大鼠内皮抑素cDNA真核表达载体的构建及C6细胞上的分泌表达

目的构建分泌型大鼠内皮抑素(endostatin,endos)基因真核表达载体,并在C6细胞上获得表达。方法利用RT-PCR法从1日龄大鼠脑组织总RNA中扩增出endos基因,并将获得的基因重组入真核表达载体pBudCE4.1上。抽取经双酶切、PCR及测序鉴定后的重组子质粒,利用脂质体方法转染C6细胞,在Zeoc in抗性下筛选稳定表达endos的C6细胞克隆,W estern-b lot法检测阳性克隆子上清液中endos,免疫细胞化学鉴定endos在阳性克隆子上的定位,MTT法鉴定阳性克隆子分泌的endos的生物学活性,ELISA方法检测阳性克隆子上清液中的血管内皮细胞生长因子(VEGF)含量。结果从1日龄SD大鼠脑组织总RNA中扩增出endos基因,构建重组质粒endo-pBud,经酶切、PCR和测序鉴定,产物大小及基因序列与预期值相符。经抗性筛选的阳性克隆子可分泌具有生物学活性的endos,同时其VEGF表达下调。结论成功获得endos基因,构建endos重组质粒,并在C6细胞上获得分泌性表达。

Construction of eukarytic vector for rat endostatin cDNA and secretive expression in C6 cells

Aim To construct eukarytic vector for rat endostatin(endos) cDNA and observe its expression in C6 cell.Methods cDNA encoding rat endostatin was amplified from newborn brain tissue with RT-PCR and inserted into the eukarytic vector pBudCE 4.1.Recombinant was identified with KpnI,XhoI double digestion,PCR and nucleotide sequencing of the target gene.After successful reconstruction of the genes of endostatin,the recombinants was transfected into C6 cells with lipofectintechniques.The positive clones were screened out through zeocin resistance test.The endostatin in supernate of the positive clones was identified with Western-blot and MTT method.With immunocytochemistry,the endostatin in the positive clones was located.The quantities of VEGF in supernate of the positive clones were quantified with ELISA assay.Results The size of the amplified endostatin gene fragment was in accord with that we expected.And the gene sequence inserted into the eukarytic vector pBudCE 4.1 was consistent with the known sequence.Endostatin was secreted from the positive clone.Down-requlation of vascular endothelial growth factor(VEGF) was found in the positive clones.Conclusion The recombinant of rat endostatin gene clone had been established and inserted into the eukarytic vector pBudCE 4.1 successfully and endostatin was expressed in C6 cells.This provides a basis for further studies of endostatin effects in vivo,and creates the conditions for final clinical trial