| There exsits the largest and most complicated natural foci of plague in China, among them, the natural foci of Marmota himalayana's plague in Qinghai-Tibet Plateau is the most important place where both animal plague and human plague outbreaks have been found. Therefore, systematic research for the character of pesticine in the natural foci of Marmota himalayana's plague becomes the main task of China's plague prevention and treatment. This study includes two parts, one is study on the character of default coding sequence discrepancy of Y. pestis, in the natural foci of Marmota himalayana, the other is study on using recombinant pesticine (Pst) to test Pst antibody in serum from animal and human.First, In order to understand the distribution and stability of genome discrepancy, we compared CDS of Tibetan Plateau's sequencing strain from natural foci of Marmota himalayana with default coding sequence in Default database, which showed the diversity on genome CDS with Z176003 and Yunan Yulong sequencing strain D106004, and verified 119 strains in Qinghai-Tibet Plateau and nearby natural foci of plague, The results showed that on the basis of six gene locus (M4,I2,3,6,8,9), all strains under experiment incurred discrepancies among types, but they were highly unified within each type. The sequecing results of the same type strains in TypeⅠareas such as Naqu and Biru in Tebetan was identical with that of Z176003, they maintained basic original condition on above locus. But typeⅡstrains from Qinghai-Tibet Plateau and strains of Qinghai Microtus maintained the same original condition with Z176003 in the locus of M4,I2 and missed of 750bp,432bp,1446bp,2040bp in the locus of 3,6,8,9.The time span for these strains'separation was more than 50 years, their distributed area included most of the natural foci of Marmota himalayana in Tibet, Qinghai, Sichuan and Gansu, this indicated that locus of 3,6,8,9 was activate during evolution, and had formed relatively stable genetic characters in these areas. TypeⅢstrains mainly included that from West Yunan mountain area and Yulong. First, point mutation occurred in M4 and 12, in the form of change of individual amino acid;at the same time, missing of 750bp,432bp,1446bp, 2040bp occurred in locus of 3,6,8,9 this showed the different characters with above two types of Y. pestis and they existed stably in natural foci of plague in North West of Yunnan.Second, the recombinant clone was selected and expressed effectively in E.coli BL21 by induction of IPTG. Using the detected method of Y. pestis F1 antibody and the indirect ELISA based on Pst antigen, which have been purified, dialyzed and concentrated by Ni-NTA affinity chromatography,375 serum samples from Himalayan marmot plague natural foci in Qinghai Provence were tested parallelly, which were obtained from different areas and hosts, and in different time. The results showed that Pst antibody appeared when the F1 antibody reached a certain level in early serum after human or animal infected Y. pestis, and increased followed by the level of Fl antibody until several months later. Therefore, Pst antigen produced by Y. pestis can induce specific Pst antibody in serum after infection. Importantly, when the Fl antibody is in low level,we can't rule out the plague infection,even if pst antibody is negative.As a new serological diagnosis of plague reagents, the recombinant Pst can work together with the classic detection method of plague, it will be helpful to solve the problems in plague monitoring, furthermore, it can make the plague antibody detection data more reliable. |
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