Study On MSI Of Microsatellite Loci On Chromosome2and3of CEF Infected With ALV-J

Microsatellite instability has been recognized as a new mechanism of formation of malignant tumor, people are increasingly concerned about the clinical application value of MSI in malignant tumor. Since1993a higher proportion of MSI was found in hereditary non-polypoid colorectal cancer, studies on MSI of human tumor diseases have had a history of20years. Avian leukosis is one of three neoplastic diseases of chickens, reports on MSI of avian leukosis have been rare at home and on abroad so far except that Wu Yanting of our laboratory did early diagnosis of the disease with MSI in2011. In this experiment, CEF was infected by ALV-J, then82microsatellite loci were amplified and detected by electrophoresis, aiming at discovering some microsatellite loci changed, which can assist clinicians in diagnosing AL by obtaining peripheral blood, bone marrow and internal organs with the combination of changing loci, and provide theoretical basis for the diagnosis of poultry diseases workers.There were23SPF chicken embryos, half of whose tissues acted as control groups, the other half was used to culture CEF. ALV-J was inoculated into cells and they were maintained for7days, CEFs were collected after avian leukosis detection kit test was positive. DNA was extracted from23groups of cultured CEFs and corresponding tissues. All the DNA templates were amplified by82pairs of microsatellite primers. Amplified products ran denaturing polyacrylamide gelelectrohoresis, the results were recorded by gel imaging system after silver staining. The genome microsatellite changes of CEFs infected with ALV-J strain were detected.The results showed that48microsatellite markers which showed different percentage of MSI accounted for58.54%of82microsatellite markers on chromosome2and3. In addition, there were34loci which did not change with12%polyacrylamide gelelectrophoresis. ABR0579and MCW0220displayed the highest change frequency, whose rate of change was30.43%. ABR0520, ABR0569, MCW0311and LEI0223showed higher change frequency, whose rate of change was26.09%,26.09%,21.74%, and21.74%respectively.All the samples of chicken embryo fibroblasts infected with avian leukemia virus showed MSI. The sample2had a highest change rate and was up to26.83%, secondly, the rate of change of sample8,7and23was19.51%,10.98%,10.98%respectively.MSI rate of change of the remaining samples was less than10%.ABR0520locus of the seventh group of sample, ABR0520and ADL0300loci of the eighth group of sample, ABR0587locus of the fifteenth group of sample were sequenced, and the results showed that core sequence bases indecreased or increased compared cells with tissues, leading to diminution and augmentation of the microsatellite strip. The sequencing results were consistent with the ones obtained with the PAGE.In this study, microsatellite instability of several loci was found on Chromosome2and3of CEF infected with ALV-J strain firstly. Multiple microsatellite loci changes took place after CEF infected by ALV, which meant that ALV would integrate into the genome of the host and cause the genome changes of the host in the early period when ALV infected CEF. MSI detection based on PCR, electrophoresis and silver staining can quickly and efficiently discover the MSI of the genome. It provided a mode for the research on the other animal tumor diseases.