Study On Fusion Expression Of The NS1 Gene Of H5N1 AIV And RNA Inhibition Against BmNPV Multiplication

Avian influenza (AI), an avian disease caused by influenza A, is one of the most severediseases in poultry industry. Previous studies have shown that H5N1-NS1 (non-structural protein1) can be used as the diagnostic reagents for immune and the naturally infected animals.Therefore, it is of great significance to research the NS1 protein for the control of avianinfluenza.In the present study, the synthetic H5N1-NS1 gene was cloned into pET-28a(+) andexpressed in E.coli BL21. The results showed a protein band at about 31 kD, which wasconsistent with the theoretical value of recombinant H5N1-NS1. However, an unexpectedprotein band appeared at about 15 kD. We presume that it might be due to the E.coli codonpreference or hydrolysis of the recombinant protein after expression. Moreover, H5N1-NS1 wasalso cloned into transfer vector pFastBacHTA and then expressed in sf-9 cells using Bae to Bacbaculovirus expression system. SDS-PAGE and Western blotting showed that the recombinantprotein was about 33 kD, which was larger than putative 30.5 kD, suggesting possible proteinpost-modifications such as glycosylation.In addition, the research of RNAi based-on DNA vector inhibiting the multiplication ofsilkworm NPV (Bombyx mori nucleo polyhedrovirus,BmNPV) was also conducted in this study.piggy-A3-lefl-neo transgenic vector was constructed using the elements of synthetic 'A3promoter-Sense lef-1-TCAAGAG-Anti-sense lef-1-TTTTT-polyA signal' sequence. BraN cellsshowed obvious inhibition on the multiplication of BmNPV after the transfection with thisconstruct. 70% positive BmN cells were obtained under the screen of g418 antibiotics for 2months. The results showed that the transgenic BmN cells could bear high concentrations ofBmNPV.