Cloning and expression of mouse Ii gene and preparation of its antibody

For cloning and sequence analysis on expression of mice Ii gene and preparation of its antibody,Ii gene was cloned from RNA by RT-PCR with a pair of designed primers;After sequence and homology analysis,this fragment was inserted into pGEX-4T-1 plasmid,and transformed into the strain BL21.Then the bacteria were induced and cultured,expressed protein was observed on SDS-PAGE;Fusion protein was extracted and purificated,which was immuned the rabbits,antiserum was collected.As a results,the length of this cloned fragment was 700 bp.After blastn in GenBank,the mouse Ii gene had a similarity of 99.2%～99.8% to the Ii gene in GenBank;A band with 495000 of molecular weight was observed on SDS-PAGE;The results of Agar diffusion assay,ELISA,Western Blotting and indirect immunofluorescent method indicated that the fusion proteins had strong antigenicity and biological activation.Ii gene was cloned successfully,the recombinant plasmid was constructed and the fusion protein was expressed in prokaryotic cells and purified,and the specific antibodies against Ii was prepared.