Expression And Purification Of AIV H7 Hemagglutinin(HA1) Gene In E.coli

Based on the published AIV H7 HA1 cDNA sequences, a pair of primers were designed which were specific to hemagglutinin(HA1) genes of H7 subtypes. A about 1.0Kb DNA fragment was amplified by reverse transcription polymerase chain reaction(RT-PCR).The DNA fragment was then successfully cloned into pGEM-T-Easy vector,which was confirmed by sequence analysis and restrict enzyme identification. The HA1gene was compared with those of other published AIV H7 subtypes, H5and H9 isolates. There were 85.1%~96.9% nucleotide sequence homology among the compared published AIV H7 subtypes; and 51.5%, 53.5% nucleotide sequence homology between H7 isolate and H5,H9 isolates respectively. The Bgl II and Sal I restricted enzyme sites were added into cloned HA1 gene, which was inserted into expressed vector pGEX-4T-2. After identification by PCR and restricted enzyme, the constructed recombinant expressed plasmid pGEX-4T-2-A was transformed into the receptive cells of E.coli ER2566 that was then induced by IPTG. Expressed protein of HA1 gene was identified by polyacrylamide gel electrophoresis and western-blot. The expressed proteint could bind to the specific antibody against of H7 subtype. Following the purification of the expressed protein, A ELISA was developed to detect reference antisera of H7, H5 and H9 subtypes. The result indicated that H7 antisera was positive, H5 and H9 antisera were negative. It is the basis for the development of AIV H7 ELISA Kit.