Screening Of Interaction Proteins Between ANK Protein 140 Of Ovipox Virus And Host Cells And Preparation Of Polyclonal Antibody

Capripox is an acute,hot and contact infectious disease caused by capripox virus.It is characterized by elevated body temperature,red plaques,papules,pustules,and finally scabs,forming nodules of different sizes.The disease is distributed all over the world.It is an outbreak or sporadic epidemic,which brings serious economic losses to sheep industry.In recent years,the range of infected hosts has expanded and the risk of transmission has increased.In order to further understand the possible host range of ANK140 gene of capripox virus and determine its biological function,the full-length and truncated protein of ANK140 gene of capripox virus were expressed and purified,and the proteins interacting with host cells were screened,and its polyclonal antibody was prepared;It also provides a new direction for the detection of capripox,diagnostic reagents and new vaccines.1.Cloning and homology analysis of ank140 gene of ovipoxvirusAccording to the reference strain of ovipoxvirus(accession number NC)₀04003.1)ANK140 gene sequence.Three fragment primers were designed for its full-length fusion expression with GST tag and His tag,and fusion expression with his tag.Five prokaryotic expression vectors were constructed by PCR amplification,ligation,transformation,bacterial liquid PCR,double enzyme digestion and sequencing.The bioinformatics analysis and genetic evolution tree analysis of ANK140 gene were carried out.Results five nucleotide sequences were amplified successfully.Two full-length expression vectors p GEX-6p-1-ANK140 and p ET-42b-ANK140 were constructed,and three segmented expression vectors p ET-42b-ANK140-1,p ET-42b-ANK140-2 and p ET-42b-ANK140-3 were constructed;The results of sequencing showed that the full length of ANK140 gene was 1500 bp,and the segments were 345 bp,378 bp and 420 bp respectively,which was consistent with the theoretical size.Phylogenetic tree analysis showed that ANK140 gene was divided into three branches: goat pox virus,sheep pox virus and bovine pimple skin disease virus.The amplified gene sequence was closely related to the capripox virus of Jaipur sheep in India.2.Prokaryotic expression and purification of ank140 protein and screening of its interacting proteinsThe constructed five recombinant plasmids were transformed into E.coli BL21 competent cells.The expression of the protein was detected by SDS PAGE,and its immunogenicity was identified by Western blot.GST fusion protein was purified by glutathione agar and his tag protein was purified by nickel affinity chromatography;The purified 95 UG protein was fully suspended overnight with nickel agarose and sheep testis cell lysate.After three times of centrifugation,the protein was precipitated for SDS PAGE.The new bands were observed and cut for MS sequencing.The results showed that the two full-length vectors were not well expressed.ANK140-1,ANK140-2 and ANK140-3 proteins with His tag were expressed,and their sizes were 12 k Da,14 k Da and 14 k Da respectively.Ank140-1 and ANK140-3 proteins were purified.The ANK140-3 protein with his tag was screened by pull-down method,and a new interacting protein band was cut and sent to the company for MS sequencing.3.Preparation of polyclonal antibodyThe purified ANK140-1 protein with His tag was used as immunogen,mixed with immune adjuvant in the same amount,injected with antigen protein 0.25 mg/kg,subcutaneously injected at 6-8 points.The New Zealand white rabbits in the experimental group were immunized 3-4 times regularly to obtain rabbit anti-His-ANK140 polyclonal antibody.The results of ELISA showed that the titer of the three antibodies was normal,and the serum titer had reached 1:64;The results showed that there was a target band between10 and 15 k Da,which proved that the polyclonal antibody was produced in serum and it could specifically recognize the prokaryotic expression of ANK140 protein.