High-Efficiency Preparation Of Surface Antigen H5HA10 Applied To Subunit Vaccine Of Avian Influenza

Avian Influenza(Avian Influenza)is caused by influenza A virus.It belongs to the Orthomyxoviridae family and is a negative-sense single-stranded RNA virus that is commonly found in poultry,humans and other animals.HPAI is caused by the H5 and H7 subtypes and is characterized by sudden death and high mortality.Once an outbreak occurs,the infected chicken population is completely lost.The genome of the bird flu virus has the characteristics of easy mutation.There are many serotypes and there is no cross-protection between the serotypes.This has brought great difficulties to the research and development of the vaccine.There are two kinds of proteins on the surface of influenza virus virus particles,namely hemagglutinin and neuraminidase.Hemagglutinin is the main component of influenza virus capsule capsule,and is commonly used in subunit vaccines.In this study,Spyligase technology was applied to the efficient preparation of the surface antigen H5HA10 of avian influenza subunit vaccine.In the experiment,H5HA10 was designed to fragment after H5N1(partially in Inner Mongolia/10/2009 part)subtype influenza virus hemagglutinin gene was designed.Fragments have an important role in hemagglutinin antigen variation.Spy Tag and KTag were added to both ends of the H5HA10 sequence,and named Spy-H5HA10,which was cyclized using Spyligase technology to increase the stability of H5HA10.Nine Spy-H5HA10 recombinant expression vectors with different soluble tags were constructed and expressed in E.coli,respectively,and recombinant proteins with high expression levels were selected.Spyligase was ligated into a His-Cherry tagged p ET21 b vector and expressed in E.coli.Firstly,The target gene Spy-H5HA10 was digested with Bam H I and Xho I,and ligated into nine vectors with different solubilizing tags and transformed into BL21 competent cells,respectively.Xho I and Nde I double enzymes After correct identification,induced by IPTG,the expression level and solubility of the fusion protein were analyzed by SDS-PAGE electrophoresis,and the soluble expression level of the fusion protein with high expression level was screened.The soluble expression level of p ET21b-His-Ars C-H5HA10 protein was screened.More than 95%.Using the same method,Nhe I and Xho I were double digested to obtain the target gene Spyligase,which was ligated to the His-Cherry soluble tagged p ET21 b vector and transformed into BL21 competent cells,Xho I and Nde I double enzymes.After the cut was identified correctly,induced by IPTG,the expression and solubility of the fusion protein were analyzed by SDS-PAGE electrophoresis,and the expression was highly soluble.Secondly,The p ET21b-His-Ars C-H5HA10 and p ET21b-His-Cherry-Spyligase were affinity-purified by Ni-NTA Agarose,and high purity H5HA10 and Spyligase recombinant proteins were obtained.Finally,After high-purity H5HA10 was mixed with Spyligase,it was reacted in PCT buffer at 4°C.It was analyzed by SDS-PAGE whether H5HA10 formed Reaction and cyclized to form monomer,dimer or even multimer.To sum up,In this experiment,we successfully constructed nine recombinant carriers of H5HA10 with different soluble labels,one Spyligase recombinant vector containing soluble labels,explored and successfully obtained high-purity recombinant proteins,and cyclized recombinant proteins;explored the practicality of Spyligase technology.It laid the experimental foundation for the follow-up study of the subunit vaccine for Avian Influenza.