本文選題：西尼羅病毒 + Nipah病毒�。� 參考：《重慶醫(yī)科大學》2009年博士論文

【摘要】： 目的: 調查西尼羅病毒(West nile virus, WNV)在中國西部地區(qū)人和動物中的自然感染及流行情況,以獲得我國西部地區(qū)WNV的流行病學資料,評估我國西部地區(qū)的生物安全現狀。預測WNV和Nipah病毒( Nipah virus, NiV)主要蛋白的二級結構和B細胞表位,為WNV和NiV的免疫預防和免疫治療提供理論依據。 方法: 1對課題組前期開發(fā)的WNV一步法實時RT-PCR檢測方法進行活病毒驗證。提取WNV、登革熱病毒(dengue virus,DENV)、日本乙型腦炎病毒(Japanese encephalitis virus,JEV)細胞培養(yǎng)液上清和WNV接種鼠腦組織的RNA。對WNV細胞培養(yǎng)液上清所提的RNA倍比稀釋,然后進行一步法Real time RT-PCR反應。對WNV接種的鼠腦組織提取的RNA倍比稀釋,并進行一步法Real time RT-PCR反應。對DEN、JEV細胞培養(yǎng)液提取RNA,進行一步法real time RT-PCR反應。 2自2007年5月起,收集重慶地區(qū)發(fā)熱患者外周血標本100份并采集重慶地區(qū)飼養(yǎng)的豬外周血標本300份,使用密度梯度離心法分離外周血單個核細胞,應用Trizol法提取細胞總RNA。收集重慶地區(qū)病毒性腦炎患者腦脊液22份,應用Trizol法提取細胞總RNA。用已建立的WNV一步法實時RT-PCR檢測方法,對RNA樣本進行WNV檢測,并提交流行病學調查報告。 3自2007年6月起,采集新疆地區(qū)驢外周血標本200份,使用密度梯度離心法分離外周血單個核細胞,應用Trizol法提取細胞總RNA。用已建立的WNV一步法實時RT-PCR檢測方法,對RNA樣本進行WNV檢測,并提交流行病學調查報告。 4自2008年6月起,采集寧夏地區(qū)羊外周血標本200份和牛外周血標本100份,使用密度梯度離心法分離外周血單個核細胞,應用Trizol法提取細胞總RNA。用已建立的WNV一步法實時RT-PCR檢測方法,對RNA樣本進行WNV檢測,并提交流行病學調查報告。 5根據WNV的E蛋白基因組序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案預測蛋白質的二級結構,采用Kyte-Doolittle親水性方案、Emini表面可及性方案和Jameson-Wolf抗原指數方案,輔以對E蛋白的二級結構中的柔性區(qū)域的分析,預測WNV的E蛋白的B細胞表位。 6根據NiV的G蛋白和F蛋白基因組序列,采用Garnier-Robson方案、Chou-Fasman方案和Karplus-Schulz方案預測蛋白質的二級結構,采用Kyte-Doolittle親水性方案、Emini表面可及性方案和Jameson-Wolf抗原指數方案,輔以對G蛋白和F蛋白的二級結構中的柔性區(qū)域的分析,預測NiV的G蛋白和F蛋白的B細胞表位。 結果: 1WNV一步法實時RT-PCR方法能檢測的WNV細胞培養(yǎng)液上清RNA的濃度范圍為103～10-2 PFU/ml,能檢測的WNV接種鼠腦組織RNA的濃度范圍為10-3～10-1(0原液起始濃度100)。該方法檢測DEN、JEV細胞培養(yǎng)液上清RNA結果為陰性。 2對重慶地區(qū)收集的人血、腦脊液和采集的豬血樣本進行WNV核酸檢測,成功進行了一步法實時RT-PCR反應,所有樣本熒光擴增曲線均無Takeoff點,曲線平坦,判為陰性結果。 3對新疆地區(qū)采集的驢血樣本進行WNV核酸檢測,成功進行了一步法實時RT-PCR反應,所有樣本熒光擴增曲線均無Takeoff點,曲線平坦,判為陰性結果。 4對寧夏采集的羊血和牛血樣本進行WNV核酸檢測,成功進行了一步法實時RT-PCR反應,所有樣本熒光擴增曲線均無Takeoff點,曲線平坦,判為陰性結果。 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區(qū)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區(qū)域形成β折疊的可能大;E蛋白的柔性結構區(qū)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區(qū)段。綜合分析推測WNV的E蛋白B細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區(qū)段,另外E蛋白N端第275-284, 313-320和396-403區(qū)段也可能存在B細胞表位。 6 Nipah病毒G蛋白N端第2-6區(qū)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區(qū)域形成β折疊的可能大;G蛋白的柔性結構區(qū)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區(qū)段。綜合分析推測Nipah病毒G蛋白的B細胞表位最有可能位于N端第140-153, 270-278和401-408區(qū)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區(qū)段也可能存在B細胞表位。 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區(qū)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區(qū)域形成β折疊的可能大;F蛋白的柔性結構區(qū)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區(qū)段。綜合分析推測Nipah病毒F蛋白的B細胞表位最有可能位于N端第95-105和519-539區(qū)段,另外F蛋白N端第42-54, 216-225, 301-307, 356-365和470-482區(qū)段也可能存在B細胞表位。 結論: 1本課題組前期開發(fā)的西尼羅病毒一步法Real-time RT-PCR方法敏感性高、特異性好,能準確定量,適用于大規(guī)模的流行病學調查和病毒性疾病檢測。 2初步流行病學研究提示我國西部重慶地區(qū)部分人群和豬群中暫未發(fā)現WNV感染。 3初步流行病學研究提示我國西部新疆地區(qū)部分驢群中暫未發(fā)現WNV感染。 4初步流行病學研究提示我國西部寧夏地區(qū)部分羊群和牛群中暫未發(fā)現WNV感染。 5應用生物信息學方法分析了WNV主要抗原蛋白E蛋白的二級結構B細胞表位,為進一步研究WNV的蛋白特征、研制疫苗和制備單克隆抗體奠定了基礎。 6應用生物信息學方法分析了NiV主要抗原蛋白G蛋白和F蛋白的二級結構B細胞表位,為進一步研究NiV的蛋白特征、研制疫苗和制備單克隆抗體奠定了基礎。
[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 .

【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R181.3

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