| Anaplsama phagocytophilum is a gram-negative obligate intracellular bacterium, it is also an important zoonotic pathogen in that it infects not only humans but also some domestic animals. Since nineteen nineties it was found in the United States for the first time, European, Australia, South Korea and China has had reported, and the upward trend in recent years. Because the disease clinical manifestations were acute fever, muscle pain, fatigue, headache and other symptoms, which was similar to virus infection. The disease was lacked clinical awareness, no better rapid diagnostic method, so the disease was easy misdiagnosis, seriously can cause multiple organ failure, and even death. Indirect indirect fluorescent-antibody (IFA) testing, nest-PCR, culture isolation, peripheral blood smear of these methods used diagnosis for HGA agent. The IFA test is the most sensitive method when both acute-phase and convalescent-phase sera are being tested. However, it requires a tissue culture system for preparation of HGA agent-infected cell antigen slides, professional people, and the result of IFA was evaluated by professional person. A.phagocytophilum infection can also be confirmed by PCR detection of bacterial DNA in acute-phase blood, but this method requires laboratory staff with specialized training and expensive equipment. As this emerging disease with nonspecific clinical manifestations, development of a specific, sensitive, and rapid diagnosis method of anaplasma phagocytophilum is necessary for evaluated antibody levels.The OMP1/MSP2/P44superfamily is the characteristic proteins of anaplama phagocytophilum that was reported by many foreign studies. These superfamily contains three omp-1, one msp2, two msp2homologs, one msp4, and113p44locis. So this superfamily can be as the candidated antigen for diagnosis of APH. We selected one msp2and two msp4three major surface proteins as the object of my study. In this experiment the full-length of msp2and msp4three surface proteins was expression in BL21(DE3), but all of three proteins was ended in failure of prokaryotic expression. We found the failed result of prokaryotic expression may associated with the existence of the signal peptide in three full-length surface proteins. Further analysis of the three full-length proteins by Signal4.1, the result of prediction of Signal4.1confirmed the signal peptide in the full-length proteins. Through bioinformatics analysis of three full-length sequence, based on the remove the signal peptide sequences, we selected the sequences which is rich antigen epitopes, by construction the prokaryotic expression vector, successfully expressed the goal sequences. The immunogenicity of the three recombinant proteins were verified by Western blot.Used of recombinant protein for the establishment of indirect-ELISA, the optimized reaction conditions were including:coating with100μg/mL antigen of recombinant proteins at4℃overnight, tested serum dilution of1:200, blocking with5%skim milk at37℃for1hour, incubating with the secondary antibody diluted in1:5000at37℃for1hour. The method showed no cross-reaction with the positive serum of brucella, lyme disease, E.coil (BL21). Coefficient of variability percent (C.V%) of intro-batch and inter-batch duplicative tests were less than11%. A total40clinical serum samples were detected by this method and the positive rate was30%.This study confirmed the existence of signal peptide sequence in full-length msp2and msp4proteins that it influenced the msp2, msp4proteins was expressed in E.coli. By Signal4.1analyzed the presence of the signal peptide, and to analyze the three major surface proteins by bioinformatics, cloned and expressed the sequences which is rich antigen epitopes, the recombinant proteins was used to establish an indirect-ELISA detection method for anaplasma phagocytophilum. This method can be effectively detected clinical serums and provided technical support for the diagnosis of anaplasma phagocytophilum. |
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