非洲猪瘟病毒P30蛋白原核表达及间接ELISA检测方法建立

doi:10.16431/j.cnki.1671-7236.2023.05.028

Abstract

Abstract: 【Objective】 This study was aimed to establish an indirect ELISA method for rapid and accurate detection of antibodies to African swine fever virus (ASFV).【Method】 The CP204L gene sequence was extracted from the whole genome of ASFV isolate collected by GenBank,without affecting the amino acid sequence,the codon was optimized,and the pCold TF cold shock expression vector was cloned to construct the recombinant plasmid pCold TF-p30.The prokaryotic expression system of Escherichia coli was used to induce IPTG expression,and the induction time and concentration of IPTG were optimized.The recombinant protein P30 was purified with His labeled nickel column.After identification by SDS-PAGE and Western blotting,the purified recombinant protein P30 was used as antigen to establish an indirect ELISA method for detecting ASFV antibodies and optimize the reaction conditions.The specificity,sensitivity and repeatability of the method were tested and compared with commercial kits.【Result】 The recombinant plasmid was successfully transferred into the competent cells of Escherichia coli BL2 (DE3) and expressed in the form of soluble protein after induced by IPTG.There was a specific band of the target protein at about 82 ku.When the induction condition was 16 ℃ and the induction time was 12 h,the protein expression was the highest.There was no significant difference in the induction effect among different IPTG concentrations.Western blotting showed that P30 recombinant protein could react specifically with ASFV positive serum,which proved that it had good reactivity.The optimal concentration of antigen coating of the established ASFV indirect ELISA antibody detection method was 1 μg/mL,using 1% BSA solution had the best sealing effect,the optimal dilution of serum was 1∶600,and the optimal working concentration of enzyme labeled secondary antibody was 1∶8 000.This method had good specificity,sensitivity and repeatability,and the total coincidence rate was 96.7% when compared with the commercial kit.【Conclusion】 P30 protein of ASFV was successfully expressed and purified,and an indirect ELISA method for detecting ASFV antibody was established,which provided a reference for the monitoring,diagnosis and development of the kit of African swine fever.

Key words: African swine fever virus (ASFV); P30 protein; prokaryotic expression; indirect ELISA

CLC Number:

S852.65^＋1

ZHANG Fangyuan, LIN Yating, YANG Dawei, CHEN Hu, LI Guimei, SHAN Hu. Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2012-2022.

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Prokaryotic Expression of African Swine Fever Virus P30 Protein and Establishment of Indirect ELISA Detection Method

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