高级搜索

淋巴瘤特异性高效基因表达载体survivin-VISA的构建与鉴定

Construction and Digestion of Lymphoma-specific and High Efficient Gene Expression Vector survivin-VISA

  • 摘要: 目的 构建含survivin启动子及VP16-GAL4-WPRE整合型系统性放大系统(VISA)的淋巴瘤特异性高效基因表达载体survivin-VISA(S-VISA)。方法 应用基因重组技术构建含survivin启动子与土拨鼠肝炎病毒转录后调控元件(WPRE)、二步转录放大系统(TSTA)和VISA系统分别构成的表达载体S-WPRE、S-TSTA和S-VISA,经PCR法及酶切鉴定。用脂质体法转染淋巴瘤细胞株Ramos、U937、 Raji及人肝细胞株Chang Liver,检测荧光素酶活性。结果 表达载体S-WPRE、S-TSTA和S-VISA通过PCR法及酶切鉴定证明构建正确,荧光素酶活性检测表明S-VISA载体在淋巴瘤细胞中实现了目的基因特异性高效表达。结论 成功构建了含survivin启动子及VISA系统的淋巴瘤特异性高效基因表达载体S-VISA,为淋巴瘤基因治疗的进一步研究奠定了良好的基础。

     

    Abstract: Objective To construct lymphoma-specific and high efficient gene expression vector composed of the survivin promoter in the VP16-GAL4-WPRE integrated systemic amplifier system (S-VISA). Methods Three vectors, S-WPRE, S-TSTA and S-VISA, were constructed by gene recombination technology, comprised of different elements of woodchuck hepatitis post-transcriptional regulatory element(WPRE), two-step transcriptional amplification(TSTA) and VISA under control of survivin promoter, respectively; PCR method and enzyme digestion were used to prove its correctness. Lymphoma cell lines Ramos, U937, Raji and hepatic cell line Chang Liver were transfected with these vectors by liposome to detect the luciferase activities. Results Expression vectors S-WPRE, S-TSTA and S-VISA were proved to be constructed correctly by PCR and enzyme digestion. Luciferase activity analysis showed that S-VISA achieved lymphoma-specific and high efficient expression of the target gene. Conclusion Lymphomaspecific and high efficient gene expression vector S-VISA is constructed successfully, which laid a good foundation for further study of lymphoma gene therapy.

     

/

返回文章
返回