内皮抑素和反义/正义内皮细胞生长因子cDNA真核共表达载体构建与表达

目的构建大鼠内皮抑素cDNA和反义/正义血管内皮细胞生长因子（VEGF）cDNA真核共表达载体,并在C6胶质瘤细胞上获得表达.方法以1 d龄大鼠脑组织总RNA为模板,利用RT-PCR法从中扩增出内皮抑素和反义/正义VEGF的cDNA,并将获得的目的cDNA依次重组入真核表达载体pBudCE 4.1上.重组子经酶切、PCR及测序鉴定后脂质体法转染C6细胞,zeocin抗性筛选.Western-blot方法、免疫细胞化学法及MTT法鉴定含内皮抑素的阳性克隆子;ELISA方法检测各阳性克隆子上清液中VEGF含量. 结果构建带有目的基因的重组子经酶切、PCR和测序鉴定,产物大小及基因序列与预期值相符.经抗性筛选的含内皮抑素cDNA的阳性克隆子可分泌具有生物学活性的内皮抑素：含反义VEGF cDNA的阳性克隆子VEGF均呈低表达,其中转染有内皮抑素cDNA和反义VEGF cDNA共表达载体的克隆子的VEGF表达最低;转染有内皮抑素cDNA和正义VEGFcDNA共表达载体的克隆子的VEGF也呈低表达.结论成功地构建大鼠内皮抑素和反义/正义VEGFcDNA真核共表达载体并在C6细胞上获得表达,内皮抑素可下调VEGF在肿瘤细胞上的表达.

Construction of eukarytic co-expression vector for rat endostatin cDNA and anti-sense /sense-VEGF164 cDNA and its expression in C6 glioma cells

Objective To construct eukarytic co-expression vector for rat endostatin cDNA and anti-sense/sense-VEGF164 cDNA and to transfect it into C6 glioma cells. Methods cDNA encoding rat endostatin and cDNA encoding anti-sense/sense-VEGF164 were amplified from newborn rat brain tissue with RT-PCR and inserted into the eukarytic vector pBudCE 4.1 sequently, recombinants were identified by restriction enzymes digestion, PCR and the nucleotide sequencing of the target gene. After successful reconstruction of the genes of endostatin and anti-sense/sense-VEGF164, the recombinants were transfected into C6 cells by lipofectin technique. The positive clones were screened out through zeocin resistance selection. The endostatin in supemate from the positive clones was identified by Western-blot and MTT method. With immunocytochemistry, the endostatin in the positive clones were located. The quantity of VEGF in supemate from the positive clones was quantitated by ELISA assay. Results The sizes of the amplified endostatin gene fragment and VEGF gene fragment were in accordance with that we expected. And the gene sequence inserted into the eukarytic vector pBudCE 4.1, were consistent with the known sequence. The endostatin with biological activities can be secreted from the clones transfected with endostatin cDNA, and the down-regulation of VEGF expression was found in the clones transfected with recombinants of endostatin cDNA or/and anti-sense-VEGF164 cDNA. Conclusion The recombinant rat endostatin gene clone has been established and cloned into the eukarytic vector pBudCE 4.1 successfully, the endostatin can expressed in C6 cells; the expression of VEGF in C6 glioma cell can be down-regulated by endostatin.