RNA Interference Research On Avian Influenza H5N1 Virus

In recent years the research results show that some little double-strand RNAs can inhibit expression of special genes through affecting target mRNA, which is defined RNA interference. Since 1998 when the mechanism was made clear preliminarily, RNAi has been the hot spot of molecular biology and genetics. It is not only an important protective mechanism against external affection in vivo, but also a gene knockout tool in genetics research.Avian influenza is a viral seriously disease of poultry by type A of influenza virus, and often causes enormous losses of economy. It is a catastrophic disease of the poultry industry, especially highly pathogenic avian influenza by H5 and H7 subtype avian influenza virus. NP (nucleocapsid protein), PA (polymerase PA) and PB1 (polymerase subunit PB1) are important protective antigens of avian influenza virus, and they are all conservative genes, which are the candidate genes for RNA interference experiment.In this article avian influenza H5N1 virus is used for RNA interference research, and the main contents included that separating and identification of H5N1 virus, and construction of RNA interference in vitro transcribed siRNAs vector, and RNA interference experiment.Firstly, a kind of virus is separated from the trachea, lung, fallopian tube, liver and spleen of dead chickens, This virus can agglutinate the red blood cell of cock and the agglutination can be inhibited by standard antiserum of avian influenza virus. Through the research of biological character, such as the agar spread experiment, regression test, EID₅0, MDT (36h), IVPI (3.5), barren-spot test, electro-microscope photograph, the result indicated that this virus is a kind of highly pathogenic virus H5N1.In the base of separating and identifying virus, three oligos (oligo-NP, oligo-PA, oligo-PB 1) were designed based on three genes NP, PA, PB1 of AIV, which were connected to pBabe-Puro vector so that three RNA interference in vitro transcribed siRNAs vector were constructed: pBabe-NP, pBabe-PA and pBabe-PB1. After cell transfection, stable transfect cells were screened. They were infected with H5N1 virus before Real-time PCR and Western Blotting experiments were preceded. The result indicated that the synthetic of mRNA and the expression of protein were obviously restrained by NP-siRNA and PA-siRNA.