The Preparation Of Rabbit Anti-TSPAN33 Antibody And Its Role On Streptozotocin-Induced Diabetes In Mice

Background & Objective:Tetraspanin 33(TSPAN 33) is a member of TM4SF. It has been reported that TSPAN33 is overexpressed on activated B lymphocyte surface of autoimmune diseases, but not expressed or low expressed on many kinds of cell surface, such as in T cells, natural killers and unactivated B lymphocytes. High expression of TSPAN33 in B cell was shown in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), and non-Hodgkin’s lymphoma, and this characteristic can be used as a clinical diagnotic and therapeutic marker for these diseases. However, whether TSPAN33 protein plays a role in autoimmune diabetes is unknown. In the present study, we investigated if the expression of TSPAN33 on B cells can be used as an immune therapeutic target.Methods:1. Cloning, expression and purification of TSPAN33:Total RNA was extracted from PBMC by Trizol. After reverse transcription of cDNAs and PCR amplification of TSPAN33 gene, the gene was cloned into pET28a. The recombinant plasmid pET28a/TSPAN33 was introduced into competent E. coli BL21 (DE3), and the expression of the protein was induced, purified with Ni2+-NTA affinity chromatography column, then further eluted with 200 mmol/L imidazole and detected by SDS-PAGE.2. The preparation, purification and identification of rabbit anti-TSPAN33 antibody:A rabbit was immunized with 200μl (500μg/ml) TSPAN33 and equivalent Freund’s adjuvant, and boostered at the day of 3,7,14,24 and 48. The rabbit serum was obtained at the day of 60, and the antibody was purified with peptide affinity chromatography elution, the specificity of antibody was detected by western blot and the titer of antibody and was evaluated by ELISA.3. Induction of autoimmune diabetes in ICR mice:Twelve ICR mice were treated by Intraperitoneal injection of 0.2 mL (6 mg/mL) STZ for 5 days, and glycose, insulin and IAA in serum were monitored to evaluate the formation of autoimmune diabetes which was comfirmed by histopathology.4. Expression of TSPAN33 on B cell surface of STZ-induced or normal mice:Peripheral blood was obtained from STZ-induced mice or normal mice, and the expression of TSPAN33 on B lymphocytes was detected by flow cytometry.5. Depletion of TSPAN33+B by injection of anti-TSPAN 33:TSPAN33+B cells were depleted with 100μl 300μg/ml TSPAN33 antibody by intravenous injection. The proportion of TSPAN33+B cells, concentration of insulin and IAA in the serum were monitored at different time points.6. Effect of TSPAN 33 antibody on insulin expression in vitro:B lymphocytes were isolated from the normal ICR mice and STZ-induced ICR by MACS. The isolated B lymphocytes were co-incubated with MIN6, an islet cell line, with or without treatment of 10μl 300μg/ml TSPAN 33 antibody, and the expression of insulin gene was detected by qRT-PCR.Results:1. The cloned TSPAN33 gene was showed to be a band about 852 bp in agarose gel electrophoresis. Sequencing the gene indicated no mutation. Expression of the protein was induced in E. coli BL21 (DE3), and the protein was purified.2. The antiserum of the rabbit immunonized with TSPAN33 was obtained at day 60, and the anti-TSPAN33 antibody was purified with ultrafiltration and beads coupled with a TSPAN33-specific polypeptide. Specificity of the antibody against TSPAN33 was detected by western blot, and the titer of purified antibody was 1:64000.3. Monitoring the common parameters for diabetes indicated that the animal model of autoimmune diabetes was successfully established in STZ-treated ICR mice.4. Significantly higher proportion of TSPAN33+B cells was found in the STZ-treated mice (4.93±0.49%) than normal ICR mice (1.82±0.19%)%, (p<0.001), indicating the animal autoimmune diabete model is very similar to that of humans.5. Depletion of TSPAN33+B cells with 100μl 300μg/ml TSPAN33 antibody significantly improved the clinic parameters for diabets in the animal model, with elevated serum level of insulin (p<0.01) and decreased serum level of IAA (p<0.001) compared with the TSPAN33+B cell un-depleted group.6. Treatment of TSPAN 33 antibody increased the expression of insulin gene in TSPAN33+B cells and MIN6 co-incubation in vitro (p<0.05), compared with the nontreatment cells.Conclusion:In the present study, we successfully prepared rabbit anti-TSPAN33 polyclonal antibody and establsished an autoimmune diabete model in the ICR mice treated with STZ. Application of the antibody in the diabet model depleted TSPAN33+B cells, and furthermore, relived clinic parameters for autoimmune diabetes. Also, treatment of the antibody increased the expression of insulin in MIN6 and TSPAN33+B lymphocyte co-incubation. These results suggest TSPAN33+B cells can be used as a therapeutic target for human autoimmune diabetes.