| β2-microglobulin (β2m) was first described and isolated from the urine of patients with tubular proteinuris by Berggard in1968. β2m is a non-glycosylated small molecular protein which can be synthetized by all nucleated cells. It has been found that β2m is a co-receptor of a major histocompatibility complex class I (MHC-I) antigen and it has been implicated in the regulation of the host immune mechanism.β2m is essential for the recognition of foreign antigens by T-lymphocytes. Recent studies indicated that β2m was involved in the survival, growth, apoptosis, and even metastasis in human cancers. Although these studies on β2m could provide a new target for cancer therapy and tumorigenic mechanism, the biological functions of β2m in cancers are still unclear.Marek’s Disease (MD) is a kind of highly contagious malignant lymphoma disease which is caused by Marek’s disease virus (MDV). MDV can cause chicken immunosuppression and T-cell lymphomas. The MDV-infected chickens are usually taken as a ideal model for the research of pathogenesis and tumorigenic mechanism for Herpesvirus. In our early studies, we have revealed that the chicken β2-microglobulin (chβ2m) proteins were abnormally expressed in MDV-infected chickens’skins and bursa by proteomics. Studies on the fuctions of chβ2m during the infection of MDV will be not only benefit to reveal the pathogenesis of MDV, but also may provide important evidence of new therapeutic targets for human cancers. 1. Cloning and Expression of Chicken β2-microglobulin in Escherichia colichβ2m gene was amplified from normal chicken peripheral blood lymphocytes by RT-PCR. It was cloned into pET-32a(+) vector and over expressed in E.coli. The expressed protein was purified and identified by Western bloting. The results showed that the sequence of chβ2m gene amplified from chickens by RT-PCR was the same as that previously reported (M84767, AY989898, Z48921) and consists of360bp encoding119amino acids (aa) with a molecular weight of12kDa; Constant-yield expression of chp2m proteins could be achieved in E.coli(DE3). Most of the soluble recombinant chp2m proteins existed in the bacterial supernatant induced with0.8M IPTG. Only a band of molecular weight of34kDa was purified by ion-exchange chromatography. Western blotling assay demonstrated that anti-His-tag monoclonal antibody could specifically recognize the recombinant protein. It indicated that we successfully constructed the recombinant vector pET-32a-chβ2m and obteined the purified recombinant chp2m proteins. This work would provid us the basis materials for further study of the structure and functions of chp2m.2. Development of Monoclonal Antibodies Against Chicken β2-microglobulin With a Synthesized PeptideWe developed a panel of monoclonal antibodies (mAb) against chicken β2-microglobulin (chβ2m) by fusions between SP2/0myeloma cells and spleen cells from mice immunized with a synthesized peptide corresponding to positions91-119of the COOH domain of chβ2m. Two of them,6E7and3D1, identified as IgG1/κ, could react with chβ2m protein from avian macrophage HD11cells and human293T cells transfected with pcDNA3.1-chp2m in immunofluorescence assays. Only a12kDaa protein band of chp2m could be detected in the HD11and293T/chβ2m cell lysates by western blot. Chicken β2m in sera and plasma could be found in western blot by mAb3D1. Moreover, mAb3D1also recognized the chp2m antigen on the cell membranes in flow cytometry. Immunohistochemical staining with these mAbs revealed that chp2m was present in chicken thymus, spleen and bursa. These mAbs will be good tools for analyzing mechanism of the chicken immune system. 3. Development of a293T cell line Stably Expressing Chicken β2-microglobulin Gene293T cells were transfected with the recombinant plasmid pcDNA3.1-chp2m by lipofectamin. Then the transfected293T cells were selected for resistant to Zeocin. After4weeks, chβ2m proteins in the transfected293T cells were detected by IFA and Western blot. The results showed that chβ2m proteins not only in the lysate of293T-chβ2m cells but also in the supernatant could be detected by western bloting. Anti-chβ2m mAbs could also recognize the chp2m protein in293T-chβ2m cells by IFA. Above of all, a293T-chβ2m cell line stably expressing chp2m was constructed. The293T-chp2m cell will provide a good tool not