Study On Soluble Expression Of Feline Parvovirus-like Particles Promoted By Four Molecular Chaperones

In recent years,due to its severe infectiousness and dangers,animal parvoviruses attracted widespread attention.With the rapid development of pet industry,the number of pet cats has increased sharply,leading to more severe challenges in the prevention and treatment.A safe and effective biological agent is urgently needed to be developed.Virus-like particle could stimulate protective antibody against disease.It was widely used in vaccine development because it did not contain viral DNA.The E.coli system was the expression of choice for exogenous proteins.The expressed protein often fails to fold correctly and forms inactive inclusions.Molecular chaperones synergistically involved in native folding of protein,which increased the soluble expression of exogenous proteins.In this study,the E.coli expression system was used to co-express soluble proteins with four molecular chaperones.The recombinant proteins could self-assemble into virus-like particles,which resembled a natural virus in size and hemagglutinating activity.The intestinal lysates of a dead cat from an animal hospital in Changchun,Jilin Province were collected and detected with Canine Parvovirus Detection Kit.F81 cells were used for isolation of virus.Negative-stain transmission electron microscopy,PCR amplification and sequencing were used to identify.The results showed that FPV strain was successfully isolated,named FPV/JL/18.The FPV VP2 gene was optimized and ligated with p ET30a to construct the recombinant plasmid p ET30a-VP2.The recombinant plasmid was transformed into E.coli ER2566 and induced by IPTG.SDS-PAGE and Western blot showed that the recombinant protein mostly appeared as inclusions(64.72 ku).In order to improve the soluble expression of VP2 protein,p ET30a-VP2 and four molecular chaperones p G-KJE7,p G-KJE8,p Gro7 and p Tf16 were transformed and co-expressed in E.coli ER2566.The results showed that four molecular chaperones could promote soluble expression of VP2protein.We explored the effects of anion-exchange chromatography,cation-exchange chromatography,hydroxyapatite chromatography,sucrose step density gradient centrifugation,hydrophobic interaction chromatography,Triton X-114 phase fractionation on the purification of VP2 protein.SDS-PAGE showed that 25%ammonium sulfate precipitation could remove most of the chaperone proteins.The VP2 protein was fractionated using a nonlinear gradient consisting of 20 mmol/L Tris,2 mmol/L Na Cl(p H 8.0).The concentration of purified VP2 protein reached 0.318mg/m L,and had recoveries of 75%.Triton X-114 isothermal extraction removed endotoxin from VP2 protein,and endotoxin levels of the purified VP2 protein assayed by the limulus test was less than 125 EU/m L.VLP was analyzed under different condition(salt concentration and p H).Dynamic light scattering showed that VP2protein was self-assembled at 50 mmol/L Tris,250 mmol/L Na Cl,p H 8.0.Under this condition,the VP2 protein self-assembled to form VLPs with uniform diameter of authentic virus(25.92 nm),which could induce high-titer hemagglutination(2¹⁹).This study showed that the obtained FPV VLP had similar shapes and hemagglutination properties with natural viruses.In this study,the optimized recombinant plasmid was used to co-express FPV VP2 with four molecular chaperones in a prokaryotic system.The VP2 protein was purified through different purification strategies.The obtained virus-like particles were similar with feline parvovirus in size,shape,hemagglutination.This research provided new ideas for the preparation of FPV VLP and other VLPs.