Construction Of Eukaryotic Expression Vector Containing Coding Region Of Human IASPP Full-length CDS And Its Expression In Colon Carcinoma Cell Lines And Identification Of Biological Function

Objective:The p53 is one of the best-known anti-oncogenes. The new discovered protein ASPP (apoptosis-stimulating protein of p53) can specific regulated the tumor suppress of p53. iASPP (inhibitory member of the ASPP family) can interfere the apoptosis of p53 through binding to the p53, so it is the important inhibitor of p53. The overexpression of iASPP was observed in several kinds of human tumors. This fact indicates that reducing the high expression of iASPP may be a new strategy to resume the abilities of p53 tumor suppressor. The iASPP was originally reported as a protein of 351 amino acids. We original cloning experiment showed that iASPP (351aa) was not the full-length iASPP. The use of bioinformatics revealed that full-length of iASPP is a protein of 828 amino acids. The short iASPP is a splice variant of full-length iASPP. Because previous studies were based on the shorter iASPP, but the full-length iASPP is the main form in vivo, so it's essential to study the function of full-length iASPP, and the work to clone full-length iASPP is the first step to do this. To investigate the function of iASPP, we use three-step PCR technique to clone the full-length CDS of iASPP. We construct the eukaryotic expression vector of full-length iASPP and transfect it into colon carcinoma cell lines by liposome. Then we observe the expression of iASPP by RT-PCR,Western Blot and detect the cell apoptosis by flow cytometry. All above studies laying a foundation for further study on the function of iASPP.Methods:1. Leukemia cell lines HL-60 was cultured, then its total DNA and RNA were extracted.We use three-step PCR technique to clone the full-length CDS of iASPP and the iASPP cDNA was amplified by PCR, and then analyze the sequence.2. The amplified PCR product was digested and inserted into pMD19-T simple vector and subcloned into eukaryotic expression vector pcDNA3.1(+).Then the recombinant eukaryotic expression plasmid containing CDS region of iASPP was constructed.3. The recombinant eukaryotic expression plasmid pcDNA3.1(+)-iASPP was transfected into colon carcinoma cell lines sw480 and lovo by liposome, the iASPP expression was analyzed by RT-PCR and Western Blot .The cell apoptosis was detected by FCM.Results:1. When we sequencing the clone product of shorter iASPP obtained by PCR repeatly,we confirmed that there are three sites were not consistent with the original report sites. By use of BLAST in PUBMED, we found there are a lots of clone fragment were consistent with our sequence, the correct sequence of full-length iASPP was discovered.2. The gene sequence of human iASPP gene's three fragments which obtained by PCR and RT-PCR was consistent with that reported in GenBank with DNA sequencing. The ligated DNA was long as 2516bp, and the sequence of ligated DNA was consistent with that reported in GenBank with DNA sequencing too.3.We obtained pMD19-T-iASPP plasmid by clone and the fragment was subcloned into eukaryotic expression vector pcDNA3.1(+), then the recombinant eukaryotic expression plasmid containing CDS region of iASPP was constructed. We named it as pcDNA3.1(+)-iASPP. Through sequence analysis, the gene squence of iASPP was consistent with that reported (gi 60457962) in GenBank .4.The mRNA expression and protein expression levels of iASPP gene of Sw480 and Lovo cell lines which transfect the positive plasmid were increased comparing with those of the cell lines which transfected the negative plasmid detected by RT-PCR and Western Blot respectively. The cell apoptosis rates of Sw480and Lovo cell lines were decreased comparing with those of the cell lines which transfected the negative plasmid detected by FCM.Conclusion:1.We confirmed that iASPP have variants in vivo and the original shorter iASPP is the splice variant of full-length iASPP. 2.The amplified reaction of iASPP cDNA was successfully. The recombination of full-length iASPP gene CDS region was successfully too. The recombinant eukaryotic expression plasmid pcDNA3.1(+)-iASPP containing CDS region of iASPP was constructed successfully.3 . The recombinant eukaryotic expression plasmid pcDNA3.1(+)-iASPP were expressed on colon carcinoma cell lines Sw480 and Lovo.4.The cell apoptosis rates of Sw480and Lovo cell lines were decreased comparing with those of the cell lines which transfected the negative plasmid detected by FCM. These results demonstrated that iASPP had some inhibitive effect to p53, These facts indicated that reducing the high expression of iASPP may be a new strategy to resume the abilities of p53 tumor suppressor.