小鼠分泌型内皮抑素原核表达载体pNZ44-ssEndostatin的构建及表达

目的: 构建带有小鼠分泌型内皮抑素(ssEndostatin)的原核表达载体pNZ44-ssEndostatin并在双歧杆菌中表达Endostatin蛋白。 方法: 利用引物设计软件oligo6.0设计引物,从pcDNA3.1-Egr1-ssEndostatin质粒上PCR扩增得到ssEndostatin基因。经测序证实获得的ssEndostatin基因序列正确后,进行粘端连接,将ssEndostatin基因链接到pNZ44原核表达载体上,构建成pNZ44-ssEndostatin重组载体,PCR、酶切鉴定正确后电转化到双歧杆菌中。然后利用ELISA法检测Endostatin表达。 结果: 测序结果证实ssEndostatin基因序列与Genebank中所公布的一致,说明ssEndostatin基因扩增正确。而且构建的质粒pNZ44-ssEndostatin的PCR、酶切鉴定结果与预测的一致,说明重组质粒pNZ44-ssEndostatin构建成功。转化后挑取阳性克隆进行菌落PCR证实重组质粒pNZ44-ssEndostatin已成功转入双歧杆菌中。ELISA法检测到转化有重组质粒pNZ44-ssEndostatin的双歧杆菌的菌体内有较高水平的Endostatin表达,且培养48h的表达量较20h的高,有氧条件下表达较缺氧条件下高,但上清中未检测到。 结论: 成功地构建了带有分泌信号的小鼠内皮抑素表达载体pNZ44-ssEndostatin,成功转入双歧杆菌中并在菌体中表达,为后续利用双歧杆菌作为工具进行Endostatin基因治疗奠定了实验基础。

Construction and Expression of Prokaryotic Expression Vector pNZ44-ssEndostatin with Secretable Form of Mouse Endostatin

Objective: To construct the prokaryotic expression vector pNZ44-ssEndostatin which contains secretable form of mouse endostatin(ssEndostatin) and to express the Endostatin protein in bifidobacterium. Methods: We used primer design software Oligo 6.0 to design appropriate primers. The ssEndostatin gene was amplified from the plasmid pcDNA3.1-Egr1-ssEndostatin by PCR and was then sequenced. When the sequencing results confirmed that the acquired gene was indeed correct, the ssEndostatin gene was ligated to the expression vector pNZ44 to construct the recombinant plasmid pNZ44-ssEndostatin. The vector was electrotransformed into bifidobacterium and the Endostatin protein expression was detected by ELISA. Results: The sequence of ssEndostatin gene acquired by PCR was in concordance with publicly available data,indicating that the ssEndostatin gene was correctly cloned. The recombinant plasmid pNZ44-ssEndostatin was successfully constructed and transformed into bifidobacterium, as shown by colony PCR. The Endostatin protein was expressed inside bifidobacterium, and the quantity was increased by hypoxia. The expression was higher in bifidobacterium cultured for 48hours than in those cultured for 20 hours. Conculsion: The recombinant plasmid pNZ44-ssEndostatin containing Endo-statin with the secretion signal sequence was constructed successfully. The endostatin protein was expressed after the transformation of recombinant plasmid pNZ44-ssEndostatin into bifidobacterium. Bifidobacterium may be used as a tool for endostatin gene therapy.