Study On The Antiserum Level Of Recombinant Middle East Respiratory Syndrome Virus Spike Protein S1 Subunit And Its Coding DNA In Mice

Middle East respiratory syndrome(MERS)is an acutely lethal respiratory disease caused by a single-stranded RNA coronavirus.In 2013,the World Health Organization(WHO)formally named the virus "MERS-Co V" and announced that the virus could spread between animals and animals,animals and humans,human and humans.Because of its high risk,the virus attracted worldwide attention.In this study,a pair of primers was designed based on the gene sequence of Spike protein S1 subunit of MERS virus.S1 subunit gene fragment of 2253 bp was amplified by PCR and cloned into the eukaryotic expression vector pEAK13CD5 LTEVhIgGFc to construct the recombinant plasmid pEAK13CD5LS1 TEVhIgGFc.Recombinant plasmid pEAK13CD5 L S1TEVhIgGFc was digested by restriction endonucleases and transfected into HEK293 E cells.Positive clone cell lines stably expressing the recombinant S1-Fc protein were selected by puromycin.The supernatant of the positive clone cell lines was collected and The S1-Fc fusion protein with a relative molecular weight of 122 kD in the supernatant was purified through protein A affinity column.The recombinant protein was identified as a target protein by Western blotting analysis.Recombinant plasmid pEAK13CD5LS1 TEVhIgGFc,recombinant S1-Fc protein,mix of recombinant plasmid pEAK13CD5LS1 TEVhIgGFc and S1-Fc proteins were inoculated into mice respectively.The antibody titer and specificity in the serum of mice was detected by ELISA and Western blotting using the purified recombinant S1-Fc as antigen.The results showed that all three methods of inoculation could induce mice to produce specific anti-S1 serum.Among them,mouse anti-S1 serum vaccinated with recombinant S1-Fc and simultaneously inoculated with recombinant plasmids and proteins produced high-titer antisera when first vaccinated,while plasmid-inoculated mice produced specific anti-S1 serum after the third inoculation,and anti-S1 serum titer was significantly lower than the others.The titers of anti-S1 serum induced by the two recombinant S1-Fc protein containing inoculation methods were basically the same,and both significantly higher than that of the plasmid-inoculated mice alone.This study demonstrated that the protein vaccine and recombinant plasmid vaccine of the S1 protein of the MERS virus could produce anti-S1 serum in mice.In the short term,the recombinant protein vaccine is more rapid and effective,resulting in a higher titer of anti-S1 serum.This study provides theoretical and technical support for the development of vaccines for the MERS virus.