Construction and identification of eukaryotic expression vector for tubulin cofactor A and its effect on proliferation of renal cell carcinoma cell line 786-0

Abstract

Objective To construct eukaryotic expression vector of human tubulin cofactor A (TBCA) and to study the effect of it on proliferation of renal cell carcinoma cell line 786-0.Methods The eukaryotic expression vectors of human tubulin cofactor A,pCMV-IRES-EGFP and pcDNA3.0-Flag,were constructed and transfected into 786-0 cells by liposome mediate method.TBCA protein level in 786-0 cells was detected by Western blotting.The effects of TBCA on proliferation of 786-0 cells were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method.Results The eukaryotic expression vectors of human tubulin cofactor A,pCMV-IRES-EGFP and pcDNA3.0-Flag,were constructed correctly,which was confirmed by restriction endonuclease digestion and DNA sequencing analysis.Western blotting indicated that TBCA was highly expression in transfected 786-0 cells (P ＜ 0.05).At 3,4,5 and 6 d after transfection,the recombinant plasmid group absorbance values were 0.52 ± 0.04,0.85 ± 0.04,1.20 ± 0.08,1.61 ± 0.11 ; no statistical significance compared with the control group (P ＞ 0.05).The colony forming efficiency in the recombinant plasmid group was higher than other groups.Conclusion The eukaryotic expression vectors of TBCA was constructed correctly and TBCA should not promote the proliferation of renal cell carcinoma cells. Key words: Renal cell carcinoma; Tubulin cofactor A; Proliferation