The Soluble Expression?Purification And Immunogenicity Analysis Of GP5/M Protein From Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome,be known as blue-ear disease is a viral infection caused by Porcine Reproductive and Respiratory Syndrome virus.The sow infected with the disease will appear anorexia,fever,abortion,mummy fetus and stillbirth.The piggy infected with the disease will appear respiratory disease.And it can also promote the secondary infection of other pathogens in infected animal particularly bacterial diseases.The disease was first reported in the United States in 1987,has been discovered all over the world,causing great harm to the national swine industry.In the recent years,the outbreak of highly pathogenic blue-ear disease in China caused a devastating blow to the swine industry in our country.GP5 and M proteins are the major structural proteins and the protective antigen of host of PRRSV,and also the hotspot of research on genes.This experiment reformed M protein and GP5 protein and named GP5/M protein,will be expressed in Escherichia coli.The soluble protein is used as an antigen to immunize rabbits to detect its antigenicity.Firstly,based on the ORF5 and ORF6 gene sequences from highly PRRSV pathogenic strain called HN07-1,we did a optimization design for escherichia coli codon bias and linear epitope,which dead against GP5 and M protein encoding nucleic acid sequence,and then take them in series named GP5/M protein.There are ten plasmids with different tages listed as follows: pEX-His6-Grifin-TEV-LIC ? pEX-His6-GST-TEV-LIC ?pEX-His6-MBP-TEV-LIC ? pEX-His6-Nus A-TEV-LIC ? pEX-His6-Sumo-TEV-LIC ?pEX-His6-Thioredoxin-TEV-LIC ? pEX-His6-?-crystallin-TEV-LIC ?pEX-His6-Ars C-TEV-LIC?pEX-His6-Ppi B-TEV-LIC?pEX-His6-Ce HSP17-TEV-LIC.We digest the ten plasmids by two enzymes Nde I and Bam HI to get the ten kinds of soluble tages fragment,then ligase each of them with pET-21 b and connected the genes of GP5/M.We constructed ten prokaryotic expression vectors fused with different fusion tags.These recombinant plasmids were transformed into Escherichia coli BL21(DE3)and induced by IPTG.SDS-PAGE analysis was used to identify the soluble expression of recombinant protein after ultrasonic decomposition.The fusion tag was selected that could significantly promote soluble expression of GP5/M protein.The results showed that the ten kinds of soluble labels can increase the amount of protein expression.From the perspective ofsoluble protein levels,Almost all proteins have improved their solubility,except Grifin-GP5/M fusion protein in the form of inclusion bodies.And the solubility of GP5/M protein connect with MBP up to 95% compared with before,so the results showed that MBP is the best tag to promote soluble expression of GP5/M recombinant protein.Secondly,we obtained high purity MBP-GP5/M recombinant proteins by Ni-NTA purified agarose affinity chromatography method.Finally,we mixed the recombinant protein(MBP-GP5/M)and the adjuvant emulsified with equal volume to make genetic engineering subunit vaccine,immunizing rabbits and detect its immunogenicity.To sum up,these results suggested that we established a method to generate GP5/M recombinant protein,which will lay a foundation for the development and high volume production of PRRSV subunit vaccine.