中文摘要：

目的：探讨插入甘氨酸（Gly）对HIVTat-TK融合蛋白功能的影响。方法：利用基因重叠（gene splicing by overlap extension，gene SOEing）PCR技术，将不同长度的甘氨酸密码子（0、2、4、6个）插入HIVTat-TK融合基因，经转染、鉴定证实后诱导表达，并经偶联Tat单克隆抗体的Sepharose CL-4B亲和层析柱纯化。4种HIVTat-（Gly）n-TK系列融合蛋白（n=0、2、4、6）、HIV Tat蛋白、TK蛋白（1μg／ml）分别与HepG2细胞在普通培养基共培养24h后，间接免疫荧光检测各自透过细胞膜效率；在加入更昔洛韦的培养基培养3d后，锥虫蓝染色计算细胞死亡率，流式细胞仪检测细胞凋亡率。结杲：精确克隆出HIV Tat-（Gly）n-TK系列融合基因，成功表达HIV Tat-（Gly）n-TK系列融合蛋白以及HIV Tat和TK蛋白。HIV Tat-（Gly）n-TK系列融合蛋白与HIV Tat蛋白透过细胞膜的效率相似，但单独TK蛋白无法进入细胞；在含有更昔洛韦的培养基中HIV Tat-（Gly）n-TK融合蛋白致HepG2细胞凋亡率最高（14．77％），其余依次为HIV Tat-（Gly）n-TK融合蛋白（12．69％）、HIV Tat-TK（8．31％）、HIV Tat-（Gly）n-TK（4．36％）和HIV Tat组（1．03％），组间均有显著性差异（P〈0．05）；细胞死亡率也发现类似的结果（分别为80．2％、65．4％、58．4％、56．7％、9．1％，组间有显著差异，P〈0．05）。结论：插入2、4、6个甘氨酸对HIV Tat-TK融合蛋白上游Tat蛋白的细胞融合穿透功能不产生影响，而对融合蛋白下游TK蛋白介导的更昔洛韦的细胞毒作用干扰较大，其中插入4个甘氨酸对TK蛋白介导的更昔洛韦的细胞毒作用影响最小。

英文摘要：

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 immunofluorescence 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