Molecular Cloning Of GSTP1 Gene Novel Variant AY887902 And Function Study

Transitional cell carcinoma of bladder was classed into 2 grades by professor Chang. In order to clarify 2 classes (grades) of bladder cancer on molecular biological characters, we used suppression subtractive hybridization technique to construct a cDNA subtractive library of human BTCC previous and got 5 anonymous ESTs, BQ135230 was one of them. For the sake of understanding the mechanism of BQ135230 and prospecting its application, we performed the experimentation described below. Objective:1: In order to obtain full length of BQ135230, identify and characterize its ORF, locating of chromosome and function by bioinformatics. Preparation of antibodies named anti-AY to AAX14401. 2: To explore the function of AY887902. 3: To study the expression and biological significance of AAX14401 in human bladder cancer. Methods:1: mRNA extracted from fresh bladder cancer tissue was amplified by smart RACE (rapid amplification of cDNA ends) to get the 5' flanking sequence and 3' flanking sequence of BQ135230 and obtained full length of AY887902 by assembling the 5' flanking sequence and 3' flanking sequence of BQ135230 (performed by Gene tool procedure). 2: AY887902 and AAX14401 was analyzed by ORF Finder procedure, e-PCR procedure, Blastn, Blastp procedure which were provide by NCBI and ExPASY-ProtParam Tool , JUFO, the result was quilted by Sequin procedureand applied for GenBank accession number. 3: AAX14401 showed significant deviations of GSTPl at amino acid residues 40 to 52 which we prefabricated a peptide and injected into mouse to make its polyclonal antibody. 4: The inserted sequence: ORF cDNA of AY887902 was derived from Reverse transcription polymerase chain reaction (RT-PCR) amplification of RNA. The upstream primer contained BamHI restriction site and the start codon ATG, whereas the downstream primer contained the stop codon TAG and Apal restriction site. The PCDNA-AY plasmid was constructed by ligating the BamH I- Apal-digested cDNA inserts into the PCDNA3 plasmid digested with the same enzymes. E. coli was transformed with the constructed PCDNA-AY plasmid and selected on ampicillin LB agar plate. The structure of E. coli-derived plasmid was verified by restriction analysis using double digestion of BamHI and Xhol 5: EJ cells were transfected with PCDNA-AY plasmid and PCDNA3 plasmid by Lipofectamine 2000, it was named EJ-PCDNA-AY cell and EJ-PCDNA cell, respectively, the transient expressive were detected by RT-PCR and immunohistochemistry. 6: The cell line: EJ cell, EJ-PCDNA cell and EJ-PCDNA-AY cell were maintained in RPMI-1640 with or without Vincristine and cultured in an incubator at 37°C and with 5% C02 , Flow cytometry analysis was used to detect cell cycle and apoptosis meanwhile MTT to detect alive cell after transfection. 7: Immunohistochemistry was carried out to detect the expression of AAX14401 and GSTPl for 75 transitional cell carcinoma of bladder and 12 normal bladder mucosa. Results:1: Finally, 774bp products were obtained by assembling the result of RACE, it has a ORF and acquired GenBank accession number: AY8879O2; Chromosome location: 1 lql3, Protein ID:AAX14401; Homologous assay showed that AY887902 was a novel variant of GSTPl gene and it exhibited T-to-A and G-to-A transitions at nucleotides 193 and 194 respectively and resulted in AAA(coding lysine) changing into TGA(stop codon). The novel produce was a protein with 52 amino acid residues comparing with the normal counter part of 210. No transmembrane regions or signal peptide were detected using bioinformatics methods. 2: ELISA results suggested that the polyclonal antibody was eligibility to detect AAX14401. 3: The ORF cDNA of AY887902 was inserted into the PCDNA3 plasmid exactly. The specific expression vector (PCDNA-AY) for AY887902 gene was constructed successfully. 4: PCDNA-AY was transfected into EJ cell successfully, and the expression of AY887902 was detected 48 hours after transfecting distinctly. The apoptosis and the number of alive cell showed no significantly difference in same group among cell line: EJ, EJ-PCDNA, EJ-PCDNA-AY. (the cells classed into two groups according to the cells cultured with Vincristine or not) 5: AAX14401 positive staining was detected in 2 tumor tissue while GSTPl expression was negative. The positive expression rate of AAX14401 and GSTPl in bladder cancer was 2. 7% and 90. 7% , respectively, and the negative correlation between AAX14401 and GSTPl in bladder cancer, the rate of AAX14401 and GSTPl positive staining in normal bladder tnucosa was 0% and 58. 3%, respectively. Conclusion:...