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目的:构建重组大鼠ΔNΔC/VEGF-C/Cys152Ser(ddVEGF-C)逆转录病毒载体,建立稳定表达ddVEGF-C的PT67包装细胞株,并验证其表达。方法:利用PCR从pSecTag-ddVEGF-C中获取大鼠ddVEGF-C基因,克隆到pLPCX逆转录病毒载体,经PCR、双酶切和测序验证,转染PT67包装细胞,嘌呤霉素筛选出稳定表达目的基因的包装细胞株,用反转录PCR(RT-PCR)和Western blot方法验证其在RAW 264.7细胞中的表达。结果:构建的pLPCX-ddVEGF-C逆转录病毒载体经双酶切和PCR鉴定正确,测序结果与ddVEGF-C序列一致,病毒滴度为2×107CFU/mL,RT-PCR和Western blot结果提示重组病毒感染RAW264.7细胞能正确表达ddVEGF-C。结论:成功构建了pLPCX-ddVEGF-C逆转录病毒载体,并建立了稳定表达ddVEGF-C的PT67包装细胞株,为以VEGF-C基础的实验研究提供了新工具。
OBJECTIVE: To construct a recombinant rat ΔNΔC / VEGF-C / Cys152Ser (ddVEGF-C) retroviral vector and establish a PT67-expressing cell line stably expressing ddVEGF-C and verify its expression. Methods: The ddVEGF-C gene was obtained from pSecTag-ddVEGF-C by PCR and cloned into pLPCX retroviral vector. The recombinant plasmid was transfected into PT67 packaging cells by PCR, double enzyme digestion and sequencing. The target gene packaging cell lines, using reverse transcription PCR (RT-PCR) and Western blot method to verify its expression in RAW 264.7 cells. Results: The constructed pLPCX-ddVEGF-C retroviral vector was identified by double enzyme digestion and PCR. The sequencing result was consistent with that of ddVEGF-C. The titer of virus was 2 × 107CFU / mL. The results of RT-PCR and Western blot suggested that recombination Virus infection RAW264.7 cells can correctly express ddVEGF-C. Conclusion: The pLPCX-ddVEGF-C retroviral vector was successfully constructed and a PT67 packaging cell line stably expressing ddVEGF-C was established, which provided a new tool for the experimental research on the basis of VEGF-C.