL63,HCoV-OC43和HCoV-HKU1,隨后取出人冠狀病毒陽性標本進行11種常見呼吸道病毒檢測。并對其流行特征進行分析。三、利用TRIzoL裂解分泌抗HAdv-3鼠源性單克隆抗體(mAb)的雜交瘤細胞株3D7提取全RNA,并通過逆轉錄為cDNA。利用多條特異性簡并引物通過PCR擴增鼠源性mAb輕鏈和重鏈可變區(qū)基因。把鼠源性mAb輕鏈可變區(qū)基因及重鏈可變區(qū)基因分別克隆至Pa-L5(含人輕鏈信號肽及人輕鏈恒定區(qū)基因)構建L鏈,克隆至Pa-H2(含有人重鏈信號肽及人重鏈恒定區(qū)基因)構建H鏈,然后把L鏈通過基因擴增酶切克隆至Pc-DNA3.1(+)中,構建pcDNA3.1-3D7L載體,把H鏈通過基因擴增酶切克隆至Pc-DNA3.1(+)中,構建pcDNA3.1-3D7H載體。把pcDNA3.1-3D7L與pcDNA3.1-3D7H質(zhì)粒以1:1比例共同轉染CHO-S細胞進行表達及構建穩(wěn)定細胞系�！窘Y果】一、在3760例患兒中,檢出RSV 392例,陽性率為10.4%;檢出MP 339例,陽性率9.0%。在RSV陽性病例中,年齡在0~9個月患兒最高,達19.6%(181/923例)。RSV流行高峰發(fā)生于2012年2月~4月和2013年1月~4月,陽性率分別為26.8%(136/507例)和18.0%(151/839例)。在MP陽性病例中,年齡在6~10歲陽性率最高,達20.1%(97/484例),MP流行高峰發(fā)生在2012年7月~10月和2013年6月~10月,陽性率分別為16.3%(92/564例)和13.4%(124/922例)。在上呼吸道癥狀中,RSV感染患兒鼻塞[28.6%(112/392例)]、流涕[33.2%(130/392例)]發(fā)生率顯著高于MP陽性患兒[15.0%(51/339例)、20.6%(70/339例)],2組比較差異均有統(tǒng)計學意義(P均0.001);RSV感染患兒咳嗽[78.1%(306/392例)]、咳痰[20.4%(80/392例)]發(fā)生率顯著低于MP陽性患兒[90.0%(305/339例)、27.1%(92/339例)],2組比較差異均有統(tǒng)計學意義(P0.001,P=0.032)。在下呼吸道癥狀中,RSV陽性患兒氣喘[33.2%(130/392例)]、氣促[13.5%(53/392例)]、喘鳴音[29.3%(115/392例)]、痰鳴音[18.9%(74/392例)]發(fā)生率顯著高于MP陽性患兒[11.5%(39/339例)、6.5%(22/339例)、11.5%(39/339例)、10.6%(36/339例)],2組比較差異均有統(tǒng)計學意義(P0.001,P=0.002,P0.001,P=0.002)。MP陽性患兒雙肺呼吸音粗[59.2%(201/339例)]較RSV陽性患兒[40.1%(157/392例)]更為多見,2組比較差異有統(tǒng)計學意義(P0.001)。二、總HCo V陽性率為4.3%(489/11399),OC43陽性率為3.0%(346/11399),229E陽性率為0.6%(65/11399),NL63陽性率為0.5%(60/11399),HKU1陽性率為0.3%(38/11399),其中一些患兒存在共感染多型HCoV的情況�？侶CoV陽性率在所有年齡組中在3.3%~5.5%之間。總HCoV和OC43在7~12個月的年齡段的患兒比例顯著最高(p0.001)�？侶CoV、OC43、229E、NL63和HKU1陽性率最高的月份分別出現(xiàn)在2012年四月(15.3%,25/163)、2012年四月(14.1%,23/163)、2011年二月(10.4%,8/77)、2009年九月(7.7%,3/39)和2010年六月(6.2%,5/81)。而在臨床癥狀上分析,只感染一種HCoV的患兒癥狀與共感染其他HCoV分型及其他常見的呼吸道病毒患兒癥狀作對比,只感染一種HCoV的患兒高熱(≥38°C)(p=0.014)和不正常肺呼吸(p=0.043)癥狀明顯比共感染其他HCoV分型及其他常見的呼吸道病毒的患兒多。另外,在HCoV 4種分型的患兒癥狀對比,咳嗽(p=0.032),肺炎(p=0.026),不正常肺呼吸(p=0.002)癥狀呈顯著差異。在HCoV陽性患兒共感染病例中,出現(xiàn)最普遍的呼吸道病毒是流感病毒A型(21.6%,50/231)和呼吸道合胞病毒(21.6%,50/231)。三、經(jīng)過3輪單克隆篩選,CLEIA結果顯示抗體表達水平達到20mg/L,通過western blotting檢測到有完整的抗體分子表達并且抗體結構正確,經(jīng)體外微量中和實驗檢測該抗體有中和活性�！窘Y論】一、廣州地區(qū)RSV的主要感染人群是0~9個月的兒童,重點在每年1月~4月;MP的主要感染人群是6~10歲的兒童,重點在每年6月~10月。預防工作要全年進行,預防因RSV和MP感染導致的住院率增高。二、廣州地區(qū)總HCo V主要感染人群是7~12個月的年齡段的兒童,重點防控時間在春季及秋季。其中,OC43主要感染人群是7~12個月的年齡段的兒童,重點防控時間在春季及秋季,229E主要感染人群是4~6個月的年齡段的兒童,重點防控時間在春季,NL63主要感染人群是7~12個月的年齡段的兒童,重點防控時間在秋季,而HKU1重點防控時間在夏季。三、本研究成功構建了抗HAdv-3鼠/人嵌合中和抗體真核表達質(zhì)粒,并在CHO-S細胞中穩(wěn)定表達,表達的抗體也被證實有中和活性。
[Abstract]:[Objective] to understand the epidemiological and clinical characteristics of four different subtypes of human coronavirus (RSV) and Mycoplasma pneumoniae (MP) in Guangzhou, and to improve the diagnostic rate of clinical diagnosis, and try to transform the anti HAdv-3 neutralizing antibody gene of 3D7 of murine monoclonal hybridoma cell line to the therapeutic effect. The research of antibody development laid the foundation. [Methods] to collect the specimen of pharynx swab in children of acute respiratory disease (less than 14 years old) in Guangzhou area during January ~2014 years, extract the specimen nucleic acid, detect RSV and MP by fluorescence quantitative PCR, and analyze its epidemic characteristics. Two, collect the Medical University in June 30th July 1, 2009, ~2016, and June 30th. The pharynx swab specimens of children with acute respiratory infection in the First Affiliated Hospital and the Sun Yixian Memorial Hospital of Zhongshan University were studied in 11399 cases. 