Application Of Homologous Recombination To M Protein Vaccine Against Influenza Virus Type B

Objective:Influenza B virus is divided into Victoria and Yamagata subtypes,and is prevalent alternatively in different regions.Because the used trivalent influenza vaccine contains only one subtype B,it couldn't completely match the epidemic strains in protective antigen.In this study,we used homologous recombination technology in the process of constructing the recombinant expression plasmid to achieve rapid and efficient connection of gene fragments.Subsequently,the recombinant M protein was expressed in the prokyrocytes system and purified.Safety and efficacy of this vaccine were evaluated by animal experiment.Methods:By investigating the characteristics of epidemic influenza B virus(wild strains in 2006-2016 in yunnan province)and considering the changing trends and difference of protective antigens of influenza B vcaccine strain(recommended by the World Health Organization(WHO)in the northern hemisphere),we chose four typical strains as experimental strains.The viral RNA genome was extracted and amplified by RT-PCR.The gel-extracted target DNA fragment was then recombined with the linearized vector at 37 ? for 30 min for homologous recombination.By transmitting into E.col,the positive clones were screened the positive plasmids were identified by PCR,enzyme digestion and sequencing.We immunized animals with purified M protein to test serum neutralizing antibodies.The challenge experiment in animal models was used to evaluate the safety and effect of this vaccine.For example,the guinea pigs were used to evaluate the systemic allergies and unexplained toxins.Results:Four typical strains of influenza B were selected as follows:Massachusetts/12/2014(By),Hongta/22/2008(By),Longyang/17/2015(Bv)and Brisbane/08/2011(Bv).With the method of homologous recombination,the linearized cloning vector(pUC57)and differently inserted fragments by PCR(PCR products from M gene and B gene from Bv and By)were combined and a positive rate of 83.3%was tested.After sequencing,there was no mutation or deletion in their gene sequences showing the sequences were correct.After intraperitoneal injection of influenza B virus recombinant M vaccine,the mouse and guinea were in good condition,the diet was normal,the skin was bright and the feces was normal.After 3 weeks post-vaccination,it was showed that the dose of recombinant M vaccine was positively correlated with neutralizing antibodies and the different subtype B virus can be cross-protected,which could make function in the challenge testing,which meant that new vaccine could be effective.Conclusions:The recombinant expressed vectors by the homologous recombination techniques could be used for construction of recombinant expression plasmids for M protein of influenza B virus.The purified M protein vaccine provides good safety and cross-immunization protection,with desirable immunogenicity.