中国西部地区WNV感染初步流行病学研究以及WNV和NiV主要蛋白的免疫信息学分析

发布时间：2018-05-12 02:43

本文选题：西尼罗病毒 + Nipah病毒　；参考：《重庆医科大学》2009年博士论文

【摘要】： 目的: 调查西尼罗病毒(West nile virus, WNV)在中国西部地区人和动物中的自然感染及流行情况,以获得我国西部地区WNV的流行病学资料,评估我国西部地区的生物安全现状。预测WNV和Nipah病毒( Nipah virus, NiV)主要蛋白的二级结构和B细胞表位,为WNV和NiV的免疫预防和免疫治疗提供理论依据。方法: 1对课题组前期开发的WNV一步法实时RT-PCR检测方法进行活病毒验证。提取WNV、登革热病毒(dengue virus,DENV)、日本乙型脑炎病毒(Japanese encephalitis virus,JEV)细胞培养液上清和WNV接种鼠脑组织的RNA。对WNV细胞培养液上清所提的RNA倍比稀释,然后进行一步法Real time RT-PCR反应。对WNV接种的鼠脑组织提取的RNA倍比稀释,并进行一步法Real time RT-PCR反应。对DEN、JEV细胞培养液提取RNA,进行一步法real time RT-PCR反应。 2自2007年5月起,收集重庆地区发热患者外周血标本100份并采集重庆地区饲养的猪外周血标本300份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。收集重庆地区病毒性脑炎患者脑脊液22份,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。 3自2007年6月起,采集新疆地区驴外周血标本200份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。 4自2008年6月起,采集宁夏地区羊外周血标本200份和牛外周血标本100份,使用密度梯度离心法分离外周血单个核细胞,应用Trizol法提取细胞总RNA。用已建立的WNV一步法实时RT-PCR检测方法,对RNA样本进行WNV检测,并提交流行病学调查报告。 5根据WNV的E蛋白基因组序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测蛋白质的二级结构,采用Kyte-Doolittle亲水性方案、Emini表面可及性方案和Jameson-Wolf抗原指数方案,辅以对E蛋白的二级结构中的柔性区域的分析,预测WNV的E蛋白的B细胞表位。 6根据NiV的G蛋白和F蛋白基因组序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案预测蛋白质的二级结构,采用Kyte-Doolittle亲水性方案、Emini表面可及性方案和Jameson-Wolf抗原指数方案,辅以对G蛋白和F蛋白的二级结构中的柔性区域的分析,预测NiV的G蛋白和F蛋白的B细胞表位。结果: 1WNV一步法实时RT-PCR方法能检测的WNV细胞培养液上清RNA的浓度范围为103～10-2 PFU/ml,能检测的WNV接种鼠脑组织RNA的浓度范围为10-3～10-1(0原液起始浓度100)。该方法检测DEN、JEV细胞培养液上清RNA结果为阴性。 2对重庆地区收集的人血、脑脊液和采集的猪血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。 3对新疆地区采集的驴血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。 4对宁夏采集的羊血和牛血样本进行WNV核酸检测,成功进行了一步法实时RT-PCR反应,所有样本荧光扩增曲线均无Takeoff点,曲线平坦,判为阴性结果。 5 WNV的E蛋白N端第41-56, 68-73, 129-139, 209, 214-220, 239-250, 261-267, 285-296, 365-371, 412-416, 468和471-472区域形成α螺旋可能大;N端第32-34, 61-65, 140-143, 157-159, 166-170, 185-189, 201-207, 210-213, 301-304, 322-328, 238-344, 354-358, 406-411, 434-436, 442-447, 450-453, 460-463和482-496区域形成β折叠的可能大;E蛋白的柔性结构区域位于N端第8, 15-17, 27, 36-38, 100-101, 103-104, 109-112, 145-147, 152-155, 173, 175, 181, 192-195, 226-230,257, 276-277, 298-299, 317-320, 334-335, 377-381, 389, 399-401, 430-432和439区段。综合分析推测WNV的E蛋白B细胞表位最有可能位于N端第35-42, 147-156, 191-198, 226-249, 329-337和375-382区段,另外E蛋白N端第275-284, 313-320和396-403区段也可能存在B细胞表位。 6 Nipah病毒G蛋白N端第2-6区域形成α螺旋可能大;N端第24-26, 47-73, 80-84, 87-95, 110-112, 117-120, 178-182, 200-210, 215-219, 229-231, 245-256, 264-269, 279-285, 290-301, 362-374, 384-386, 398-401, 406-409, 427-430, 435-436, 450-456, 462-472, 483-485, 511-517, 519-526, 560-567, 573-581, 586-589, 592-596和600-602区域形成β折叠的可能大;G蛋白的柔性结构区域位于N端第13-19, 76-78, 106-107, 140-146, 151-153, 162-165, 174-175, 185-186, 194, 227, 240-243, 257, 259-260, 275-277, 288, 309-312, 324-329, 343-346, 352-354, 379-382, 390-394, 402-404, 419-421, 423-424, 432-434, 440-441, 481-482, 488-490, 494-500, 527-530, 541-543, 555-558和582-585区段。综合分析推测Nipah病毒G蛋白的B细胞表位最有可能位于N端第140-153, 270-278和401-408区段,另外G蛋白N端第7-23, 72-79, 162-175, 192-198, 255-262, 340-348, 373-394, 416-424, 432-448, 479-489, 527-534, 540-547和552-561区段也可能存在B细胞表位。 Nipah病毒F蛋白N端第92-96, 108-111, 122-127, 131-139, 157-165, 194-203, 251, 352, 355, 467-469和474-479区域形成α螺旋可能大;N端第1-4, 11-19, 37-41, 44-47, 57-60, 78, 82-88, 102-107, 112-117, 121, 128-130, 170-179, 184-190, 209-214, 226-231, 240-246, 263-271, 276-285, 292-299, 308-311, 316-321, 324-331, 338-340, 368-377, 382-386, 390-391, 396-401, 408-412, 416, 421-429, 447-460, 480-484, 491-515和541-546区域形成β折叠的可能大;F蛋白的柔性结构区域位于N端第52, 66-67, 98-100, 216-218, 222, 236-239, 303-306, 334-336, 343, 348-351, 357-359, 380-381, 402-405, 439, 462-463, 470-472, 523-524和537-539区段。综合分析推测Nipah病毒F蛋白的B细胞表位最有可能位于N端第95-105和519-539区段,另外F蛋白N端第42-54, 216-225, 301-307, 356-365和470-482区段也可能存在B细胞表位。结论: 1本课题组前期开发的西尼罗病毒一步法Real-time RT-PCR方法敏感性高、特异性好,能准确定量,适用于大规模的流行病学调查和病毒性疾病检测。 2初步流行病学研究提示我国西部重庆地区部分人群和猪群中暂未发现WNV感染。 3初步流行病学研究提示我国西部新疆地区部分驴群中暂未发现WNV感染。 4初步流行病学研究提示我国西部宁夏地区部分羊群和牛群中暂未发现WNV感染。 5应用生物信息学方法分析了WNV主要抗原蛋白E蛋白的二级结构B细胞表位,为进一步研究WNV的蛋白特征、研制疫苗和制备单克隆抗体奠定了基础。 6应用生物信息学方法分析了NiV主要抗原蛋白G蛋白和F蛋白的二级结构B细胞表位,为进一步研究NiV的蛋白特征、研制疫苗和制备单克隆抗体奠定了基础。
[Abstract]:Purpose :

