Objective: To establish p53 trancated muteins group and analyse their expression and effects on p53 related downstream genes in HeLa cells. Methods: Using PCR-based site-directed mutagenesis technique, a group of P53 muteins with the N- and/or C-terminals differently trancated were set up. The plasmids with p53 mutein genes were used to transfect HeLa cells, the p53 muteins and some p53 related downstream genes' expression were analyzed by RT-PCR. Results: The p53 muteins were expressed in HeLa cells and they had effects on some of p53 related downstream genes. Conclusion: The P53 muteins may be a good tool to analyze P53 function and has effects on p53 related downstream genes' expression model.

Aim To analyze THANK gene expression in peripheral blood mononuclear cells(PBMC) stimulated with different stimulators and to clone whole length human THANK gene. Methods PBMC were conventionally isolated and cultured in RPMI1640 containing 10％ FCS. After stimulated with LPS,TNF α ,IL 2,IFN γ ,PHA or PMA,the THANK gene expression in PBMCs was analyzed by RT PCR and THANK cDNA was cloned. Result RT PCR detection showed that THNAK gene expressed in PBMCs after stimulated with interferon γ for 3 days, whereas THANK'expression could not be detected after stimulated with LPS,TNF α ,IL 2,IFN γ ,PHA or PMA respectively. Then THANK gene was cloned by cloning PCR product and sequenced. Conclusion Human THANK gene is cloned successfully, thus providing the possibility for further research of THANK'function.

An recombinant vector pCSV-EPO for expression of EPO cDNA in mammalian cells was constructed by the techniques of gene recombinant, PCR amplification and region-specific mutagenesis. The pCSV-EPO was introduced into COS7 cells by DEAE-dextran-mediated transfection. The expression of the EPO was demonstrated by EPO-ELISA assay. At 48h post transfection, the EPO level was 25ng/ml and 72 h was 17ng/ml.

Objective: To prepare highly purified THANK protein. Methods: THANK was efficiently expressed in E.coli as inclusion bodies. After bacteria were lysed under ultrasonication, TE buffer,1% TritonX and 2 mol /L urea was used to efficiently extract inclusion bodies. After denaturation with 8 mol/L urea,THANK was partially purified by Sephacryl S-200 chromatography, and then subsequent refolding step was optimized for a maximal recovery of biological active protein. The renatured THANK was purified to homogeneity with Q Sephadex Fast Flow gel filtration and then desalted by Sephadex G-25 chromatography. Results: The purity of biologically active THANK was above 97% in SDS-PAGE densitometric studies. Conclusion: An effective method of denaturation, renaturation and purification of recombinant THANK is established.

Objective: To clone THANK gene and express its extracelluar fragment. Methods: Using RNA isolated from HL 60 cell lines, THANK cDNA was amplified by RT PCR. The fragment was linked to pMD18 T vector and sequenced, and then the extracellular fragment of THANK was subcloned into pET vector and THANK protein expression was induced.Results: A 858 bp DNA fragment was amplified and the cDNA sequence was identical with the published sequence encoding THANK gene. Western blot showed that THANK protein with a relative molecular weight of 2.6?10 4 was expressed. Conclusion: Human THANK gene was cloned and expressed successfully, which provides a base of further research of THANK gene. [