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| 本項目應(yīng)用分子生物學(xué)、反向遺傳學(xué)實驗技術(shù)和動物感染試驗?zāi)P,針對目前犬瘟熱病毒大?guī)模感染靈長類動物的現(xiàn)狀,通過對病毒的分離及生物學(xué)特性鑒定并利用實驗室現(xiàn)有的VSV△G*假病毒和含有雞的β-actin 啟動子和CMV 增強子的真核高效表達(dá)質(zhì)粒pCAGGS和pHW2000平臺,從犬瘟熱病毒H蛋白和其不同動物細(xì)胞的SLAM受體兩方面著手,分別構(gòu)建表達(dá)犬瘟熱病毒H/F蛋白的假病毒、多種不同動物的Vero-SLAM細(xì)胞系。并且在犬瘟熱反向遺傳平臺的基礎(chǔ)上,通過定點突變等技術(shù)手段來研究犬瘟熱病毒H蛋白與SLAM受體結(jié)合的關(guān)鍵性氨基酸位點,及不同動物的細(xì)胞SLAM受體與H和F蛋白作用的特異性氨基酸位點,進而通過雪貂、猴動物感染試驗來驗證特定位點改變后對動物的致病力。從而闡述犬瘟熱病毒跨種傳播的分子機制,為揭示犬瘟熱病毒對宿主細(xì)胞的選擇性感染、病毒與宿主的相互依賴性進化等一系列重大理論問題的研究奠定基礎(chǔ)。 |
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Regarding the current situation where primates are affected by Canine distemper in a large scale, the project applies molecular biology and reverse genetics test model, isolates the virus and identifies its biological characteristics, and makes use of the existing VSV△G* pseudovirus in the lab, as well as pCAGGS, the eukaryotic expression vector, and the platform of pHW2000 that contains Chicken β-actin promoter and CMV enhancer. It starts from Canine distemper protein H and various animal cells SLAM acceptor to construct respectively speudovirus of expression Canine distemper protein H/F and various Vero-SLAM cell lineage for different animals. Based on the platform of Canine distemper reverse genetics and by using site-directed mutagenesis, the project studies the key amino acid site, where Canine distemper protein H and SLAM acceptor combine, as well as the idiosyncratic amino acid site, where various animal cells SLAM acceptor and protein H/F functions. Further, the pathogenicity to animals after changed specific locus is to be checked by affection tests on ferrets and monkeys, The aim of the study is to demonstrate the molecular mechanism of Canine distemper cross-species affection, to discover the selective affection to the host cell, and to set the foundation for a series of theories concerning the interdependency of virus and its host. [ Last edited by 愛與雨下 on 2012-2-21 at 15:45 ] |

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