| Macrophage migration inhibitory factor (MIF), a cytokine controlled by the hypothalamus-pituitary axis, is almost expressed in all tissues and mainly secreted by pituitary anterior lobe, activated T lymphocytes, mononuclear phagocytes, and so on. hMIF cDNA is a sequence of 353bp, and subsequently translated into a non-glycosylated protein of 115 amino acid residues, 12.5kD in molecular weight. hMIF has multiple functions which are similar to those of cytokines, hormones, growth factors, stress reaction proteins, glycosylated inhibitory factors (GIF), and what's more, it plays an important role in the regulation of immunity. Many studies have proven that hMIF can improve the incidence and development of tumor and some inflammatory and autoimmunity diseases such as septic shock, rheumatic arthritis, etc.. In this way, monoclonal antibody against hMIF must have potential effects on the treatment of above-mentioned diseases. Besides, hMIF was identified to accelerate tissue reparation in trauma disease. So hMIF and its corresponding monoclonal antibody have extensive application in following aspects: the repair of tissue lesion, the regulation of immunologic system, and the diagnosis, therapy and prognosis of tumor and infective, autoimmunity diseases, etc..Following is main content and results of our research:1. Gene cloning and prokaryotic expression of hMIF(1) After a couple of primers were designed according to the published hMIF cDNA sequence, the cDNA was amplified from human breast cancer cell line MDA-MB453 and human breast cancer tissue with RT-PCR technique. After sequencing was used to confirm the cloned 353-bp sequence is identical with that in GenBank, the sequence was inserted into prokaryotic expression vector pET11b.(2) The E.coli. BL21 transformed with the recombinant vectors were cultured and induced with IPTG to express hMIF protein. Tricine SDS-PAGE electrophoresis proved thatan about 12.5kD soluble recombination protein was expressed and the proportion of the protein was about 30% of the total protein of induced bacteria.(3) The E coli. BL21 haboring the recombinant vector of pETllb/hMIF was fermented with IPTG induction and lysated with sonic wave.After purified by cation exchange chromatography, and then hydrophobic chromatography, 53mg hMIF with purity of 95.40% and concentration of 1.145mg/ml was obtained from lg wet bacterium. The results of Western blotting verified that specific reaction was observed between the purified hMIF and the gifted mouse anti-hMIF monoclonal antibody.2. Preparation of anti-hMIF monoclonal antibody(1) The purified hMIF was used to immunize Balb/c mice. After 5 times of cell fusion between the spleen cells of the immunized mice and the myeloma cell SP2/0, two stains of hybndoma cells hMIF-A2 and hMIF-D, stably secreting the antibody were screen with the aid of indirect ELISA and cloning. The titers of the supernatants of these two strains were 1:3200 and 1:6400 respectively.(2) The karyotype analysis of these two strains indicated that the chromosomes of fused cells had the features of their two parent cells, and its number ranged from 80 to 102, which confirming that the strains were absolutely fused from two parent cells, mouse splenocytes and SP2/0 cells. Antibody subtypes was identified to be Igd and IgG3 in their heavy chain, and k in their light chains. The reaction of specificity and the reaction with special antigen showed that these two antibodies had strong specificity, and only reacted to hMIF without any cross- reaction with other proteins.In this study, a large amount of hMIF with high purify and two strains of hybridoma secreting anti-hMIF monoclonal antibody were obtained, which may found a basis for further studv on MIF mechanism and the diagnosis and treatment of relative diseases. |
