Cloning And Expression Of FHIT Gene And Anticancerous Effects Of FHIT Protein

Tumour, intrinsically a kind of gene disease, grows long-termed, multi-phased and multi-gened. Lung cancer is a common malignant tumor in China, many oncogenes and / or tumor suppresser genes are involved in the initiation and progression of lung cancer. FHIT (Fragile histidine triad) gene, a newly discovered tumor-suppresser gene, main functions of anticancer are FHIT protein and its combination with other proteins. The study cloned and expressed FHIT gene and observed the anticancerous effects of FHIT protein. The main tasks and results are as follow:1.Cloning and identification of ORF(Open reading frame) of FHIT gene.To clone and isolate the coding sequence ORF of FHIT gene by RT-PCR, we extracted total RNA from normal fresh thyroid gland's organization. A FHIT gene recombinant prokaryotic expression plasmid was constructed by cloning the coding sequence into a prokaryotic expression vector PET-28a(+) at the sites of Ndel and Sail, and named PET-28a(+)-FHIT. After the recombinant was amplified by transforming into DH5a, it was cleaved with appropriate endoneucleases and sequenced . The results showed that the orientation ofthe insert was found to be correct, while no rearrangement or mutation was found.2.Expression and identification of FHIT protein.The recombinant was transformed into E.coli BL21(DE3). The expression of the protein in the E.coli was induced with IPTG. The results showed that the recombinant was stable in the E.coli BL21(DE3), that the aim protein induced by IPTG could be expressed efficiently and the highest production of expression at 5 hour after inducement, that the aim protein existed in the form of "inclusion bodies", and that the aim protein was identified by Western blot.3.Purification and renaturation of FHIT protein.The aim protein was purified successfully with 6 the column of His, and the renaturation of the protein was completed by dialysis.4. The growth inhibition of FHIT protein on human lung adenocarcinoma cells Anip973.The cells were cultured with the fusion protein. The viable cells were quantitated by MTT assays, from which we found that the fusion protein significantly suppressed the growth of Anip973 cells and that The effect of the fusion protein increased with the increasing FHIT fusion protein .The above results showed that FHIT protein's expression by prokaryotic expression system is a reliable, effective and handy way. FHIT protein has a dramatically suppressed effect on the proliferation of human lungadenocarcinoma cells Anip973 in vitro. It proved that FHIT protein can suppress tumor growth. Our study provided experimental and theoretical data for the basic and clinical study on carcinoma .