Recombinant Expression Of A-FMDV VP1 Protein & Fusion Expression Of Three O-FMDV Capsid Proteins

Foot-and-mouth disease(FMD)is a highly-transmitted cross-border infectious disease caused by the foot-and-mouth disease virus(FMDV)that infects cloven-hoofed animals and causes them to develop acute fever and other symptoms.The disease has a strong ability to infect,a variety of transmission routes and a wide range of areas.It has experienced outbreaks in many countries and regions.FMD continues to be the viral disease posing the greatest economic threat to agriculture.An unusually fast replication rate,extreme transmissibility,broad species tropism and antigenic diversity have made its etiologic agent,FMD virus,a difficult pathogen to defeat.At present,FMDV has been found to exist in 7 different serotypes,and there is no cross-immune response between each serotype.Today,Type A,Type O,and Type Asia 1 are the most prevalent serotypes of foot-and-mouth disease in China.From the beginning of 2013,A-FMD of pigs began to occur in China.Until July 2013,China reported to OIE more than 10 times about the epidemics of A-FMD.These epidemics have brought tremendous losses to the local animal husbandry and even the local economy.Over the last 70 years,use of an inactivated virus vaccine has played a key role in disease control and eradication was possible in certain regions of the world.However,a rapidly changing environment,increased trade,population growth,international travel and migration,contribute to disease resurgence,challenging the capabilities of any available vaccine.Therefore,with the support of new technologies,we need to develop new vaccines as possible alternatives to control and eradicate FMD.In order to obtain active A-FMDV VP1 protein.Recombinant plasmid p ET-28a-MM-VP1 was constructed,and this plasmid was then transferred into E.coli BL21(DE3)for expression.SDS-PAGE showed that VP1 protein was highly expressed in the form of inclusion bodies.To increase the soluble expression of VP1 protein,the recombinant plasmid p ET-28a-MM-VP1 and TF16 were co-transfected into E.coli BL21(DE3)for expression and expression.Conditions are optimized.The results showed that the final concentration of IPTG was0.1mmol/m L,the final concentration of arabinose was 2mg/ml,the temperature was 18°C,and after 20 hours of induction,the soluble expression level was higher.SDS-PAGE electrophoresis and Western-Blot results showed that TF16 significantly increased the expression ofrecombinant A-VP1 protein,and the recombinant protein was recognized by the positive serum of anti-FMDV.Expression of high activity A-VP1 protein is foundation for further development of genetic engineering vaccine and diagnosis reagent of foot-and-mouth disease.To obtain SUMO fusion proteins of three FMDV capsid proteins(VP0,VP1 and VP3).The recombinant plasmid p E-VP0-VP3-VP1 was constructed by using p E-SUMO vector.The p E-VP0-VP3-VP1 was then transferred into E.coli BL21(DE3)for induction and the induction temperature was optimized.SDS-PAGE showed that the optimal induction temperature was30°C and the target protein was highly expressed.Western-Blot results demonstrated that the recombinant protein can specifically bind to His monoclonal antibody.Obtaining the fusion protein can lay the foundation for the subsequent production of virus-like particle vaccine.