【Abstract】 Objective To construct the recombinant plasmid of pUC57 for matrix protein expression. Methods We analyzed the trend and features of influenza B virus vaccine strains recommended by the World Health Organization from 2006 to 2016 in the northern hemisphere. Furthermore, we studied the epidemic of influenza B virus in Yunnan Province. Four strains were selected in this experiment. After extraction of virus RNA, it collected the purification of RTPCR production. The recombinant plasmid was constructed at 37℃ for 30min with the pUC57 and M gene. Then a series of identification experiments were carried out, including cloning of the fragments by PCR, digestion experiment with EcoRⅠand neutralization experiment. Results Taken plate colony counting, the positive rate of homologous recombination obtained by this method was 833%. It was at the right size of the PCR production of positive bacteria. After digestion with EcoRⅠ, it was also in the correct size. All the correctness of the base pair was verified by sequencing, and there was no meaningful mutation in the recombinant plasmid. The total time spent on this method was 34h. Furthermore, the protection titers within the same lineage was 1024, but from 256 to 512 in different lineages. Conclusion This method of homologous recombination could provide a fast and efficient way to construct expression vector with influenza B universal vaccine in upstream vaccine development, which could harvest a good immune protection effect.