Screening Of Potential Diagnostic Targets To Yersinia Pestis

Plague, a kind of natural focal disease caused by Yersinia pestis, is ancient but remains a serious threat to human so far. Historically, three global pandemics of plague deprived millions of lives of human beings. In recent years, there are still thousands of plague cases around the world. Plague is still a major public health problem for human being.Immunological diagnosis is an important mean in plague surveillance and clinical diagnosis. Immunological diagnosis of plague was based on detection of Fl specific antigen of Yersinia pestis. Strains with F1 antigen deficient exists in nature, so the current diagnostic methods targeting F1 antigen have the possibility of missed detection. Meanwhile, F1 antigen expression is controlled by temperature, with high expression at 37℃while micro or no expression under 28℃. The temperature-controlled expression of F1 antigen makes the current immunological diagnosis can not be used to strains living blow 28℃.In recent years, positive antibodies to F1 antigen were detected in some normal human in Zhejiang and Guangzhou province, China. It is also a challenge to current immunological diagnosis with F1 antigen.In this study, a immunological diagnosis was developed basing on recombinant F1 antigen (rF1), and 2 B cell epitopes of F1 antigen were detected out by cutting F1 with hydroxylamine, cloning, expression and chemical synthesis of F1 fragments. They are "QFTTKVIGKDSRDFD" and "TGSQDFFVRSIGSK", which can be used to distinguish cross-reactions of F1 antigen.In addition to F1 antigen, the 21 related proteins of Yersinia pestis were cloned, and purified recombinant proteins were evaluated by anti-Yersinia pestis EV76 rabbit sera. Two potential diagnostic targets, CaflM and Pla were determined. And new diagnostic methods were developed with rCaf1M and rPla, which provide assistant means of plague diagnosis.Positive sera to F1 of Zhejiang normal human can react with rCaf1M, rPla and two B cell epitopes of F1 antigen. It indicates that the F1 antibodies may be associated with exposure of Yersinia pestis, vaccination or infection of Yersinia pestis.In order to find out biomarkers between vaccine strains and natural isolates of Yersinia pestis, proteome and genome comparison were performed between vaccine strain EV76 and Yunnan isolates of Yersinia pestis.No significant protein marker was found by proteome comparison, while there are two other valuable findings. The first, glycerol kinase and sulfate transport protein were highly expressed in Dehong region isolates comparing with vaccine strain EV76 and other Yunnan isolates. Second, GroEL chaperone protein were highly expressed in Yunnan isolates carrying 6Kb plasmid (pYC) comparing with vaccine strain EV76 and other Yunnan isolates,In addition to the 102Kb pgm pathogenicity island, we found 3 genes of betaine synthesis (GI:4121108-4121110008150>) and 3 other genes (GI:4121112-4121114008150>) were deficient in Yersinia pestis EV76 by comparative genomics. These genes could serve as potential biomarkers between vaccine strain EV76 and natural isolates of Yersinia pestis.In conclusion, two B cell epitopes of Fl antigen were confirmed in this study, and Caf1M and Pla were selected out as new diagnostic targets of plague. Positive sera of Zhejiang normal human to Fl can react with the above diagnostic targets. Moreover, the genes of GI:4121108-4121110008150> and GI:4121112-41211104008150> may be the potential biomarkers between vaccine strain EV76 and natural isolates of Yersinia pestis.