Mouse Foxp3 Recombinant Protein Expression, Preparation Of The Poly-antibody And Research On The Expression Of Foxp3 In Model Of Type 1 Diabetes Mellitus

Objectives The Foxp3 fragment was amplified by polymerase chain reactions(PCR) and cloned into the prokaryotic expression vector pGEX-6p-2. The recombinant plasmid pGEX-6p-2/ Foxp3 was transformed into E.coli BL21(DE3) to be expressed. The antibody was obtained by immunizing New Zealand rabbits with the purified fusion protein as antigen. To induce type 1 diabetes mellitus of mouse by STZ. To detect the Foxp3 expression in mouse's spleen cells by Western blot with the antibody. To provide foundation for further study of Foxp3.Methods (1) Expression, purification of mouse Foxp3 recombinant proteinand and preparation of the poly-antibody The segment (532～1008bp) from mouse Foxp3 gene was selected as target gene by software analyses and the target gene encodes 159 amino acids that has better antigenicity and high specificity. The Foxp3 segment was amplified from recombinant plasmid pMIGR1/Foxp3 by polymerase chain reactions(PCR) and cloned into the prokaryotic expression vector pGEX-6p-2. The recombinant plasmid pGEX-6p-2/Foxp3 was transformed into E.coli BL21(DE3). The expressed fusion protein GST-Foxp3 was analyzed by SDS-PAGE and Western-blot, and was purified with GST Sepharose F.F. affinity chromatography. The concentration of the purified protein was determined by the BCA protein assay kit, and the fusion protein was used to immunize New Zealand rabbit. The anti-serum was evaluated by indirect-ELISA and the immunogenicity of the fusion protein was analyzed. The specificity of poly-antibody was analyzed with the NIH3T3 cells transfected by pMIGR1/Foxp3 or pMIGR1. (2) Research on the expression of Foxp3 in the model of type 1 diabetes mellitus. Type 1 diabetes mellitus of mouse was induced by STZ. The status of mouse with food and drink was observed, and body weight, urine glucose and serum glucose was detected in regulary, CD4+T and CD4+CD25+T cells of mouse's spleen cells were detected by Flow cytometry. The Foxp3 expression of mouse's spleen cells was detected at the mRNA level by RT-PCR and at protein level by Western-blot.Results The PCR amplification product was about 477bp. Restriction enzyme digestion analysis and sequencing showed that the amplified sequence have 100% homogeneity and the amino acids were 100% similarity with the published sequence by GenBank. Constructed recombinant plasmid was transformed into E.coli BL21 and the fusion protein was expressed by IPTG induction. The expression products were analyzed by SDS-PAGE and Western blot. The results showed that the molecular weight of the expressed protein was about 45kDa similar to the deduced value. After immunization for 4 times to New Zealand rabbits with recombinant protein, the specific antibody titer above 1∶12 800 was obtained. The Western-blot results showed that total protein of NIH3T3 cells transfected by pMIGR1/Foxp3 was identified specificly by preparated antibody, but the total protein of NIH3T3 cells with pMIGR1 did not be recognized by Foxp3 antibody. The model induced by STZ showed a representative manifestation of type 1 diabetes with polydipsia, polyphagia, urorrhagia and body weight loss. The level of serum glucose in the model was stepping up remarkably after inducing with STZ one month, and the areas of Langerhans show different degree insulitis. CD4+T cells of mouse's spleen cells had been proliferating more significantly in the model than the other groups. CD4+CD25+T cells had been proliferating slightly in the model. But the ratio of CD4+CD25+T/CD4+T was lower in model than the other two groups. In the model, the mRNA ratio of Foxp3/β-actin and the protein ratio of Foxp3/G3PDH were slightly higher. Conclusions(1) Prokaryotic expression vector pGEX-6p-2/Foxp3 was constructed successfully and the target protein was expressed in E.coli BL21;(2) The purified recombinant protein could induce the New Zealand rabbits to produce high titer specific polyclonal antibodies;(3) In the model, the expression of Foxp3 was slightly higher but not still to remain the normal ratio of CD4+CD25+Treg/CD4+T to inhibit generation of responsive CD4+T cells.