Screening And Analysis Of Anti-Avian Influenza Virus Immune-Related Genes

This study was carried out to research the molecular mechanism of inactivated influenza virus vaccines immunity and find new anti-avian influenza virus genes. in addition,this study may provide candidate genes for producing anti-avian influenza virus transgenic chicken and lay the foundations for invention of anti- avian influenza virus medicine. In the research, mRNA differential display method was used. 40 ARBOR ACRES chickens(AA) were selected randomly in this study, 20 of which were injected H5 subtype inactivated influenza virus vaccines ,the other 20 were controls. The 40 chickens were killed, and Spleen samples were collected and saved in liquid nitrogen. The total RNA was isolated from the spleen samples using TRIZOL Kit according to the manufacturer's protocol,and digested with RNase-free DNase I to eliminate chromosomal DNA.The concentration of RNA was determined by spectrophotometer.A RNA pool containing equal amounts RNA of 20 chickens samples was established. Each RNA pool was reverse transcribed with M-MLV reverse transcriptase using one of the three anchor primers. The cDNA of the two samples were amplified with random primer and corresponding anchor primer. For the DDRT-PCR,combinations of the 3 anchor primers together with 18 random primers. The DNA bands were separated on a polyacrylanlid gels and identified by silver staining. The differentially expressed bands were extracted from the gel and reamplified using the same primers set previously.The PCR products were identified by reverse Northern dot bloting with the DIG-labeled cDNA using DIG High Prime DNA Labeling and Detection Kit according to the manufacturer's protoco1. The positive DNA fragments were cloned into pGEM-T easy vector and then transformed into E.coli DH5αcompetent cells. After identification by enzyme digestion,the positive clones from the ampicillin positive LB plate were selected and sequenced. Uncertain fragment were identified by semi-quantitative RT-PCR.13 anti-avian influenza virus immune-related ESTs were found. DD1 and DD2 were expressed only in A group, DD3 and DD4 were expressed higher in A group than B group; DD5, DD6, DD7, DD8 and DD9 were expressed only in A group; DD10, DD11, DD12 and DD13 were expressed higher in B group than A group.All 13 ESTs were compared with nucleotide sequences deposited in the nr database and the dbEST database of GenBank via BLASTn tool. DD10 and DD13 were found similar to gallus mRNA for ribosomal protein L7a. ribosomal protein L7a play a important role in tumour and disease process. We assume...