Expression And Examination Of A Novel LAPTM4B Gene In Tumor Cells, Cloning And Analysis Of LAPTM4B Gene Putative Promoter Region

LAPTM4B, a novel oncogene candidate,encodes two isoforms of proteins with molecular weights 35 kDa and 24 kDa, respectively. LAPTM4B-35 is the product of translation initiating from the first (nt 157) ATG in LAPTM4B ORF, but LAPTM4B-24 is the product of translation initiating from the second (nt 430) ATG in frame. The expression levels of LAPTM4B-35 protein in HCC tissues were dramatically upregulated, but LAPTM4B-24 protein not. A 297bp sequence at the 5'end of LAPTM4B cDNA was obtained by PCR and inserted into prokaryotic expression vector pGEX-KG. Then the constructed recombinant plasmid pGEX-KG-N1-99 was transformed into E.coli JM109 to express GST-fusion protein. The recombinant expression system was confirmed by restriction endonuclease digestion and DNA sequencing. E.coli cells were lysed by ultrasonication and the fusion protein was purified by Glutathione SepharoseTM 4B agrose. The purified GST-LAPTM4B-N1-99 was characterized by SDS-PAGE, and used to immunize Balb/c mice . The titer and specificity of antisera were detected by ELISA and Western blot, respectively. Western Blot analysis showed that the monoclonal antibody reacted specifically to the fusion protein of LAPTM4B and its nature protein of tumor cells.The expression of LAPTM4B in tumor cells was examined by technology of Western-blot and immuno-histologic chemistry(IHC). The expressions of LAPTM4B-35 in other 5 cancer cell lines were confirmed via immunohistochemical analysis. To explore the possible mechanisms underlying deregulation of LAPTM4B in HCC, the promoter of LAPTM4B was cloned and analyzed. The LAPTM4B cDNA was used as the template in the polymerase chain reaction to amplify its various upstream regions from the translation initiation codon. The PCR products were directly cloned into the Luciferase reporter vector pGL3-Basic. BEL7402 and HLE Hepatocellular carcinoma cell lines were transiently transfected with above recombinant plasmids, and the activities of putative promoters were analyzed by Luciferase assay. Restriction enzyme digestion and nucleotide sequencing confirmed that the recombinant plasmid was complete correct without any base mutation and deletion. Our results showed that the highest promoter activeity of LAPTM4B gene is within the 231bp DNA fragment. Transient transfected assay revealed the basic promoter within 231bp upstream of transcription initiation codon, and it was the core promoter of LAPTM4B. Luciferase reporter assays showed a distinct difference in the promoter activities of LAPTM4B gene between BEL7402 and HLE cell lines. It has been suggested that the transcription factors responsible for regulation of the gene in the two cell lines are different, and that possible negativeregulatory cis-elements may exist upstream the promoter region. In conclusion, the recombinant plasmid PGE-KG-N1-99 containing 297bp sequence at 5' end of LAPTM4B cDNA has been constructed, fusion protein GST-LAPTM4B-N1-99 is successfully expressed and purified.The monoclonal antibody LAPTM4B-N1-99(LAPTM4B-N1-99 McAb) directly to N-terminal region of LAPTM4B-35 rather than to LAPTM4B-24 is prepared with genetic engineering techniques. LAPTM4B-N1-99 McAb could specically reacted with LAPTM4B expressed by prokaryotic and eukaryotic cells, so that it could be used in the exploration of biological function and assay of LAPTM4B. In other side, the possible promoter of LAPTM4B is primarily identified, which paves the way to further investigate the function of LAPTM4B-35 .