生育酚结合蛋白基因重组腺相关病毒的制备及抗癌作用

目的 制备生育酚结合蛋白(TAP)重组腺相关病毒,并探讨其对前列腺癌生长的作用.方法 构建重组腺相关病毒质粒pSNAV-TAP-EGFP,酶切、聚合酶链反应(PCR)、测序.225 cm2细胞培养瓶中,接种293T细胞,以重组质粒转染.扩增、纯化病毒,并转染DU-145细胞,噻唑蓝(MTT)比色法观察第2、4、6天的细胞增殖.结果 成功构建pSNAV-TAP-EGFP质粒,并制备滴度6.4×1011vg/ml、纯度95%的rAAV2-TAP.该病毒转染DU-145细胞后TAP表达量明显上升.转染后第4天,TAP转染组的细胞吸光度(A值)0.546±0.072,对照组以及空载体转染组的A值分别为0.673±0.015、0.638±0.051,生长明显受到抑制(P＜0.05).至第6天各组细胞生长差异更显著.结论 成功制备高滴度的rAAV-TAP-EGFP病毒,并可抑制前列腺癌细胞的生长.

Abstract：

Objective To produce recombinant rAAV2 virus carrying α-tocopherol associated protein (TAP) and to observe its effect on prostate cancer cells. Methods The pSNAV-TAP-EGFP plasmid was constructed and digested with restriction enzyme. Polymerase chain reaction (PCR), and sequence analysis were performed to identify the correct recombinant clones. The plasmid was transfected into 293T cells to produce virus. The virus was amplified and purified. DU-145 cells were infected by rAAV-TAPEGFP. Cell growth was detected by MTT assay. Results The pSNAV-TAP-EGFP was constructed successfully. The titer of rAAV2-TAP-virus was 6. 4 × 1011 vg/ml and the purity was 95%. The rAAV-TAP-EGFP was infected into the DU-145 cells with high efficiency. The expression of TAP was increased significantly by real-time PCR. MTT assay showed differences at the 4th day. The cellular absorbance value in the TAP transfection group, control group and empty vector transfection group was 0. 546 +0. 072, O. 673 +0. 015,and 0. 638 + 0. 051 (P ＜ 0. 05 ). The differences became more significant after 6 days. Conclusion High tier recombinant adeno-associated virus rAAV2-TAP-EGFP was obtained, which was able to inhibit the proliferation of prostate cancer cells.     

中国汉族人群apoBVNTR等位基因的多态性分析及其分型标准物的克隆制备

目的对中国汉族人群的apoB VNTR等位基因进行分布频率、序列结构等多态性分析,并通过分子克隆技术制备apoB VNTR等位基因分型标准物。方法通过PCR扩增并结合电泳、测序等方法分析人群apoB VNTR等位基因的多态性,并将该人群中得到的所有类型等位基因片段插入pUC重组质粒,经转染、扩大培养、扩增及再鉴定后,制备出apoB VNTR等位基因分型标准物。结果所检测人群中共检测出16种apoB VNTR等位基因,发现核心序列存在变异体,并成功制备出分型标准物。结论对apoB VNTR的多态性分析应结合等位基因的分布频率与核心序列的结构,所制备的apoB VNTR等位基因分型标准物使apoB VNTR电泳法分型更加准确。