[The prokaryotic expression and the establishment of the putative indirect ELISA assay for the HA gene for avian influenza virus (AIV) H5N1 subtype].
Using a pair of specific primers designed according to the relevant nucleotide sequence from GenBank, the HA1 gene of H5N1 subtype AIV was amplified with PCR method. The PCR product was cloned into pET-32a(+) to get a prokaryotic recombinant plasmid pET-HA1. The target gene was successfully expressed in the host cell BL21 (DE3) when induced with IPTG. The expression was optimized with proper inducing conditions of 0.8 mmol/L IPTG and 3 hours induction. The highest expression of the target protein added up to 32.7% of the total bacterial protein. Western blot analysis proved the recombinant protein has good reactive ability against H5N1 subtype AIV positive serum. The optional working circumstances for the iHA-ELISA assay (antigenicity concentration: 4 microg/mL; serum dilution: 1:200) was tried out with chess titration. The positive criterion of this ELISA assay is OD(the tested serum) > 0.5 and OD(the tested serum)/OD(the negative serum) > 2.0.
Zheng
Qi-sheng
QS
Key Laboratory of Animal Disease Diagnosis and Immunology, Nanjing Agricultural University, Nanjing 210095, China.
Zhang
Xiao-yong
XY
Liu
Hua-lei
HL
Li
Peng
P
Chen
Pu-yan
PY
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D002645:Chickens; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics* / Q000276:immunology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology
D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins; C109963:hemagglutinin, avian influenza A virus