| β2-microglobulin (β2-M) is a light molecular weighted non-glycosylated protein which consists of99amino acid residues and it can be synthesized by all kinds of nucleated cells. As the β-strand of MHC-I, β2-M can bind with MHC-I to form a complex which is very important to maintain MHC-I molecule’s spatial structure and normal functions. Besides being involved in hosts’immunity responses, P2-M may be also related to the proliferation and spread of multiple tumor cells. P2-M’s level in the serum is associate with tumor burden and may be a nonspecific marker of malignant tumor.The Marek’s Disease (MD) is a chicken malignant lymphatic tumor disease which is caused by Marek’s Disease Virus (MDV). It is characterized with symptoms of peripheral nerve damage and diffused lymphoma in multiple organisms (except the bursa). Since the successful application of MD vaccine, it is also a very valuable model for research on human viral lymphatic tumor diseases. Based on the MAbs we developed, we studied the level of β2-M in different stages of MD to find the relationship between β2-M and lymphatic tumor.Part1:Development and identification of MAb against chicken β2-microglobulinWith synthesized polypeptide which covers the major antigenic epitope of P2-M as immunizing antigen, five MAb strains of hybridoma cells chβ2-M-lE2、chP2-M-2E9、 chβ2-M-3D1、chβ2-M-6E7and chβ2-M-7D5have been developed. chβ2-M-3D1chβ2-M-6E7, identified as IgG1/κ, had an ascites titers of1:25600and1:204800respectively. Indirect ELISA results indicated that MAbs have a good reactivity with the TF-29and His-β2-M fusion protein. Western blot and IFA analysis indicate that chβ2-M-3D1could interact with β2-M protein expressed in E.coli and293T cells specifically. Moreover, it could interact with P2-M in HD11cells, chicken blood, but no cross-interaction with β2-M from other animals.Part2:Dynamics of P2-M level in sera of chickens infected with MDVIn order to study the dynamics of β2-M level in sera of chickens during MDV infection,80one-day old SPF chickens were divided into two groups randomly. Chickens in experimental group were infected with1000PFU of RBIB strain of MDV, while the chicks in control group were injected with DMEM. Samples were collected from both experimental and control groups at4,7,14,21,28,35and42days post infection (DPI). Sera were isolated and organs like skin, thymus, spleen and bursa were collected. The results showed that virus could be recovered from peripheral blood lymphocytes of the experimental chicks at7DPI. Meq gene (1020bp) could be amplified by PCR. The chickens post infection2-3weeks showed depression and splits in clinical symptoms. Results of morphology showed an enlarged spleen and the atrophy for thymus and bursa. The skin tumors began to appear after4-5weeks. Histopathology showed atrophy of thymus and bursa and lymphocytes massive necrosis. β2-M level in experimental and control groups at4、14、28、42DPI were analyszed by Western blot. The expression levels of β2-M in the thymus and bursa were detected by immunohistochemistry. Results indicated that β2-M was high expressed in cell and the level of β2-M was increased significantly (p<0.05) at42DPI. These results suggested that β2-M may be related to the development of lymphatic tumor and it could be the tumor maker. |
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