Bioinformatics analysis and preparation of polyclonal antibody against African Swine Fever Virus p30
[Objective]The mutated African swine fever virus(ASFV)currently circulating in China shows the characteristics of low virulence,strong infectivity and strong concealment.The clinical manifestations of African Swine Fever(ASF)are not obvious,and the early diagnosis is difficult.Rapid and sensitive early diagnostic kits are very important for the prevention and control of African swine fever,and the study of p30 and the preparation of polyclonal antibody can provide an important theoretical basis for the development of early diagnostic kits.[Method]According to the separation of Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,African swine fever virus(ASFV)Pig/Heilongjiang HRB1/2020(GenBank:MW656282)provided p30 amino acid sequence.By using bioinformatics software,physical and chemical properties of the sequence,hydrophilic/hydrophobic,across the membrane area,the signal peptide,as well as the secondary and tertiary structures were analyzed and predicted,and the codon was optimized and the recombinant plasmid pET-28a-p30 was synthesized by the biological company.p30 was cloned into pET-32a(+)prokaryotic expression vector and transformed into E.coli BL21(DE3)receptor cells.Then,the induction concentration,induction temperature and induction time of isopropyl galactoside(IPTG)were optimized.The purified p30 protein was used to immunize mice for three times.Polyclonal antibody was thus obtained and titer was detected.[Result]The results of bioinformatics analysis showed that p30 protein contained 194 amino acids,the molecular formula was C100 1H157 5N259O308S7,p30 protein was hydrophilic protein and did not contain transmembrane region,no signal peptide was found,and there contained a lot of α-helix in the secondary and tertiary structure.The protein expression results showed that when the induction time was 6 h,the final IPTG concentration was 0.05 mmol/L;when the induction temperature was 34℃,the recombinant protein expression level of p30 reached the best;when the concentration was higher than 0.9 mg/mL,1 L bacterial solution could produce 53 mg recombinant protein.The titer of mouse serum antibody was as high as 1:100 000 00 by indirect ELISA.Western Blotting results showed that the prepared p30 protein had good specificity.[Conclusion]After optimized expression,the production of p30 protein was significantly increased,and polyclonal antibody with high titer and good specificity was obtained.This study provides an important theoretical basis for developing a more sensitive diagnostic kit for p30.
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