Study On The Inhibition Of PB1 Gene Of Aain Influenza Virus By Antisense RNA

According to thetheoryofantisenseRNAandsequenceofPB1geneofA/Goose/Guang dong/1/96 (H5N1)(GD1),two different lenghth cDNA of PB1 gene of H5N1 were amplified by transription-polymerase chain reaction(PCR).we named individually them PB1A(365bp),PB1B(931bp).We obtained PB1C (843bp) which digested by neucleotide restriction emzyme EcoRI.The two forer fragments were indentified by neucleotid restriction emzyme .Then the three fragments were cloned reversely and separetly into the responding site of retrovirus expressing vector-pLXSN.Each fragment located under the strong promoter of 5'long terminal repeat sequence (LTR).So we constructed re-combinant plasmids of antsense such as pLXSN-PB1A,pLXSN-PB1B,pLXSN-PB1C.After the recombiant plasmids were indentified by PCR and neucleotide restriction emzyme and purified, they were packed by packing cell lines PA317 using liposome-mediated and selected by resistant medicine G418 whose concentration was 400ug/ml.We gained 98 positive clones. Then we expanded each positive clone and harvested the cell's supernant.The cell's supernant was concentrated bysupercentrifugal.The titer of each recombinant virus were detected by magnifying method of fibroblast of little rat and PT-PCR and Nested-PCR.We compared with the two methods .The result showed that the former was simple ,quick and sensitive After that we selected the higher titer of recombinant virus to infected the target cells-MDCK and incubated with the total culture media including G418 whose concentration is 400ug/ml.We extracted DNA and total RNA of the target cells to examine the conformity of foreign gene fragments and Neomycin resistant gene NeoR in DNA and RNA by PCR and RT-PCR.The antisense RNA all expressedstably. Finally we examined the inhibition of each antisense RNA with the eryth- rocyte agglutinate assay and the plaque assay.The result indicated that the inhibition of PB1A fragment was the highest(58.3-83.3%) ;While that of PB1B fragment took the second placeand (41.7-75%);That of PB1C fragment was the lest (16.7-50%).The difference of inhibition was probably caused by the position and the length of antsense fragments.