To investigate the natural infection and epidemic situation of West nile virus ( WNV ) in people and animals in western China , to obtain epidemiological data of WNV in the western region of China , and to assess the present situation of biological safety in the western region of China . The secondary structure and B cell epitopes of major proteins of WNV and NiV virus were predicted to provide theoretical basis for the prevention and immunotherapy of WNV and NiV .

Method :

A one - step real - time RT - PCR method was used to detect the RNA of WNV and JEV cell culture medium supernatant and WNV in mouse brain tissue . The RNA was diluted with the RNA to be extracted from WNV cell culture fluid . The RNA was extracted from the mouse brain tissue inoculated with WNV and the real time RT - PCR reaction was carried out .

From May , 2007 , 100 parts of peripheral blood samples were collected and 300 parts of peripheral blood samples were collected from Chongqing area . The total RNA of peripheral blood was isolated by density gradient centrifugation . The total RNA of cells was extracted by Trizol method . The total RNA of cells was extracted by Trizol method .

3 From June 2007 , 200 samples were collected from donkey blood samples in Xinjiang . Using density gradient centrifugation to separate mononuclear cells from peripheral blood , total RNA of cells was extracted by Trizol method . WNV was detected by the established WNV one - step RT - PCR method , and an epidemiological investigation report was submitted .

From June 2008 , 200 samples of peripheral blood samples from Ningxia and 100 parts of bovine peripheral blood samples were collected . Peripheral blood mononuclear cells were isolated by density gradient centrifugation , and total RNA was extracted by Trizol method .

5 According to the E - protein genome sequence of WNV , the secondary structure of protein was predicted by using the Garnier - Robson protocol , Chou - Fasman protocol and Karplus - schulz protocol . Using Kyte - Doolittle hydrophilic scheme , Emini surface accessibility protocol and Jameson - Wolf antigen index scheme , the B - cell epitopes of E protein of WNV were predicted by the analysis of the flexible region in secondary structure of E protein .

