Cloning And Expression Of Mouse Ii Gene And Preparation Of Their Antibodies

Invariant chain (Ii), a nonpolymorphic typeâ…¡transmembrane glycoprotein and chaperone of MHCâ…¡molecule, plays a key role in presenting exogenous antigen reaction. Firstly, it can facilitate the construction of the MHCâ…¡molecule. Secondly, it can prevent peptide from associating prematurely with MHCâ…¡molecule; Thirdly, it can provide a targeting signal for endosomal/lysosomal compartments. Research indicated that Ii molecule has a close relation in proliferation and maturation of CD4⁺T cell and B lymphocytes cell. In this experiment, the Ii gene of mouse was cloned and expressed, then the polyclonal antibodies was prepared in order to elucidate the function of Ii in the immune reaction and its mechanism.Firstly, according to mRNA of mouse Ii gene sequence registered in GenBank and multi-cloning restriction enzyme sites of vector pGEX-4T-1, a pair of specific primers(P₁,P₂) were designed and synthesized. Using total RNA from mouse spleen, one specific gene was amplified by RT-PCR. The endonuclease digesting result showed that the target gene has the same restriction enzyme site as the known mouse Ii. Then the gene was inserted into vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1-Ii was identified by endonuclease EcoRâ… +Salâ… digestion and PCR. Then, the positive recombinant clone was sequenced and analyzed. These results showed that the gene of mouse Ii has been successfully cloned from mouse spleen. The target gene was 700bp in length, and contained a complete ORF of mouse Ii which encodes 213 amino acids. The homologous of the nucleotide sequence cloned by this study with the reported mouse Ii gene is higher than 99.2%.Secondly, the constructed recombinant plasmids pGEX-4T-1-Ii was induced by IPTG at 37℃after transformed into E.coli BL21. The MW of the expressed protein was about 49.5KD as analyzed by SDS-PAGE. The identity of the fusion protein solubility showed the target protein GST-Ii located in inclusion bodies. Those results suggest that the gene of mouse Ii has been successfully expressed in prokaryotic expression system.Finally, we determined the optimum inducement time and concentration of isopropylthio-β-D-galactoside (IPTG) after optimizing expression conditions. The recombinant plasmid pGEX-4T-1-Ii was induced to express large-scale GST- Ii fusion protein, the induced recombinant bacteria were lysed by freeze-thaw and sonication. We obtained the GST-Ii inclusion body protein, which could be solubilized by sonication after the detergent lauroylsarcosine was added. At last, the polyclonal antibodies against mouse Ii chain was collected after rabbits were immunized with the purified GST-Ii proteins. The results of Agar diffusion assay, ELISA and Western Blotting indicated that the fusion proteins GST-Ii had strong antigenicity and biological activation.In conclusion, in this study the gene of mouse Ii was cloned successfully, the recombinant plasmid of pGEX-4T-1-Ii was constructed and the GST-Ii fusion protein was expressed in prokaryotic cells and purified, and the specific polyclonal antibodies against GST-Ii was prepared. All these would provide some experimental materials for the further studies on the immunity function of mouse Ii chain.