Expression of p30 protein of African swine fever virus in Nicotiana benthamiana and establishment of indirect ELISA detecting its antibody
To establish an indirect ELISA method for detecting antibodies to African swine fever vi-rus(ASFV),ASFV p30 protein was expressed in Nicotiana benthamiana using plant bioreactor mediated by potato virus Y(PVY),and the expressed proteins were used as coated antigen to opti-mize the reaction conditions of indirect ELISA.According to the CP204L(p30)gene sequence of the ASFV SY-18 strain and the preference of the codon of Nicotiana benthamiana,the nucleotide sequence of p30 gene(231-536 bp)was optimized,synthesized,and cloned.The amplified p30 gene was inserted between P1 and HC-Pro genes in the PVY genome by using the In-Fusion connec-tion technology to construct the PVY-p30 recombinant viral plasmid.The plasmid was inoculated into the leaves of Nicotiana benthamiana by friction.After 14 d,the transcription of the target gene and the expression of the recombinant protein were analyzed by RT-PCR and Western blot.The p30 recombinant protein expressed in Nicotiana benthamiana was purified by Ni-NTA affini-ty chromatography column.The purification effect and reactivity of p30 recombinant protein was analyzed by Western blot.An indirect ELISA method for the detection of ASFV antibody was es-tablished with the purified p30 recombinant protein as the coating antigen.The results showed that the constructed PVY-p30 recombinant virus can systematically infect Nicotiana benthamiana;the p30 recombinant protein could be expressed in the leaves of Nicotiana benthamiana with good re-actogenicity;the purified p30 recombinant protein had a good effect and could be recognized by p30 protein monoclonal antibodies.The optimal reaction conditions of the established ELISA method were 0.5 mg/L antigen concentration coated at 37 ℃ for 1 h;the dilution ratio of serum was 1∶400 and incubation at 37 ℃ for 0.5 h;HRP-conjugated secondary antibody was incubated at 37 ℃ for 0.5 h;TMB color was developed at room temperature and protected from light for 10 min;the negative and positive critical value was 0.461.When the dilution of ASFV positive serum was 1∶2 560,it was still detected as positive and had high sensitivity.This method did not cross react with other common disease positive serum and could specifically detect ASFV antibodies.The repeat-ability experiments showed that the intra-assay coefficient of variation was less than 5％and the inter-assay coefficient of variation was less than 10％.The coincidence rate between the method es-tablished in this study and the ASFV commercial detection kit was 85.0％.The recombinant PVY-p30 virus constructed in this study can mediate the expression of p30 recombinant protein in the Nicotiana benthamiana.In conclusion,the indirect ELISA detection method for ASFV antibodies established using plant expressed p30 recombinant protein as antigen can be used for clinical detec-tion of diseases and epidemic monitoring,while providing basic materials for the development of feed vaccines.
indirect ELISAAfrican swine fever viruspotato virus Yplant bioreactorp30 protein
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