摘要
目的:构建人Flt3配基的胞外区表达载体pCDNA3FL,并在体内外对其表达率及活性进行检测。方法:从健康人单核细胞中反转录PCR扩增FLcDNA胞外区片段,并进行测序,构建表达载体pCDNA3FL,测定pCDNA3FL转染H22细胞后及经小鼠尾静脉注射后hFL表达量。结果:测序结果表明,所扩增、克隆的靶序列正确,Western结果证实pCDNA3FL能正确表达hFL蛋白。ELISA结果表明表达载体pCDNA3FL在体外H22细胞中的瞬时表达量为5.9ng/ml。小鼠经尾静脉注射pCDNA3FL载体后血浆中的hFL蛋白最高含量为36μg/ml,外周血中CD3+、CD4+、CD8+、CD34+细胞亚群的比率均有不同程度的提高。结论:成功构建了表达载体pCDNA3FL,为构建肿瘤细胞瘤苗创造了条件。
Objective: To clone human Fit3 ligand gene,and construct pCDNA3FL and detect the eukaryotic expression vector expression of hFL protein both in vivo and in vitro; To construct H22 - pCDNA3FL and detect the role of the anti -tumor both in vivo and in vitro. Methods: A cDNA encoding soluble FL was cloned through RT PCR from the total RNA extracted from human peripheral blood mononuclear cells and identified by analyzing the nucleotide sequences. The human FL gene was subcloned into the expressing vector pCDNA3. The recombinant plasmid pCDNA3FL was transformed to BHK cells or H22 cells, and then Flt3 ligand was detected. Western -blot was employed to identify the expression of human Flt3 ligand. After injection the plasmid pCDNA3FL into mice tail vein with large volume within short period of time, the FL expression in plasma of mice blood was detected continuously by ELISA and the ratios of CD3^+ ,CD4^+ ,CD8^+ ,CD34^+ cell populations in peripheral blood were examined by flow cytometry. Results: FL gene with a length of 593bp was isolated from human peripheral blood mononuclear cells. Sequence analysis revealed the sequence of FL gene was consistent with relevant reports. FL gene was cloned into the expressing vector pCDNA3 identified by enzyme digestion of EcoR and Hind enzyme. Wenstern - blot showed the FL protein could be recognized by anti - FL antibody. After injection the plasmid pCDNA3FL into mice tail vein with large volume within short period of time, the results showed that there was high level expressions of hFL in mice plasma and the highest point reached 33μg/ml. The expression lasted about 2 weeks. The ratios of CD3^+ , CD4^+ , CD8^+ , CD34^+ cell populations in peripheral blood increased obviously. Conclusion: The FL gene was obtained by RT - PCR amplifycation. The expressing vector of FL was successfully constructed and expressed in pCDNA3FL. The study will provide necessary conditions for using this in the treatment of tumor.
出处
《现代肿瘤医学》
CAS
2007年第1期1-4,共4页
Journal of Modern Oncology
基金
陕西省科技攻关基金资助项目(编号:2003K10-G63号)