【摘要】 目的构建人内皮抑素(humanendostatin,hES)原核表达质粒pET28a/hES并与简化人纤溶酶原饼环区5(predigestedhumanplasminogenkringle5,predhPK-5)在大肠杆菌中实现共表达。方法RT-PCR法从人肝癌组织中扩增人内皮抑素基因,利用基因克隆技术构建原核表达质粒pET28a/hES。在卡那霉素与氨苄青霉素的选择压力下,将其与pGEX-1λT/predhPK-5共同转化E.coliBL21(DE3),并诱导表达重组蛋白。同时从连续培养时间与传代次数两方面检测了共转化子的稳定性。结果成功构建了重组质粒pET28a/hES,其与pGEX-1λT/predh-PK-5的共转化子在双抗生素压力下可以共存,并在E.coliBL21(DE3)中成功共表达了人内皮抑素及简化的人纤溶酶原饼环区5,其表达量分别占蛋白总量的20%和21%。在双抗性培养基中培养16h及传代120代,共转化子存活率均大于75%,具有较好的稳定性。结论不相容质粒pET28a/hES与pGEX-1λT/predhPK-5在大肠杆菌中的成功共表达,对基因共表达方法作了有益探索,验证了不相容质粒共表达方法的可行性。更多还原

【Abstract】 Objective To construct the recombinant plasmid pET28a/hES and coexpress the human endostatin (hES) and predigested human plasminogen kringle 5 (predhPK-5) in E.coli. Methods The mRNA was extracted from human liver tissue, and the endostatin gene was amplified by RT-PCR, which then was cloned into pET-28a(+). Under screening pressure by ampicillin and kanamycin simultaneously, E.coli BL21(DE3) was cotransformed with pET28a/hES and pGEX-1λT/predhPK-5 and induced with IPTG to express the recombinant proteins.The stability of cotransformants existing in E.coli was measured through two aspects in the serial culture time and passage number of bacterium. Results The two incompatible plasmids could be coexisted under the pressure of two antibiotics (ampicillin and kanamycin). After induced with IPTG, both human endostation and predhPK-5 gene were coexpressed, and the recombinant proteins comprised about 20% and 21% of total cell proteins, respectively.The two incompatible plasmids could still be maintained in over 75% E.coli cells for at least 16 hours or after 120 passages under the pressure of two antibiotics. Conclution The two incompatible plasmids pET28a/hES and pGEX-1λ T/predhPK-5 may coexpress the recombinant proteins in E.coli.A new method for coexpression of proteins in E.coli containing two incompatible plasmids is proved to be feasible.更多还原