Prokaryotic expression and purification of measles virus N proteins and preparation of rabbit antisera
Objective To express and purify the N proteins of measles virus(MV)in prokaryotic cells,immunize rabbits to prepare rabbit antisera against MV N proteins,and identify the specificity.Methods Using the genome of MV-S191 vaccine strain as template,three fragments of N gene were amplified by PCR to construct the recombinant expression plas-mids pGEX-MV-N1,pGEX-MV-N2 and pGEX-MV-N3,which were then transformed into E.coli BL21(DE3)and induced to express the fusion proteins GST-MV-N1,GST-MV-N2 and GST-MV-N3 with GST tags on the N-terminal,respec-tively.After purification,the fusion proteins were mixed with equal volumes of Freund's complete adjuvant(for initial immu-nization)and Freund's incomplete adjuvant(for booster immunization),separately,and were used to immunize female rabbits for six times after evenly emulsification,with one rabbit for each protein.The whole blood was collected to isolate the rabbit antisera against MV N proteins,and the specificity of rabbit antisera was identified by Western blot and indirect immunofluorescence assay(IFA).Results The recombinant expression plasmids pGEX-MV-N1,pGEX-MV-N2 and pGEX-MV-N3 were constructed correctly as identified by double enzyme digestion,PCR and sequencing.The fusion proteins GST-MV-N1 and GST-MV-N3 mainly existed in the form of inclusion bodies,with a relative molecular mass of about 42 000 and 39 000 as well as a purity of 96％and 85％,respectively.However,GST-MV-N2 fusion protein was not detected in the supernatant and precipitate.The rabbit anti-MV-N3 antisera specifically recognized the N protein expressed by MV-S191 infected cells,and the specificity was higher than that of rabbit anti-MV-N1 antisera.Conclusion In this study,rabbit anti-MV-N antisera was successfully prepared,which provides technical support for the development of recombinant MV vaccine and the preparation of MV structural protein antibody.
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