Abstract: The aim of this study was to obtain a highly purified antigenic peptide of Batai virus (BATV) G1 protein and to establish an indirect ELISA method for detecting sheep serum antibodies against BATV. Firstly, the codon of 189-239 amino acids strong antigen peptide encoded by 51 amino acids of G1 protein were optimized. After gene synthesis, a recombinant expression plasmids pET-32a-G1_189aa-239aa was constructed, and transformed into E. coli BL21 competent cells to induce expression. A large number of rHis-G1_189aa-239aa peptides were obtained by optimizing the expression conditions, which were purified by nickel column affinity chromatography, and the protein concentration was determined by BCA kit. Then the antigen specificity of rHis-G1_189aa-239aa peptide was analyzed by Western blotting. Using rHis-G1_189aa-239aa peptide as antigen and positive serum of BATV sheep as antibody, the reaction conditions were optimized by square titration, and the indirect ELISA detection method of BATV was established. SDS-PAGE result showed that the rHis-G1_189aa-239aa peptide existed mainly in the form of inclusion bodies in E. coli, the protein molecular weight was 24.5 ku, and the expression of rHis-G1_189aa-239aa peptide reached the peak after induced by 0.1 mmol/L IPTG for 5 h. After purification by nickel column affinity chromatography, the protein concentration was 0.296 mg/mL. Western blotting results showed that the specificity of rHis-G1_189aa-239aa peptide was good. The indirect ELISA method using rHis-G1_189aa-239aa peptide as antigen was established. Through the optimization of conditions, the optimum antigen coating concentration was 2.5 μg/mL, serum and the dilution of the second antibody were 1:60 and 1:10 000, respectively. When the sample D_{450 nm} value ≥ 0.367 was positive, D_{450 nm} value ≤ 0.319 was negative. This method had no cross reaction with the positive serum of Goatpox virus and Brucella abortus, the coefficient of variation within and between batches was less than 10%, and the positive serum could be diluted to 1 600 times at most. The method had the characteristics of strong specificity, high sensitivity and good repeatability. This method was used to detect 120 sheep serum samples in Yunnan, and the results showed that the serum positive rate was 21.67%. The rHis-G1_189aa-239aapeptide with high specificity and purity was obtained in this study, and the indirect ELISA method was established, which could be applied to the seroepidemiological investigation of sheep BATV and laid a foundation for the study of epidemic prevention and control and pathogenic mechanism.

Key words: Batai virus (BATV); G1 protein; recombinant peptide; prokaryotic expression; protein purification; ELISA