| Asparagine Synthetase belongs to the family of aminotransferase, and it can be used for the catalytic synthesis of asparagine (ASN), which provides ingredient for synthesis of protein. Comparing with normal cells, the enzymatic activity of Asparagine Synthetase in some kinds of malignant lymphoblasts is too low to synthesize enough ASN, that makes these malignant cells rely on extracellular enviorenment. This study used prokaryotic expression plasmid PMS-ASNSalready constructed to express fusion protein MS2-ASNS, then we prepared anti-ASNS monoclonal antibody with cell conjugation and hybridoma technique.The result indicated that:1. MS2-ASNS fusion protein was expressed in 42℃and its molecμLar weight is 55 kDa. After SDS-PAGE electrophoresis separation, electricity elution, PBS dialysis purification, fusion protein was used to immunize BALB/C mouse.2. The efficiency of confluent cells with 50% PEG (Mr 3350) was 88%, and the proportion of positive clone was 2.3%. After screened for 3 times, three hybridoma cell strains producing anti-ASNS monoclonal antibodies (McAb) were obtained and named D10-9,F4-15,F4-16, respectively. Three strains of hybridoma secreting McAbs to ASNS can secret stably after vitro transfer of generation and recovery. Monoclonal Antibodies'subtypes were IgG1 and IgG2a respectively.3. The specificity of McAb and titer were identified by ELISA methods. The result shows that McAb has high specificity and the titer is: D10-9: 1:2×102 ;F4-15: 1:5×105 ;F4-16:1:5×105.ASNS is detected by western blot method in K562 and Hela .IHC technology was used to examine ASNS cytoplasmic expression in each kind of tumor cell line:liver cancer cell lines SMMC-7721,BEL-7402,HepG2 and stomach cancer cell lines SGC-7901,SGC-823 and lung,esophageal carcinoma,thyroid cancer tissue。In the research the anti-ASNS monoclonal antibodies was obtained and the expression of ASNS was examined in tumor cell lines, which established a basis for the following research of nasal type NK/T cell lymphoma. |
PDF Full Text Request