A Preliminary Study Prokaryotic Expression And Cellular Distribution Of AIF-1 Genes

Allograft inflammatory factor-1(AIF-1), also known as ionized calcium-binding adapter molecule1(Ibal), was initially cloned from rat cardiac allografts undergoing chronic rejection. So far, the AIF-1gene was cloned from many species, such as human, mouse, carp, sponge and disk abalone. The AIF-1protein was presented at high levels in the spermatids of rat and low levels in some other tissues and serum. AIF-1has actin-crosslinking activity, which plays an important role in cell skeleton’s rearrangement of macrophage/microglia. The AIF-1could be induced in vascular smooth muscle cells (VSMCs) when stimulated by artery injury or inflammation, which promoted the proliferation and migration of VSMCs. In addition, upregulated expression of AIF-1was detected in the ascites of endometriosis patients and synovial fluid of rheumatoid arthritis patients. The distribution mechanism and correlation with pathological process of AIF-1need further research.Firstly, AIF-1gene was obtained from the testis cDNA library of the tufted deer (Elaphodus cephalophus). Including5’and3’untranslated regions, the cDNA spans an open reading frame from nucleotide81to518, encoding a145-aminoacid protein. Primers were designed according to cDNA sequence, and the gene was cloned into E.coli expression vector pET-28a (+) and induced to express, then the recombinant protein was purified. Results showed that, after induced by lmmol/L IPTG for4hours, a clear band which was similar to the theoretical size at20kDa was detected by SDS-PAGE. Through Western Blot, the recombinant protein could be recognized by anti AIF-1antibody, which indicated that it was expressed successfully. The recombinant protein was detected mainly in the supernatant after ultrasonication and centrifugation, and could be used to prepare antibody for subsequent experiments.Secondly, the AIF-1mRNA and protein levels in different cell lines was analysed by RT-PCR and Western Blot. The result showed that, the expression of AIF-1is higher in HL-60cell line, while lower in Jurkat cell line. The AIF-1cDNA was cloned from the HL-60cell line, and used to construct the eukaryotic expression vectors including pEGFP-C3-AIF-1, pEGFP-N1-AIF-1. To trace the distribution of AIF-1protein by EGFP, different cell lines including Hela,293and MCF-7were transfected. After fixing and staining, the cells were observed under fluorescent microscope.The results showed that, compared with the empty vectors, over expression of exogenous fusion protein affected the distribution of AIF-1in the cytoplasm and the nucleus.Finally, Sequences comparative analysis confirms that AIF-1of tufted deer is a member of AIF-1family and has high evolutional conservation among different species. The phylogenetic analysis shows that the tufted deer has close genetic relationship with cattle and goat, which is consistent with the traditional classification method.