The Role Of The Immunosuppressive Domain Of Transmembrane Protein In ALV Infection

Avian leukosis virus (ALV) is defined as alpha-retrovirus in poultry, it is a transmissible neoplastic viral groups which can induce hematopoietic cells hyperplasia. ALV can be divided into A-J 10 subgroups, in which subgroup J avian leukosis (ALV-J) is endogenous recombinate exogenous ALV, since the group has been found to cause significant economic losses.The envelope protein of ALV was consist of Surface protein (SU) and Transmembrane protein (TM). SU had receptor combine function, which induced host to produce antibody, but SU is easy to occur mutation, so virus can escape the neutralization of antibody successfully. Therefore, the vaccine and drug which were prepared by virus SU as target were not effective so far. However, the pivotal role of TM in the fusion of virus envelope and host cell membrane was paid attention to scientists. According to some reports, ALV adopts virus-host cell membrane fusion mechanism in entry process and the subsequent conformation change of transmembrane protein after virus binding to cell receptor play an important role in this mechanism. Therefore TM plays an important role in the process of fusion between avian leukosis virus (ALV) and cells. In addition, after invading host cell, ALV can trigger serious immunosuppression which may induce tumors. It was found in the research that the pivotal protein which can induced immunosuppression may come from highly conserved region of TM.In order to analyze the effect of highly conserved sequences in virus infection. In this study, peptide that contain twenty-seven amino acid sequences (LQNRAAIDFLLLAQGHGCQDVEGMCCF) derived from highly conserved region of TMs of ALV-J(HRPS-103), ALV-J(NX0101), ALV-J(ADOL-7501), ALV-A(MAV-1), ALV-B(MAV-2), ALV-C(RSV-Prague C), ALV-D(RSV-Schmidt-Ruppin D) and ALV-E(RAV-0) was synthesis. The peptide had helical secondary structures and located in between fusion peptide of TM protein and transmembrane domain. The molecular weight of the peptide was 2 953.46 Da and the peptide was analyzed and purified by HPLC, which was named ISU. In vitro immunocompetence test, lymphocytes activity was detected by flow cytometry and displayed that the ISU was similar with ALV-J inhibition the growth and mitosis of lymphocytes obviously in vitro.In order to attest the immunosuppression of ISU peptide, we checked it by the method that ISU peptide polyclonal antibody removed immunosuppression of ALV-J. In this study, the peptide was conjugated with keyhole limpet hemocyanin (KLH), then polyclonal antibody was gained after rabbits were immunized by peptide-KLH. The titers (sensitivity) of polyclonal antibody were detected by ELISA, the specificity was detected by Western blot, the interaction site between polyclonal antibody and ALV-J-infected cells / normal cells was observed by indirect immunofluorescence (IFA), The immunosuppression of ALV-J was removed by polyclonal antibody which was detected by fluorescent quantitative real time PCR. These results indicated that the peptide (ISU pepetide) composted of twenty-seven amino acids was able to stimulate rabbits to produce the high titers of polyclonal antibody which was able to identify ISU peptide specifically after conjugating with KLH; IFA detection discovered, polyclonal antibody was competitive binding nucleus with ALV-J; and polyclonal antibody was able to inhibit the replication of ALV-J completely detected by fluorescent quantitative real time PCR for viral RNA. Moreover, inhibition of virus depended on the dose of polyclonal antibody. The study not only proved the immunosuppressive domain of transmembrane protein in ALV possessed intense immunosuppressive function, but also did provide new effective way and referential method to prevent and cure ALV and another related retroviruses.