Study On The Expression And Identification Of VP1 Protein Of O-type Foot-and-mouth Disease Virus

Foot-and-mouth disease(FMD)is a potent infectious disease caused by foot-and-mouth disease virus(FMDV).It mainly infects more than 70 cloven-hoofed animals such as pigs,cattle and sheep.The disease has the characteristics of many transmission routes,rapid transmission speed and wide range of transmission.It is often widespread in the affected areas,leading to a serious decline in the quality of livestock products,which seriously affects the development of the local animal husbandry industry.The World Organization for Animal Health(OIE)ranks it as the first of a class A zoonotic disease.Based on the characteristics of the spread and disease of FMD,the prevention of FMDV has become the main means of controlling FMD.At present,inactivated vaccines are used in many countries to prevent FMD.Although inactivated vaccines have achieved remarkable results in the prevention and treatment of FMD,there are also many shortcomings,such as incomplete vaccine inactivation and the risk of living virus exogenous in the production process.The development of a safe and efficient new FMD vaccine is very urgent.FMDV is a single-stranded positive-strand RNA virus.Its capsid is composed of four proteins,VP1,VP2,VP3 and VP4,and encapsulates RNA to form icosahedral virions,without capsules.VP4 is located in the capsid and binds to the RNA core.VP1,VP2 and VP3 are exposed outside the capsid.VP1 contains the main antigenic site of FMDV,which is a key antigenic protein that sti Mulates cellular immunity and humoral immunity in animals.In this study,the VP1 protein of O-type FMDV was expressed and purified by E.coli expression system and Baculovirus expression system using genetic engineering technology.Provide technical reference for the development of FMDV subunit vaccine and the development of foot-and-mouth disease related kits.The experiment obtained the following results:(1)In the prokaryotic expression of VP1,the recombinant plasmid p ET28a(+)-T7-VP1 was constructed,and the recombinant VP1-6×His protein was induced by Rosetta(DE3)strain,and the expression conditions were optimized.The results of SDS-PAGE showed that the changes of IPTG concentration and temperature had little effect on the expression of recombinant VP1-6×His protein,which were in the form of a large number of inclusion bodies.The inclusion bodies were denatured and renatured and purified by NGC Mediumhigh Pressure Chromatography System.The reconstituted VP1-6×His protein was subjected to Ni-chelating affinity chromatography and identified by SDS-PAGE and Western blot.The results showed that soluble recombinant VP1-6×His protein was successfully obtained.(2)In the eukaryotic expression of VP1,the recombinant plasmid p F-ph-VP1-p10-e GFP was constructed,and the patented strain asd-deficient r Sw106-inv modified on the Bac-toBac Baculovirus expression system was used,and through the Tn7 zero background transposition,the Ac Sw106-ph-VP1-p10-GFP recombinant strain and the Bm Sw106-phVP1-p10-GFP recombinant strain were successfully obtained;the infection of Sf9 cells and Bm N cells was successful.Ac BV-ph-VP1-p10-GFP recombinant Baculovirus and Bm BVph-VP1-p10-GFP recombinant Baculovirus were constructed,and Sf9 cells were infected by Ac BV-ph-VP1-p10-GFP recombinant Baculovirus.The expression of soluble recombinant VP1-6×His protein was detected in Sf9 cells;Bm N cells and silkworm silkworms were infected by Bm BV-ph-VP1-p10-GFP recombinant Baculovirus,and both in Bm N cells and silkworm silkworms.The expression of soluble recombinant VP1-6×His protein can be detected.This study induced the expression of recombinant VP1-6×His protein by prokaryotic expression system,which was mainly present in the form of inclusion bodies,which can be solubilized by optimizing the process of denaturation and renaturation,and affinity through nickel column.A highly purified sample of soluble recombinant VP1-6×His protein was obtained by chromatography.It provides a theoretical basis and a practical basis for the subsequent production of subunit vaccines and related scientific research work,and has certain practical significance.At the same time,this study also provides a new method,using the laboratory's patented strain asd-deficient r Sw106-inv and zero background transposition technology,combined with silkworm bioreactor,can directly express soluble recombinant VP1-6× His protein.It is efficient,low-cost and easy to operate,and can be used as an alternative to production and research.