Establishment And Application Of MHC Haplotype Duck Embryo Liver Mesenchymal Stem Cells

In order to solve the practical problem that a variety of duck disease viruses do not have suitable passage cell lines,this paper used ducks with specific pathogens free?SPF?to carry out research on duck embryo liver mesenchymal stem cells?DuLMSC?.Transporter associated with antigen processing?TAP?genes?Tap1 and Tap2?and MHC class I heavy chain coding gene?UAA?are located in the core region of MHC and are adjacent to each other in ducks.In this study,UAA coding region sequences of four duck strains with homozygous Tap1 and Tap2sequences were determined,and UAA sequences homology in each strain were determined.Using the four strains,DuLMSC were isolated from duck embryo liver with type IV collagenase digestion method.Primary DuLMSC were sub-cultured to passage 50 in vitro.The cells morphology was stable and showed long fusiform adherent growth.The cells did not express molecular markers of hematopoietic stem cells,and were able to differentiate into osteoblasts and adipocytes.The first 35 generations of cells were not tumorigenic to nude mice.The four cell lines were named DuLMSC/1,DuLMSC/2,DuLMSC/3 and DuLMSC/4,respectively.To compare the adaptability of duck enteritis virus?DEV?to four cell lines,DEV C-KCE strain was inoculated on four cell lines separately and passed for 10 generations.The results showed that cells inoculated with the F1 to F10 generation viruses all produced typical cytopathic effect?CPE?.The F1 to F10 generation viruses could specifically amplify the UL2 gene fragment of DEV,and the nucleotide sequences of the UL2 gene of the F1?F5 and F10 generation viruses were completely homologous to the original virus.Typical herpes virus-like particles were observed in the cytoplasm,nucleus and intercellular space of the cells inoculated 24 h.The one-step growth curve of DEV on four cell lines showed that the virus titers corresponding to each time point on DuLMSC/4 were higher than or close to the other three cell lines.In addition,the virus titers of F1 to F10 tended to be stable from F5,the virus titer on Du LMSC/3 or DuLMSC/4 was about 0.5 TCID₅₀0 higher than that on Du LMSC/1 or DuLMSC/2,and the average virus titers of the first five generations on the four cell lines were about 0.5 TCID₅₀higher than that on CEF cells.When analyzing the adaptability of Duck hepatitis A virus?DHAV?type 1 attenuated strain and DHAV-3 attenuated strain to DuLMSC/4,the results showed that CPE was produced when DHAV-1 was serially passaged to F8 generation,the F1 to F15 generation viruses could specifically amplify the 3D gene fragment of DHAV-1,and nucleotide sequence of 3D gene of the F1?F7 and F15 generation viruses were completely homologous to the original virus;CPE was produced when DHAV-3 was serially passaged to F3 generation,the F1 to F15 generation viruses could specifically amplify the VP1 gene fragment of DHAV-3,and nucleoside sequences of VP1 gene of the F1?F7 and F15generation viruses were completely homologous to the original virus.The viruses proliferation curve showed that the relative copy number of DHAV-1 nucleic acid ranged from 1 to 30,while the relative copy number of DHAV-3 nucleic acid ranged from 10 to 120.Duck MHC class I molecule-specific murine monoclonal antibody G1 was prepared using the E.coli expression product of the partial?2 and complete?3 regions of the B3 line duck UAA.The heavy chain of G1 was IgM,and the light chain was Kappa type.The results of flow cytometry of G1 on peripheral blood cells of B4 duck and SPF Bailaihang chicken were 70.3%and 19.4%,respectively.After DEV infected DuLMSC/4,the expression level of MHC class I molecules on the cell surface were down-regulated and then up-regulated,while the transcription level of MHC class I molecules were always up-regulated.It was speculated that the early stage of DEV infection could inhibit the presentation of MHC I.DuLMSC/4 established in this study could support the proliferation of DEV and DHAV an d could be used for basic research related to MHC I.It is a good experimental material for th e development of duck disease prevention and control technology.