Prokaryotic Expression of Anaplasma phaigocytophilum MSP4 Protein and Preparation of Its Polyclonal Antibodies
[Objective]The purpose of this study was to express the protein MSP4 of Anaplasma phagocytophilum(AP),then prepare the polyclonal antibody against MSP4 protein,so as to provide experimental materials and theoretical basis for the establishment of plasma free detection method for phagocytic cells.[Method]According the gene sequence published in GenBank,MSP4 gene was optimized and synthesized.Then the MSP4 gene was inserted into pET-30a vector to construct recombinant plasmid pET-30a-MSP4 and then was transformed into Escherichia coli BL21(DE3)competent cells.The factors affecting the expression effect(temperature and IPTG)were optimized to select the best induced expression conditions.The expression of protein was analyzed by SDS-PAGE.The recombinant protein was purified by Ni-IDA affinity chromatography,and was identified by SDS-PAGE and Western blotting.The polyclonal antibody against MSP4 was prepared by immunizing New Zealand White rabbits with the purified recombinant protein MSP4,and then the titer and specificity of the polyclonal antibody were determined by indirect ELISA and Western blotting.[Result]The results showed that the pET-30a-MSP4 recombinant plasmid was successfully constructed.The protein expression level was high under the induction conditions of 37 ℃ and 0.8 mmol/L IPTG,and it was mainly expressed in the form of inclusion bodies with molecular weight of about 28 ku.Western blotting showed that it had good immunogenicity.The titer of rabbit-derived polyclonal antibodies were 1∶64 000(rabbit 1)and 1∶128 000(rabbit 2),respectively and the prepared polyclonal antibodies had high specificity.[Conclusion]In this study,MSP4 recombinant protein was obtained,and polyclonal antibody was successfully prepared,which could provide reference for the establishment of a method for detecting antigen and antibody of AP and the evaluation of vaccine efficacy,and also laid the foundation for the study of MSP4 protein function.
Ana plasma phagocytophilumMSP4 proteinprokaryotic expressionpolyclonal antibody
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