Citation: ZHENG Qi-sheng, LIU Hua-lei, ZHANG Xiao-yong, ZHOU Bin, CAO Rui-bing, REN Piao-xue, LI Peng, CHEN Pu-yan. Prokaryotic Expression and the Establishment of a Putative Indirect ELISA Assay for the HA gene of Avian Influenza Virus H9N2 Subtype .VIROLOGICA SINICA, 2005, 20(3) : 293-297.

Prokaryotic Expression and the Establishment of a Putative Indirect ELISA Assay for the HA gene of Avian Influenza Virus H9N2 Subtype

  • Available online: 20 June 2005
  • The complete HA gene of AIV H9N2 subtype was amplified with PCR method using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank. The PCR product was successively cloned into pMD18-T and pET-32a (+) to get a prokaryotic expression vector pET-HA. The target gene was successfully expressed in the E.coli BL21(DE3) host cell in inclusion body when induced with IPTG. The expression was optimized with proper inducing conditions of 0.7 mmol/L IPTG and 3 hours induction. The highest expression of the target protein was up to 30.8% of the total bacterial proteins. Western-blot analysis proved that the recombinant protein had good immunoreactivity against H9 subtype AIV antibody. The indirect ELISA method for the detection of H9 subtype AIV antibody in chicken serum was established after the optional working circumstance for the ELISA assay (antigen concentration:4μg/mL;serum dilution: 1:80 ) was tried out with chessboard titration . The positive criterion of this ELISA method

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    Prokaryotic Expression and the Establishment of a Putative Indirect ELISA Assay for the HA gene of Avian Influenza Virus H9N2 Subtype

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    Abstract: The complete HA gene of AIV H9N2 subtype was amplified with PCR method using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank. The PCR product was successively cloned into pMD18-T and pET-32a (+) to get a prokaryotic expression vector pET-HA. The target gene was successfully expressed in the E.coli BL21(DE3) host cell in inclusion body when induced with IPTG. The expression was optimized with proper inducing conditions of 0.7 mmol/L IPTG and 3 hours induction. The highest expression of the target protein was up to 30.8% of the total bacterial proteins. Western-blot analysis proved that the recombinant protein had good immunoreactivity against H9 subtype AIV antibody. The indirect ELISA method for the detection of H9 subtype AIV antibody in chicken serum was established after the optional working circumstance for the ELISA assay (antigen concentration:4μg/mL;serum dilution: 1:80 ) was tried out with chessboard titration . The positive criterion of this ELISA method

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