Prokaryotic Expression Of Goatpox Virus G9 Protein And Establishment Of Indirect ELISA Detection Method

Goat pox(GTP)is an acute,hot and highly contagious infectious disease caused by Goat pox virus(GTPV).GTPV mainly infects goats.Infected goats show decreased appetite,increased body temperature,and acne on the skin and mucous membranes,resulting in decreased immunity.If a secondary infection occurs during this period,it can directly cause the death of the goat and cause huge economic losses to the goat industry.There are reports that goat pox can infect humans,indicating that the disease has potential public health hazards.G9 protein is a membrane protein associated with the invasion of host cells by vaccinia virus.Its function and antigenicity indicate the potential value of G9 protein in the development of diagnostic methods.By conducting research on GTPVG9 protein,applying bioinformatics methods to analyze the structure and function of G9 protein,obtaining purified G9 recombinant protein through an in vitro expression system,we tested its biochemstry properties and established an indirect ELISA detection method,which laid the foundation for the specific,sensitive and convenient diagnostic technology of GTPV.1.Cloning and bioinformatics analysis of G9 geneSequencing was performed after cloning the G9 protein encoding sequence by PCR from GTPV.We used bioinformatics technology to predict and analyze the nucleic acid sequence of G9 gene and the translated protein sequence.The results show that the G9 protein has stable physicochemical properties.The secondary structure is mainly a-helix and random coil,accounting for 36.61%and 42.86%respectively.It has 17 epitope sites,including 34 phosphorylation sites.There is a transmembrane area at C-terminal of the sequence.In the predictional tertiary structure,a domain with apoptosis and transmembrane function was found.2.Prokaryotic expression,purification,and identification of G9 proteinThe recombinant plasmid vector pET-30a-G9 was constructed and transformed into strain BL21 to induce the expression of G9 recombinant protein.The optimal expression conditions were determined by exploring the concentration and induction time of the inducer.After SDS-PAGE analysis,G9 recombinant protein was successfully expressed with a protein size of 39kDa,which mainly exists in the form of inclusion bodies.Western blot test confirmed that G9 recombinant protein has good reactogenicity.3.Establishment of indirect ELISA method based on G9 recombinant proteinAn indirect ELISA method for detecting GTPV antibody was established using the recombinant G9 protein as a coating antigen,and the optimal conditions of indirect ELISA were as follows:G9 antigen concentration of 0.1?g/100?L/well,reacting for 2 h;the blocking time was 1.5 h;serum samples diluted to 1:80,reacting for 1 h;anti-goat IgG of HRP conjugated diluted to 1:10000,reacting for 1 h.When the critical value OD450nm is determined to be?0.251,it is judged as positive;when it is less than 0.204,it is judged as negative;when 0.25>OD450nm>0.204,it is judged as suspicious and needs to be retested;The ELISA method detected the positive sera of common pathogens of goats such as Mycobacterium paratuberculosis,Foot-and-mouth disease virus,Ruminant pestivirus,Brucella,etc.The results were 0.066,0.052,0.048,and 0.059,without cross-reaction,indicating that it has strong specificity;GTPV positive goat serum was diluted to 1:320 and the OD 450nm value was 0.386,indicating that the method has high sensitivity;The variation coefficient of the intra-batch and inter-batch repeatability tests was less than 10%.The positive coincidence rate of samples with commercial ELISA test kits has been 88.9%,the negative coincidence rate has been 97.3%,and the total coincidence rate has been 97.8%.,Indicating that the established indirect ELISA test method has high accuracy.has beenBioinformatics technology was used to analyze the unique epitope and structure of goat pox virus G9 protein,we obtained recombinant proteins with good reactivity,and established an indirect ELISA method successfully,providing a powerful tool for the accurate diagnosis of goat pox infection.