Biological Properties Of SIV A/Swine/HeNan/703/2001 (H3N2) And Expression Of NA Gene In Baculovirus System

SIV is infectious not only to pigs and poultry but also to human. Neuraminidase (NA) is an important protein on the surface of virion and plays an important role in immune and lethality. Based on the biological properties of SIV A/Swine/HeNan/703/2001 (H3N2), the NA gene was cloned, sequenced, analyzed and expressed in the baculovirus expression system. It is a good base for development of SIV subunit vaccine, the further study for function of NA and prediction of pandemic influenza.One strain SIV of H3N2 subtype was isolated from the pig flocks with respiratory system syndrome in HeNan province. Its morphological and biological properties were studied. The observations demonstrated that the virions displayed various shapes of spherical, oval, elliptic and elongated forms with diameters in the range of 80-120 nm. Fifty percent of Egg Infective Doses (EID50) and 50% Lethal Doses (LD50) to small white mice of the strain were 10-956/0.2ml and 10-1.45/0.1ml respectively. Hemagglutinin of the strain was unstable to heat. Red blood cells of horse, cattle, pig, donkey, pig, sheep, cock, rabbit, guinea pig, small white mouse and human O-type could be agglutinated by the isolate.The complimentary DNA of NA gene was amplified by RT-PCR and was cloned into pMD18-T vector. The sequence indicates that the cDNA amplified by RT-PCR has a complete Open Reading Frame (ORF) with 1410 nucleotide acids encoding 470 amino acids. After analyzed in the BLAST, its sequence of nucleotide acids was compared with that of A/Guangzhou/ass/1999 (H9N2) and A/Chicken/Hong Kong/G9/1997(H9N2) published in the GenBank, and the homology of nucleotide acids was 97.9% and 96.3 % repectively. The result showed that their relationships ofevolution were closely related. It is worth researching further.The NA gene was subcloned into the transfer vector pMelBacB after being digested with HindIII. The recombinant baculovirus transfer vector was verified by PCR and being digested with Hind III and Sail. According to the manual of Bac-N-Blue Transfection Kit, the purified pMelBacNA was co-transfected tolog-phase sf9 insect cells with the linear DNA of baculovirus(Bac-N-Blue DNA)" and Cellfectin?reagent. The recombinant baculovirus were purified by three cycles of plaque assay with the chromogenic substrate x-gal in the agarose overlay. After that, the purified recombinant baculovirus was confirmed by PCR that it was not contaminated with field baculovirus. The results of SDS-PAGE indicated that NA gene was expressed successfully in sf9 insect cells. Western blot and NA assay indicated that the NA expressed by recombinant baculovirus in sf9 insect cells has good immunity and biological activity. The research is a good base for development of SIV subunit vaccine, the further study for the function of NA and prediction of pandemic influenza.