Study On DF-1 Cells Infected With ALV-A Part Of Microsatellite Instability And Mismatch Repair Related Gene

Avian Leukemia is one of the three major neoplastic diseases in poultry with a worldwide distribution.Avian Leukemia is a class of avian neoplastic diseases induced by ALV/RSV in chickens.An increasing body of studies were performed based on the avian leukemia virus in domestic and foreign,while there are few reports regarding the genomic levels in hosts infected with ALV.In order to provide new clues for the early diagnosis of avian leukemia and the molecular mechanism of cancer from the perspective of the host genome mutations,the studies on the Microsatellite Instability(MSI)and the expression of Mismatch repair(MMR)genes were performed after the inoculation of DF-1 cells with ALV-A.First experiment,the amplification of ALV-A virus and the detection of cell cycle,cell apoptosis in the DF-1 cells inoculated with ALV-A.ALV-A(RAV-1 strain)virus lyophilized powder was dissolved and diluted for the inoculation of DF-1 cells.After 7d maintained in culture medium,the virus content was determined to 10-4 75 TCIDso/0.1ml;and the cell cycle was out of control,the G1 was shortened,the S and G2 was extended,the DNA replication was influenced;while the apoptosis rate was not significantly changed,and it blocked the DNA synthesis.These findings provide research foundation for the following studies of microsatellite instability and mismatch repair genes.Second experiment,the total DNA of the DF-1 cells infected with ALV-A were extracted,and 16 microsatellite sites were selected.PCR reaction,the denaturing polyacrylamide gel electrophoresis,silver staining and sequencing were used to detect the microsatellite instability.There was no difference of the DNA sequence between sequencing inoculation group and control group;and there was no mutation detected.Third experiment,after the inoculation of DF-1 cells with ALV-A,the total DNA was extracted.16 pairs and 19 pairs of exon primers of MSH2 and MLH1 were respectively designed.The PCR reaction and the 8%non-denaturing polyacrylamide coagulation gel electrophoresis were used to detect gene mutations.Beta-actin were used as an internal control and the mRNA transcription of MSH2,MSH3,MLH1 and PMS1 were measured by RT-PCR analysis.Our results showed that there was no genes mutation of the exons of MSH2 and MLH1 after the infection of DF-1 cells with ALV-A;and the mRNA transcriptions were significantly increased on the MSH2(P<0.05)and MLH1(P<0.01);while the mRNA transcription of MSH3 and PMS1 were not significantly changed(P>0.05).