Establishment Of Immortalized Duck Embryo Muscle Cell Line And Exploration Of Virus Tropism

Cell substrates is an important material of viral vaccine manufacturing.Because of its advantages of low cost,definited composition and high virus yield.Cell substrates have gradually replaced chicken embryo.The research on poultry cell substrates started late,and the research of duck derived cells is few.Duck derived cells play an important role in the research of waterfowl virology and vaccinology.Therefore,the exploitation of duck derived cells is of great significance for the research of waterfowl virological.At present,Goose parvovirus disease and duck parvovirus disease are the main diseases that seriously damage waterfowl breeding industry.The pathogens of waterfowl severe infectious diseases include the newly discovered pathogen of duck short beak and dwarf syndrome virus(SBDSV)and Muscovy duck parvovirus(MPV).The susceptible population of these two diseases are ducklings,and virus isolation research has been carried out in the muscle cells of infected ducks.Therefore,in this study,we introduced human telomerase reverse transcriptase(hTERT)into duck embryo muscle cells by lentivirus system to solve the problem that primary duck embryo muscle cells could not be passaged,and realized the immortalization of duck embryo muscle cells.In this study,we obtained the protein sequence information of human telomerase reverse transcriptase(hTERT)and three kinds of poultry telomerase reverse transcriptase and their key domains: telomere RNA binding domain and reverse transcriptase domain.By comparison,we found that the key domains of hTERT were very conservative in poultry.The immortalized duck muscle cell line(iDEM)was obtained by the second generation lentiviral expression system.Then,iDEM was identified by RT-PCR,PAS staining and ganciclovir(GCV)-induced apoptosis.It was found that hTERT was stably expressed in iDEM,and the expression of exogenous gene was confirmed by GCV induced apoptosis test.The results of PAS staining showed that iDEM was rich in glycogen.The results of qPCR showed that iDEM was an hTERT dependent immortalized duck embryo muscle cell line rather than a muscle satellite cell line.Basic fibroblast growth factor(bFGF)and small molecule compound Y27632 were selected from 21 kinds of additives.The combination of b FGF and Y27632 greatly increased the proliferation rate of iDEM.It was found that Y27632 in the optimized system could solve the problem of anoikis during iDEM culture.Duck short beak short syndrome virus(SBDSV)and Muscovy duck parvovirus(MPV)were tested on iDEM.Compared with primary duck embryo fibroblasts,although there was no obvious pathological changes in iDEM group,PCR results showed that Muscovy duck parvovirus could effectively reproduce on iDEM.In this study,we determined the conservation of key domains of hTERT in poultry.After introducing hTERT into duck embryo muscle by Lentivirus Expression System,we obtained immortalized duck embryo muscle cell line,and screened out the suitable culture conditions.This study proved that iDEM could effectively reproduce Muscovy duck parvovirus.