4 kinds of human coronavirus (Coronavirus, HCoV) were detected by fluorescein quantitative PCR, including HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, and then taken out. The positive specimens of the coronavirus were tested for 11 common respiratory viruses. And their epidemic characteristics were analyzed. Three, the hybridoma cell line, 3D7, which was secreted by TRIzoL lysis and secreted the HAdv-3 murine monoclonal antibody (mAb), was used to extract all RNA, and by reverse transcription to cDNA., multiple specific degenerate primers were used to amplify the mouse derived mAb light chain and weight by PCR. Chain variable region gene. Clone the mouse derived mAb light chain variable region gene and heavy chain variable region gene to the Pa-L5 (the human light chain signal peptide and the human light chain constant region gene) to construct the L chain and clone to the Pa-H2 (the human heavy chain signal peptide and the human heavy chain constant region gene) to construct the H chain, and then clone the L chain through gene amplification enzyme to Pc-DNA3.1 (+). The pcDNA3.1-3D7L vector was constructed and the H chain was cloned through gene amplification enzyme to Pc-DNA3.1 (+) and pcDNA3.1-3D7H vector was constructed. PcDNA3.1-3D7L and pcDNA3.1-3D7H plasmids were transfected to CHO-S cells with 1:1 ratio to express and construct a stable cell line. [results] 1. In 3760 cases, 392 cases were detected, the positive rate was 10.4%; MP was detected in MP. In 339 cases, the positive rate of 9.0%. in RSV positive cases was the highest in 0~9 months, up to 19.6% (181/923 cases).RSV epidemic peak occurred in February 2012 ~4 month and January 2013 ~4 month, the positive rate was 26.8% (136/507 cases) and 18% (151/839 cases) respectively. In MP positive cases, the positive rate of age at 6~10 age was the highest, up to 20.1% (97/484 cases), peak epidemic peak. The positive rates were 16.3% (92/564 cases) and 13.4% (124/922) in July 2012 ~10 month and June 2013 June 2013. In the symptoms of upper respiratory tract, the incidence of [28.6% (112/392 case) in children with RSV infection, and the incidence of runny [33.2% (130/392 case)) was significantly higher than [15.0% of MP positive children (51/339 cases), 20.6% (cases)], and the 2 groups had statistical differences. Meaning (P 0.001); RSV Infection Children's cough [78.1% (306/392 case)], expectoration [20.4% (80/392 case) incidence rate is significantly lower than MP positive children [90.0% (305/339 case), 27.1% (92/339 case)], the 2 groups are statistically significant (P0.001, P=0.032). The incidence of phlegm sound [29.3% (115/392)], phlegm sound [18.9% (74/392 case) was significantly higher than that of [11.5% (39/339 cases), 6.5% (22/339 cases), 11.5% (39/339 cases), and 10.6% (36/339 cases)]. 