only for obtaining chp2m protein expressed by eukaryotic cells, but also benefit to further research of interaction between β2m protein and host cell receptor.4. Different expression levels of chicken β2-microglobulin and MHC Class I in Marek’s disease virus-infected cells in vitro and vivoMHC expression in different MDV-infected cells is not completely understood. Therefore, we investigated the expression of the MHC-I, β2m, and Meq genes in response to different MDV infection time points by real-time PCR. The results showed that the expression of chp2m gene was significant upregulated in MSB1cells cultured with Budr for24h (P<0.01). Although the expression of MHC-I gene was upregulated at this time (P<0.01), it was downregulated with a increasing expression of Meq gene. Especially at48h, the expression of MHC-I gene was significantly downregulated (P<0.01) while the expression of chp2m was still a little upregulated; In the MDV infected-CEFs, the expression of MHC-I gene was significantly upregulated at24hours post-infection (hpi)(P<0.01) and then gradually downregulated with the increasing expression of Meq gene, especially at120and168hpi it was significantly downregulated (P<0.01). The expression of chp2m was upregulated in the early stage of MDV infected-CEFs and was only downregulated at168hpi; We also detected the expression levels in peripheral blood lymphocytes from MDV RBIB-infected chickens, the expression of MHC-I and chp2m genes were increased during early MDV infection and then decreased as the Meq gene was accumulated in the latent stages (14dpi and28 dpi). Interestingly, at35dpi, both MHC-I and P2m gene expression levels increased again in vivo. In conclusion, the results showed that the expression of chβ2m was abnormally upregulated and it prompted that chp2m may pay some potential functions during the infection of MDV. Moreover, the machanism of the difference expression of MHC-I and chp2m need to further study.5Anti-chicken β2-microglobulin Monoclonal Antibody Induces the Apoptosis in MSB1cellsTo investigate the growth of MSB1cells when chp2m was inhibited by mAbs, The MSB1cells, a chicken T-lymphoblastoid cell line, were co-cultured with chp2m mAbs. Then cell viability assays were determined by CCK-8. The changes of apoptotic morphology and DNA fragmentation were analysed by flow cytometry, electrophoretic analysis of DNA and nuclear stained by DAPI. The expression levels of caspase-3,8,9genes in MSB1cells cultured with chp2m antibody were detected by Real-time PCR. The results showed that anti-chβ2m mAb strongly suppressed the growth of MSB1cells, also in a dose and time dependent manner. At48h of cultures, flow cytometry assays certified that51.7%Annexin V+of primary tumour cells cultured with80μg/mL chp2m mAb were detected; Changes of DNA ladder and irregular nuclear in MSB1cells were observed at48h of cultures with addition of chp2m mAb (80μg/mL); The expression of caspase-3and caspase-9genes were upregulated in MSB1cells cultued with3D1by Real-time PCR. These results demonstrated that inhibition of chp2m by mAbs induced apoposis of MSB1cells by overepression of caspase-3and caspase-9genes. Therefore, such mAbs provide reference for development of human μ2m antibodies for a therapeutic approach to lymphoma.6. Effect of chp2m overexpression on the replication of MDV in Chicken embryo fibroblastsIn our earlier studies, aberrant expression of chp2m has been found in MDV infected-CEFs, therefore we investigated the effects of chp2m overexpression of CEFs on the replication of MDV and analyzed the expression of chp2m、gB and Meq genes by Real-time PCR. Compared with MDV-infected CEFs trasnfected with0.4μg pcDNA3.1-β2m or4μg pcDNA3.1vectors for controls, the expression of chβ2m gene was progressively increased from24hour post-transfection(hpt) to a peak level at72hpt in4μg pcDNA3.1-p2m transfected CEFs. In the meanwhile, the expression of gB and Meq genes were higher in4μg pcDNA3.1-β2m transfected-CEFs than other vector transfected-CEFs for controls. The results indicate that the chβ2m overexpression influenced MDV’s replication in CEFs, and upregulation of chβ2m promoted the expression of MDV gB and Meq genes. This work may lay knowledge-based construction for the research of host-virus interaction. |
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