目的 构建单顺反子表达人 GM- CSF和 B7.1融合基因真核表达载体。 方法 应用巨引物 PCR技术对 h GM- CSF基因 c DNA进行定点突变 ;应用 PCR重叠延伸法 ,将 h GM- CSF和 h B7.1基因的 c DNA编码序列通过一 linker序列拼接 ,构建 h GM-...目的 构建单顺反子表达人 GM- CSF和 B7.1融合基因真核表达载体。 方法 应用巨引物 PCR技术对 h GM- CSF基因 c DNA进行定点突变 ;应用 PCR重叠延伸法 ,将 h GM- CSF和 h B7.1基因的 c DNA编码序列通过一 linker序列拼接 ,构建 h GM- CSF与 h B7.1融合基因 h GM- B7.1,将其插入 pc DNA3真核表达载体 ,测定核苷酸序列。 结果 序列分析表明 :(1) h GM- CSF突变体基因 c DNA编码序列的 178位核苷酸由 C变为 A,其余核苷酸序列均未发生变化 ;(2 ) h GM- CSF、linker、h B7.1的连接顺序、方向及序列完全正确。 结论 构建 pc DNA3- h GM- B7.展开更多
This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the hu...This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.展开更多
文摘目的 构建单顺反子表达人 GM- CSF和 B7.1融合基因真核表达载体。 方法 应用巨引物 PCR技术对 h GM- CSF基因 c DNA进行定点突变 ;应用 PCR重叠延伸法 ,将 h GM- CSF和 h B7.1基因的 c DNA编码序列通过一 linker序列拼接 ,构建 h GM- CSF与 h B7.1融合基因 h GM- B7.1,将其插入 pc DNA3真核表达载体 ,测定核苷酸序列。 结果 序列分析表明 :(1) h GM- CSF突变体基因 c DNA编码序列的 178位核苷酸由 C变为 A,其余核苷酸序列均未发生变化 ;(2 ) h GM- CSF、linker、h B7.1的连接顺序、方向及序列完全正确。 结论 构建 pc DNA3- h GM- B7.
文摘This work was supposed by CMB (No. 96—635) This is one of papers of the special issue on gene therapy research (Chin J Cancer Res Vol. 9 No. 4 December, 1997). Although cervical carcinoma cells may express the human papillomavirus protein E6 and E7, they fail to induce an effective specific cytotoxic T lymphocyte response. Recent studies suggest that expression of CD 80 (B7 1) on tumor cells is effective to induce antitumor immune responses. 1,2 In our study, CD 80 gene was transfected into human Hela cell line with a CD 80 expression plasmid (B7 1 +pcDNA 3) by electroporation, then the immunogenecity of the modified Hela cell was tested in TLMC (tumor lymphocyte mixed culture) system. Thymidine lymphocyte proliferation assays showed that the response of human peripheral blood lymphocytes (PBLS) to CD 80 positive Hela cells demonstrated a substantial increase in cell proliferation compared to the response to control cells. Cocultivation of allogeneic PBLs with CD 80 positive tumor cells for three days can induce an increased secretion of IL 2. Our results demonstrate an immunostimulatory effect of CD 80 expression on cervical cancer cells, which provides a basis for the development of a therapeutic tumor vaccine.
