人颗粒酶B真核表达载体的构建与表达分析

• 作 者: 李秀英 夏良平 谢金卫 赵肃清 曾宗渊 张海涛 吉琼梅 李民友 朱振宇
• 机 构: 中山大学中山医学院生物化学教研室,中山大学肿瘤防治中心头颈外科,中山大学中山医学院生物化学教研室,中山大学中山医学院生物化学教研室,中山大学肿瘤防治中心头颈外科,中山大学中山医学院生物化学教研室,中山
• 出 处: 中国病理生理杂志
• 发表日期: 2004年12期
• 关键词: 颗粒酶B 淋巴细胞 Hep2细胞

目的构建能在Hep2细胞中表达的颗粒酶B的表达质粒pVAX1-GrB。方法用淋巴细胞分离液分离肿瘤组织中的淋巴细胞,并抽提RNA,用RT-PCR方法扩增其分泌蛋白颗粒GrB的全部外显子片段,利用基因重组技术将其定向插入pVAX1多克隆位点。脂质体介导转染Hep2细胞,用间接免疫荧光法观察目的蛋白在细胞中的表达。结果限制性内切酶酶切和DNA测序分析证实成功获得GrB基因片段,GrB准确克隆入pVAX1的多克隆位点,未改变读码框架。转染Hep2细胞后,检测到目的蛋白的表达。结论成功构建了VAX1-GrB,并在Hep2细胞中得到表达。

Objective To construct plasmid pVAX1-GrB expressing granzyme B expressed in Hep2 cells. Methods Lymphocytes from tumor tissues were isolated by lymphocyte separation and RNA was extracted. All the exon fragments of GrB, secreted protein granules were amplified by RT-PCR and inserted into pVAX1 polyclonal locus by gene recombination technique point. Lipofectamine was transfected into Hep2 cells, and the expression of the target protein was observed by indirect immunofluorescence. Results Restriction endonuclease digestion and DNA sequencing confirmed that the GrB gene fragment was successfully obtained. GrB was cloned into the multiple cloning site of pVAX1 without changing the reading frame. After transfected Hep2 cells, the expression of the target protein was detected. Conclusion VAX1-GrB was successfully constructed and expressed in Hep2 cells.