呼吸道合胞病毒与肺炎支原体，四型人冠状病毒流行病学分析及抗人3型腺病毒人源化中和抗体研发

发布时间：2018-05-01 05:27

本文选题：肺炎支原体 + 呼吸道合胞病毒　；参考：《广州医科大学》2017年硕士论文

【摘要】：【目的】深入了解广州地区呼吸道合胞病毒(RSV)及肺炎支原体(MP),四种不同亚型人冠状病毒的流行病学情况及临床特征,提高临床诊断的确诊率,并尝试对鼠源单克隆杂交瘤细胞株3D7的抗HAdv-3中和抗体基因进行人源化改造,为治疗性抗体的研发奠定基础。【方法】一、采集2012年1月~2014年1月期间在广州地区急性呼吸道疾病患儿(≤14岁)咽拭子标本,提取标本核酸,利用荧光定量PCR进行RSV与MP检测;并对其流行特征进行分析。二、采集2009年7月1日~2016年6月30日广州医科大学附属第一医院及中山大学孙逸仙纪念医院患有急性呼吸道感染的儿科住院患儿的咽拭子标本,共11399例。采用了荧光定量PCR方法对上述标本进行4种人类冠状病毒(Coronavirus,HCoV)检测,包括HCoV-229E,HCoV-NL63,HCoV-OC43和HCoV-HKU1,随后取出人冠状病毒阳性标本进行11种常见呼吸道病毒检测。并对其流行特征进行分析。三、利用TRIzoL裂解分泌抗HAdv-3鼠源性单克隆抗体(mAb)的杂交瘤细胞株3D7提取全RNA,并通过逆转录为cDNA。利用多条特异性简并引物通过PCR扩增鼠源性mAb轻链和重链可变区基因。把鼠源性mAb轻链可变区基因及重链可变区基因分别克隆至Pa-L5(含人轻链信号肽及人轻链恒定区基因)构建L链,克隆至Pa-H2(含有人重链信号肽及人重链恒定区基因)构建H链,然后把L链通过基因扩增酶切克隆至Pc-DNA3.1(+)中,构建pcDNA3.1-3D7L载体,把H链通过基因扩增酶切克隆至Pc-DNA3.1(+)中,构建pcDNA3.1-3D7H载体。把pcDNA3.1-3D7L与pcDNA3.1-3D7H质粒以1:1比例共同转染CHO-S细胞进行表达及构建稳定细胞系。【结果】一、在3760例患儿中,检出RSV 392例,阳性率为10.4%;检出MP 339例,阳性率9.0%。在RSV阳性病例中,年龄在0~9个月患儿最高,达19.6%(181/923例)。RSV流行高峰发生于2012年2月~4月和2013年1月~4月,阳性率分别为26.8%(136/507例)和18.0%(151/839例)。在MP阳性病例中,年龄在6~10岁阳性率最高,达20.1%(97/484例),MP流行高峰发生在2012年7月~10月和2013年6月~10月,阳性率分别为16.3%(92/564例)和13.4%(124/922例)。在上呼吸道症状中,RSV感染患儿鼻塞[28.6%(112/392例)]、流涕[33.2%(130/392例)]发生率显著高于MP阳性患儿[15.0%(51/339例)、20.6%(70/339例)],2组比较差异均有统计学意义(P均0.001);RSV感染患儿咳嗽[78.1%(306/392例)]、咳痰[20.4%(80/392例)]发生率显著低于MP阳性患儿[90.0%(305/339例)、27.1%(92/339例)],2组比较差异均有统计学意义(P0.001,P=0.032)。在下呼吸道症状中,RSV阳性患儿气喘[33.2%(130/392例)]、气促[13.5%(53/392例)]、喘鸣音[29.3%(115/392例)]、痰鸣音[18.9%(74/392例)]发生率显著高于MP阳性患儿[11.5%(39/339例)、6.5%(22/339例)、11.5%(39/339例)、10.6%(36/339例)],2组比较差异均有统计学意义(P0.001,P=0.002,P0.001,P=0.002)。MP阳性患儿双肺呼吸音粗[59.2%(201/339例)]较RSV阳性患儿[40.1%(157/392例)]更为多见,2组比较差异有统计学意义(P0.001)。二、总HCo V阳性率为4.3%(489/11399),OC43阳性率为3.0%(346/11399),229E阳性率为0.6%(65/11399),NL63阳性率为0.5%(60/11399),HKU1阳性率为0.3%(38/11399),其中一些患儿存在共感染多型HCoV的情况。总HCoV阳性率在所有年龄组中在3.3%~5.5%之间。总HCoV和OC43在7~12个月的年龄段的患儿比例显著最高(p0.001)。总HCoV、OC43、229E、NL63和HKU1阳性率最高的月份分别出现在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)。而在临床症状上分析,只感染一种HCoV的患儿症状与共感染其他HCoV分型及其他常见的呼吸道病毒患儿症状作对比,只感染一种HCoV的患儿高热(≥38°C)(p=0.014)和不正常肺呼吸(p=0.043)症状明显比共感染其他HCoV分型及其他常见的呼吸道病毒的患儿多。另外,在HCoV 4种分型的患儿症状对比,咳嗽(p=0.032),肺炎(p=0.026),不正常肺呼吸(p=0.002)症状呈显著差异。在HCoV阳性患儿共感染病例中,出现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、经过3轮单克隆筛选,CLEIA结果显示抗体表达水平达到20mg/L,通过western blotting检测到有完整的抗体分子表达并且抗体结构正确,经体外微量中和实验检测该抗体有中和活性。【结论】一、广州地区RSV的主要感染人群是0~9个月的儿童,重点在每年1月~4月;MP的主要感染人群是6~10岁的儿童,重点在每年6月~10月。预防工作要全年进行,预防因RSV和MP感染导致的住院率增高。二、广州地区总HCo V主要感染人群是7~12个月的年龄段的儿童,重点防控时间在春季及秋季。其中,OC43主要感染人群是7~12个月的年龄段的儿童,重点防控时间在春季及秋季,229E主要感染人群是4~6个月的年龄段的儿童,重点防控时间在春季,NL63主要感染人群是7~12个月的年龄段的儿童,重点防控时间在秋季,而HKU1重点防控时间在夏季。三、本研究成功构建了抗HAdv-3鼠/人嵌合中和抗体真核表达质粒,并在CHO-S细胞中稳定表达,表达的抗体也被证实有中和活性。
[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.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R725.6

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