40.1% (157/392 cases) were more common, the 2 groups were statistically significant (P0.001). Two, the total HCo V positive rate was 4.3% (489/11399), the positive rate of OC43 was 3% (346/11399), the positive rate of 229E was 0.6% (65/11399), NL63 positive rate was 0.5% (60/11399), and the HKU1 positive rate was 0.3%. The positive rate was between 3.3%~5.5% in all age groups. The proportion of total HCoV and OC43 at 7~12 months was the highest (p0.001). The highest positive rates of HCoV, OC43229E, NL63 and HKU1 were found in April 2012 (15.3%, 25/163), April 2012 (14.1%, 23/163), February 2011 (10.4%, 8/77), September 2009 (7.7%,). And in June 2010 (6.2%, 5/81). In clinical symptoms, the symptoms of children infected with only one HCoV were compared with other HCoV types and other common respiratory virus symptoms. The symptoms of high fever (p=0.014) (p=0.014) and abnormal lung breathing (p=0.043) in children infected with one kind of HCoV were significantly more than that of other HCoV types. Other common respiratory viruses were found in more children. In addition, the symptoms of HCoV 4 types of children, cough (p=0.032), pneumonia (p=0.026), and abnormal lung breathing (p=0.002) were significantly different. Among the cases of HCoV positive children, the most common respiratory tract virus was influenza virus A (21.6%, 50/231) and respiratory syncytial virus (SR) virus. (21.6%, 50/231) three, after 3 rounds of monoclonal screening, CLEIA results showed that the antibody expression level reached 20mg/L. The antibody was detected by Western blotting and the antibody structure was correct. The neutralization activity of the antibody was detected by microneutralization test in vitro. [Conclusion] 1, the main infection population of RSV in Guangzhou area is 0~9 The focus of the month's children is ~4 month of January; the main infection population in MP is 6~10 year old children, and the focus is on the year of June. The prevention work is to be carried out throughout the year to prevent the increase in the rate of hospitalization caused by RSV and MP infection. Two, the main infection population of the total HCo V in Guangzhou area is the year old children of 7~12 months, and the key prevention and control time is in spring and autumn. Among them, the main infection population of OC43 is the children of 7~12 months of age. The key prevention and control time is in spring and autumn. The main infection population of 229E is the age group of 4~6 months. The key prevention and control time is in the spring. The main infected people of NL63 are the children of 7~12 months of age. The key prevention and control time is in the autumn, while the key prevention and control time of HKU1 is in the time. In summer. Three, the anti HAdv-3 mouse / human chimeric neutralization antibody eukaryotic expression plasmid was successfully constructed and expressed steadily in CHO-S cells, and the expressed antibody was also confirmed to have neutralization activity.

【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R725.6

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