Expression And Application Of Subgroup J Avian Leukosis Virus SU Protein Fused To The Fc Of Rabbit IgG

Avian leukemia virus (ALV) is a group of avian retroviruses that cause a variety of tumor in poultry. The viruses are classified into ten subgroups including A-J. ALV-J was first isolated from meat-type chickens in1980s and identified as a new subgroup of ALV. In contrast to the other ALV strains, ALV-J predominantly induced myeloid leukosis in commercial meat-type chickens and caused huge economic losses to the poultry industry. It has been found that the host ranges of ALV-J were changed from meat-type chickens to commercial layer chickens and Chinese local breeding. Differences of the infection characteristics and host ranges may associate with the antigen variation of ALV-J. The envelope protein is a key factor to determine the host range of ALV-J. The gp85of surface glycoprotein (SU) of Env mediates the binding of virus and receptor. It has been demonstrated that the sequence of ALV-J SU was highly variant which may result in the changes of cell tropism, receptor types and the host ranges. How to recognize the unknown receptor or co-receptor of ALV-J is focused by many scientists. In this study, SUJ-IgGFc fusion proteins were expressed in two eukaryotic expression systems and used to capture the receptor in DF1cells by co-immunoprecipitation. These works will provide basic materials for the further study of interaction between ALV-J SU protein and cell receptor during the infection of ALV-J.1. Expression and application of subgroup J avian leukosis virus SU protein fused to the Fc of rabbit IgG in sf9cellThe ALV-J SU and rabbit IgG Fc genes were amplified by PCR and cloned into pFastBacl to construct the recombinant plasmid pFastBac1-SUJ-IgG Fc. Then, pFastBacl-SUJ-IgG Fc was transformed into DH10BacTM competent cells to construct the recombinant shuttle plasmid which was transfected to sf9cells to get recombinant baculovirus. Immunofluorescence assay showed that the fusion protein could be recognized by JE9monoclonal antibody and antibodies to rabbit IgG. Western-blot results showed that the molecular weight of fusion protein was about95kDa. The expression of fusion protein provides a powerful tool to study ALV-J receptor on the surface of chicken cells.Total proteins of DF1cells susceptible to ALV-J were extracted and co-immunoprecipited with SUJ-IgGFc binded to protein A/G agarose. The proteins interacted with the SU proteins were analzed by SDS-PAGE. Two proteins were obtained, which molecular weights were different from chNHEl. The two proteins were identified as centrosomal protein keratin and type II cytoskeletal1-like by mass spectrometry. The results suggested that other proteins combined with SU may exist in the DF1cell except chNHE-1. However, whether the proteins are the receptor of ALV-J is needed further research.2. Expression and identification of subgroup J avian leukosis virus SU protein fused to the Fc of rabbit IgG in adenovirus expression systemThe expression protein may not be able to maintain the natural state because of protein modification of insect cell expression, which will affect the interactions of proteins. In order to obtain more natural protein, we express SUJ-IgGFc fusion protein in adenovirus expression system; pcDNA3.1-SUJ-IgGFc plasmid was digested with Kpn I and Xho I. Then, SUJ-IgGFc gene was cloned into pShuttle-CMV to construct the recombinant shuttle plasmid pShuttle-CMV-SUJ-IgG Fc. The linearized recombinant plasmid was transformed to BJ5183-AD-1cells with pAdEasy-1. Homologous recombination occurred between linearized pShuttle-CMV-SUJ-IgGFc plasmid and pAdEasy-1vector in E.coli BJ5183after electrotransformation and recombinant adenovirus plasmid pAd-SUJ-IgGFc was constructed by resistance screening. Recombinant adenovirus plasmid linearized with Pac I was transfected to293T cells with LipofectamineTM2000to get recombinant adenovirus which was named to be rAd-SUJ-IgGFc. The expression of SUJ-IgGFc gene was analysed by RT-PCR, FA and Western-blot. The results showed that fusion protein could express in293T cells. Immunofluorescence assay (IFA) showed that the fusion protein could be recognized by monoclonal antibody JE9to ALV-J and antibodies to rabbit IgG. Western-blot results showed that the molecular weight of fusion protein was about95kDa. The expression of fusion protein provides a powerful tool for the research of ALV-J receptor or co-receptor. Total proteins of DF1cells susceptible to ALV-J were extracted and co-immunoprecipited with SUJ-IgGFc binded to protein A agarose. The proteins interacted with the SU proteins were analyzed by SDS-PAGE. Five proteins were obtained, which molecular weights were different from chNHE1. However, whether these proteins are the receptor of ALV-J will be investigated in the further.