Influence of inserting glycines on biological properties of HIV Tat-(Gly)n-thymidine kinase fusion proteins / 第二军医大学学报

ABSTRACT

Objective: To study the influence of inserting glycines(Gly) on biological properties of HIV Tat-(Gly)n-thymidine kinase (-TK) fusion proteins. Methods: Different fragments containing 0, 2, 4 or 6 Gly were inserted between the HIV Tat gene and TK using gene splicing by overlap extension (SOEing) PCR, and the products were cloned into PBK vector. The vectors were then transferred into E. coli after sequencing. After IPTG induction, bacilli were collected and destructed by ultrasound; the fusion protein was collected and identified by monoclonal antibody of HIV protein. HepG2 cells were incubated with DMEM supplemented with 1 μg/ml fusion protein containing 0, 2, 4 or 6 Gly for 24 h. HepG2 cells of different groups were detected by immunofluoreseence assay with HIV Tat monoclonal antibody; the apoptosis rate of HepG2 cells was determined by cell flow cytometry after they were incubated with gencilovir (10 μg/ml) for 3 d and the survival rate of cells was recorded by trypan blue in different groups. Results: The recombined genes containing 0, 2, 4 or 6 Gly were successfully constructed, inserted into PBK vectors, and expressed into E. coli. Their proteins were obtained and purified. The level of fluorescence in different groups was similiar, but the cell survival rate and apoptosis rate were different. The highest apoptosis rate was 14.77%, which was found in the group containing 4 Gly, followed by 12.69% in 2 Gly group, 8.31% in HIV Tat-TK group, 4.36% in 6 Gly group, and 1.0% in group containing no Gly. Significant differences were found between each 2 groups (P<0.05). Trypan blue showed similar results in the cell death rate of different groups the highest cell death rate was 80.2%, which was found in the group containing 4 Gly, followed by 65.4% in 2 Gly group, 58.4% in HIV Tat-TK group, 56.7% in 6 Gly group, and 9.1% in the group containing no Gly. Conclusion: The number of Gly inserted into HIV Tat-TK protein does not alter the transcellular function of upstream Tat protein, but does substantially influence the TK protein-mediated cytoxic effects of gencilovir, and the influence is the smallest when 4 Gly are inserted.