Construction Of Survivin And Its Truncates' Expression Vectors And Functional Role Of Related Proteins

Survivin is a novel member of inhibitor of apoptosis (IAP) protein family. The Survivin gene spans 15 kb, and is located on chromosome 17 at band q25, and consists of four exons and three introns and encodes a protein of 142 amino acids, with a molecular weight of approximately 16.5 kDa. Survivin is expressed in embryonic tissues as well as in the majority of human cancers, but is not expressed in most normal adult tissues. The cancer-specific expression of Survivin, coupled with its importance in inhibiting cell death and in regulating cell division, makes it a useful diagnostic marker of cancer and a potential target for cancer treatment.Recently, there is emerging evidence that Survivin has attracted growing attention. There are multiple strategies to utilize Survivin gene as a cancer therapeutic target as Survivin is overexpressed in cancer but undetectable in normal, differentiated adult tissues.It has been demonstrated that the expression of Survivin in cancer associates with poor prognosis, cancer progression/recurrence, drugresistance, and a short patient Survival rate. These observations triggered enthusiasm in the scientific community for using Survivin itself as a target for cancer therapeutics. The current approaches are summarized below: Survivin dominant negative mutant approaches.;Survivin ribozyme approach.;Survivin RNA interference (RNAi) approach;Approaches using small organic compounds or other small antagonists such as small peptides. Survivin-derived peptide-specific cancer immunotherapy.Comparison with the strategies above, we sought to an approach not only safety but also efficiency, in this study, we made two truncates of Survivin at its N- terminal depending on its structure and function characterization. First, the three residues Leu6, Pro7? TrplO of the N-terminal is the key residues of the dimmer, so if cut off part of them may be fail to form a dimmer. Second, Survivin5-14 is an epitope for HLA-A2. According to the two points we decide to make a truncate of SurvAN7 and as all know Thr34 is a site of CDC2/cyclinBl-specific phosporylation, so if cut off it may be gain a truncate of survivin no function of inhibition of apoptosis. In addition, the 38th residue's codon is ATG, So, we decide to make other truncate of SurvAN37. We anticipate that the two truncates SurvAN7 and SurvAN37 can not inhibit apoptosis, at the same time, we mutated amino acid Thr34 to Ala as a control.Surviving, SurvAN7, SurvAN37 and dominant negative mutant SurvT34A were cloned into the VR1012 mammalian expressive vector. The activity of Survivin, SurvT34A, SurvAN7 andSurvAN37 to affect cell apoptosis was assayed by transfecting Hela cells and using Trypan Blue exclusion method, DNA fragment assay and caspase-3 activity assay. The result of this three method confirmed that SurvAN7 and SurvAN37 just as SurvT34A can not inhibit apoptosis.In conclusion, in this study we gained two dominant negative truncates SurvAN7 and SurvAN37. We think they are can be used for tumor gene therapy in the future.At the same time, we constructed another plasmid of pRSET B-Surv for protein expression, and gained high liter anti-Survivin mAb by immunization rabbit using purification recombinant Survivin protein, and used it in this study for prime antibody.