Research On Marek’s Disease Meq Gene After Transfection Of CEF Part Of Microsatellite Instability And Mismatch Repair Gene

Marek’s disease(MD)is caused by Marek’s disease virus serotype I(Marek ’s Disease Virus,MDV)T lymphocytes neoplastic disease,and it is highly infectious.MDV is the only known alpha herpesvirus that can be acute transformated.MDV can not only cause chicken T cell lymphoma paralysis,blindness and nerve dysfunction,but also can induce strong immune suppression.Microsatellite(MS)is a DNA sequence which has specific sites,Their nucleotide sequences are short(repeat unit of 1-6 base pairs),nucleotide sequences tandem repeat 10-60 times,the flanking sequence is unique.In organism’s genome,MS is a specific DNA marker of high abundance.Microsatellite instability(MSI)is due to replication errors(RER)causing the increase or loss of new microsatellite alleles,and leading to changing the length of microsatellite DNA.manifested as(CA)n dinucleotide electrophoretic mobility changes with different allele with the normal length of the gene.Mismatch Repair(MMR)is an excision repair that removes unpaired or mispaired bases and replaces them through unscheduled DNA synthesis with correct base pairs.This experiment is focused on the study that after transfecting Marek’s disease meq gene to the chicken embryo fibroblasts(CEF),to test part of microsatellite instability and mismatch repair gene expression.Research aims to provide clues to Marek’s disease meq gene tumorigenic mechanism.The experiment includes three parts,the first part is to build the pvaxl-meq eukaryotic expression vector,and verify that it can express in the chicken embryo fibroblasts.Transfect the constructed pvax-l-meq eukaryotic expression plasmid to the CEF,And 24h,48h,72h later after transfecting,extract adherent cells’ total RNA,and reverse transcription into cDNA.According to meq gene mRNA primers designed MEQ mRNA F/R,B-actin as an internal control to make real-time PCR reaction,detect meq gene mRNA expression in cells successfully.After transfection of meq gene,cell apoptosis rate did not change significantly,cell cycle lost control,G2 and S phase arrest,DNA replication process is blocked,using MTT assay that we found the cell activity decreased.After transfection of meq gene to CEF cells did not make CEF cells transformed,but it will cause the CEF DNA synthesis blocked.The second part of the experiment is to transfect the pVAX-1-meq eukaryotic expression plasmid with transfection reagent to CEF,and set the empty vector control group,liposome control group and the control group,and 24h,48h,72h later after transfecting,extract adherent cells’ total DNA.Select our previous preliminary screening of Marek’s disease changes in the higher frequency of tumor microsatellite markers,according to the microsatellite loci design the 46 pairs of primers,and then for PCR amplification.Investigated by denaturing polyacrylamide gel electrophoresis to detect microsatellite instability,according to the results after silver staining of view,we did not find that the experimental group and the control group had significant differences in microsatellite bands.The third part of the experiment is to transfect the pVAX-l-meq eukaryotic expression plasmid with transfection reagent to CEF,and set the empty vector control group,liposome control group and the control group,And 24h,48h,72h later after transfecting,extract adherent cells’ of total RNA,using reverse transcription kit to reverse the RNA into cDNA,select P-actin as internal control primers to detect mRNA expression levels of MSH2,PMS1,MLH1 gene changes.After detection and analysis we found that after transfecting meq gene 24h,mismatch repair genes MLH1,MSH2 and PMS1 relative expression levels were higher than that in control group,while after transfecting meq gene 48h,72h mismatch repair genes MLH1,MSH2 and PMS1 relative expression levels were lower than in the control group.The results showed that 24h after transfection mismatch repair genes perform its functions,while 48h and 72h after transfection this function weakened or absent,it suggested that meq mismatch repair genes engaged in the regulation of gene expression,which is important to the study of Marek’s disease pathogenic genes meq tumorigenic mechanism.