6 According to the G protein and F protein genome sequence of NiV , the secondary structure of protein was predicted by using the Garnier - Robson protocol , Chou - Fasman protocol and Karplus - schulz protocol . Using Kyte - Doolittle hydrophilic scheme , Emini surface accessibility protocol and Jameson - Wolf antigen index scheme , the analysis of flexible region in secondary structure of G protein and F protein was used to predict the B cell epitopes of G protein and F protein of NiV .

Results :

1 WNV one - step RT - PCR method can detect the concentration of RNA on WNV cell culture medium . The concentration range of RNA from WNV cell culture medium can be detected as 10 - 3 - 10 - 1 ( initial concentration of 0 raw solution 100 ) . The method can detect DEN and JEV cell culture supernatant RNA as negative .

2 . WNV nucleic acid detection was carried out on blood , cerebrospinal fluid and collected pig blood samples collected in Chongqing . The real - time RT - PCR reaction was carried out successfully . All the sample fluorescence amplification curves had no Takeoff point , the curve was flat , and the negative result was judged .

3 . The samples of donkey blood collected in Xinjiang region were detected by WNV nucleic acid , and the RT - PCR reaction was carried out in one step . All the sample fluorescence amplification curves had no Takeoff point , the curve was flat , and the negative result was judged .

4 . WNV nucleic acid detection was carried out on blood samples from sheep blood and bovine blood collected in Ningxia . One - step real - time RT - PCR reaction was carried out successfully . All the sample fluorescence amplification curves had no Takeoff point , the curve was flat , and the negative result was judged .

5 WNV E - protein N - terminal Nos . 41 - 56 , 68 - 73 , 129 - 139 , 209 , 214 - 220 , 239 - 250 , 261 - 267 , 285 - 296 , 365 - 371 , 412 - 416 , 468 and 471 - 472 ; 317 - 320 , 334 - 335 , 377 - 381 , 389 , 399 - 401 , 430 - 432 , and 439 . The comprehensive analysis suggests that the E - protein B cell epitopes of WNV are most likely to be located in the N - terminal 35 - 42 , 147 - 156 , 191 - 198 , 226 - 249 , 329 - 337 and 375 - 382 segments , and B - cell epitopes may also be present in the N - terminal 275 - 284 , 313 - 320 and 396 - 403 sections of E protein .

the regions of the flexible structure of the G protein are located at the N - terminal 13 - 19 , 76 - 78 , 106 - 107 , 140 - 146 , 151 - 153 , 162 - 165 , 174 - 175 , 185 - 186 , 194 , 227 , 240 - 243 , 257 , 259 - 260 , 275 - 277 , 288 , 309 - 312 , 324 - 329 , 343 - 346 , 352 - 354 , 379 - 382 , 390 - 394 , 402 - 404 , 419 - 421 , 423 - 424 , 432 - 434 , 440 - 441 , 481 - 482 , 488 - 490 , 494 - 500 , 527 - 530 , 541 - 543 , 555 - 558 and 582 - 585 .

N - terminal , N - terminal , 92 - 96 , 108 - 111 , 122 - 127 , 131 - 139 , 157 - 165 , 194 - 203 , 251 , 352 , 355 , 467 - 469 and 474 - 479 regions ; 66 - 67 , 98 - 100 , 216 - 218 , 222 , 236 - 239 , 303 - 306 , 334 - 336 , 343 , 348 - 351 , 357 - 359 , 380 - 381 , 402 - 405 , 439 , 462 - 463 , 470 - 472 , 523 - 524 and 537 - 539 .

Conclusion :

The one - step real - time RT - PCR method developed in the early stage of the study group has high sensitivity , good specificity and accurate quantification , and is suitable for large - scale epidemiological investigation and viral disease detection .

2 Preliminary epidemiological studies suggest that WNV infection is not found in some population and herd in the western part of China .

3 Preliminary epidemiological studies suggest that WNV infection is not found in some donkey populations in the western Xinjiang region .

4 Preliminary epidemiological studies suggest that WNV infection is not found in some cattle and cattle in Ningxia region of western China .

5 . The secondary structure B cell epitopes of WNV main antigen protein E protein were analyzed by bioinformatics methods , which laid the foundation for further research on the protein characteristics of WNV , the development of vaccine and the preparation of monoclonal antibodies .

The secondary structure B cell epitopes of the main antigen protein G protein and F protein of NiV were analyzed by bioinformatics method , which laid the foundation for further studying the protein characteristics of NiV , developing vaccine and preparing the monoclonal antibody .

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R181.3

【参考文献】