| Purpose and background The most part of HCC( hepatocellular carcinoma) cases have the background of liver cirrhosis, the differential expressed genes between the tissues of HCC and liver cirrhosis may be the cause of HCC developing and progressing. SSH ( suppressive subtractive hybridization, SSH) is a quick, effective and sensitive method to obtain these genes, P02 EST (Expressed sequence tag ) is one of these genes highly expressed in HCC tissue through SSH screen between HCC and liver cirrhosis tissues. Northern blot confirmed that this result is authenic. Furthermore, RT-PCR demonstrated that P02 is highly expressed in almost all the HCC cases. BLAST analysis found that P02 is highly homologous with gene TPT1. It is demonstrated that there are a lot of pseudogenes of TPT1 in mammalian genome, also, these pseudogenes show transcription activity in some degree. The protein product of TPT1 gene is called TCTP( translationally controlled tumor protein) , the initial study showed that TCTP is a protein which can promote cell growth. Recently, some researchers reported that the anti-apoptosis effect of this gene and the inversed effect on the maligant phenotype of tumor cells when it is down-regulated. Western blot showed that fortilin, the highly homologous analog of TCTP, is strongly expressed in tumor cell lines comparing with normal cell lines. The origin of P02 and theresearch results of its homologous analogs imply: the high expression of P02 relates closely with tumor, but until now, there is no direct evidence demonstrated that the high expression of P02 and its homologous analogs resulted in the developing and progressing of tumor. To test the carcinogenesis of P02 and find new therapy target for HCC, we plan to clone the full length cDNA of P02 based on the known sequence of P02 EST, and find whether there are pseudogenes or mutation of P02 in HCC. Then we insert the ORF (open reading frame) of P02 full length cDNA into the eukaryotic report expression vector pEGFP-N3, this construct is called pEGFP-N3P02, and the pEGFP-N3P02 is transfected into cell line SMMC-7721 and L-02, respectively, the changes on the biological behaviors of liver cell lines were observed when P02 is overexpressed.Methods According to the polyA structure and the adding tail signal of the P02 EST, we think that the P02 EST is located at its 3' end, and the primers for 5' and 3' RACE (rapid amplification of cDNA ends) were designed accordingly. SMART (Switching Mechanism At 5' end of RNA Transcript) RACE was used to amplify the 5' and 3' ends of P02 in the HCC tissue. TA clone was used to ligate the amplifying products into pT-Adv vector, the clones were double selected with aminobenzylpenicillin and colour screen. Five clones were selected, their plasmids were extracted by miniprep, checked to contain the inserter by PCR and restricted map, and then send for sequencing. The sequences were analysed through online biosoftware VecScreen, BLAST was used to find out the overlapping region between the known P02 EST and the inserter, after being omitted the overlapping region, the inserter was ligated with the known P02 EST together , this resulting sequence was analysed according Kozak rule to determine whether it is a full length cDNA, and sent to Genbank to find its homologous sequences through BLAST, then biosoftware is used to find its ORF and predict its structure, function,subcellular localization, physical and chemical properties of its coding protein. The specific recognize sites of EcoRI and BamHI were introduced respectively into the two ends of P02 ORF through PCR method, this P02 ORF PCR products and the vector pEGFP-N3 were then doubled digested with EcoRI and BamHI restriction enzyme respectively and then ligated together, kanamycin was used to screen the positive reconstituted vectors, plasmids were extracted by miniprep and checked through PCR and digestion with restriction endonuclease , and then sequenced using Sanger method. VecScreen and BLAST were used to analyse the sequence to confirm that the P02...
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