人促黄体激素释放素-血管生成素重组毒素的构建表达及其复性的实验研究  

Construction, expression and refolding of recombinant luteinizing hormone releasing hormone-angiogenin toxin

作　　者：倪志立[1] 张秋航[1] 曲秋懿[1] 吕海丽[1] 范舒雅[2] 蔡超[3] 

机构地区：[1]首都医科大学宣武医院耳鼻咽喉头颈外科,北京100053 [2]吉林省人民医院耳鼻咽喉科 [3]北京地坛医院耳鼻咽喉科

出　　处：《中华耳鼻咽喉头颈外科杂志》2010年第8期680-684,共5页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

摘　　要：目的 研究在大肠杆菌中高效表达人促黄体激素释放素-血管生成素（luteinizing hormone releasing hormone-angiogenin,LHRH-Ang）重组毒素及其复性的方法.方法 利用分子生物学技术构建pET28a/LHRH-Ang表达载体,序列测定验证融合基因的正确性.将测序正确的质粒转化在大肠杆菌BL21（DE3）中,异丙基-β-D-硫代半乳糖苷（Isopropyl-β-D-Thiogalactoside,IPTG）诱导观察重组毒素的表达.采用梯度透析法对以包涵体形式表达的重组毒素进行复性研究.结果 利用基因工程技术,成功构建了表达LHRH-PⅡ-Ang重组毒素的原核表达载体,转化大肠杆菌BL21（DE3）后经IPTG诱导,重组毒素以包涵体的形式获得了表达,其表达量占菌体蛋白总量的18.43%.通过细胞裂解、8 mol/L尿素的变性溶解和二氨基乙基琼脂凝胶（diethyl-aminoethyl DEAE）纯化过程,获得纯度达85%的目的蛋白.用梯度透析法对变性蛋白进行复性,复性重组毒素的质量浓度为1.27 mg/ml.结论 应用墓因工程技术能够成功构建LHRH-Ang重组毒素,并在大肠杆菌中获得高效表达,以包涵体形式存在的重组毒素经梯度透析可以获得有效的复性.Objective To express, purify and refold recombinant luteinizing hormone releasing hormone-angiogenin（LHRH-Ang） toxin using E. coli. expression system. Methods Recombinant LHRHAng expression vector was constructed by replacing of EGF fragment in plasmid pET28a/EGF-Ang with LHRH-P Ⅱ fragment amplified from plasmid pET28/MSH-PEA0. DNA sequencing would be used to verify the correction of fused LHRH-P Ⅱ -Ang gene. Then, E. coli strain BI21 （DE3） was transformed by pET28a/LHRH-Ang vector. Expression of recombinant LHRH-Ang toxin was induced by Isopropyl-β-D-Thiogalactoside（ IPTG ）. Refolding effects of gradient dialysis was evaluated by SDS-PAGE. Results Prokaryotic expression vector pET28a/LHRH-Ang, containing LHRH-P Ⅱ -Ang fusion gene, was constructed by PCR amplification, restriction enzyme digestion and ligation method. Sequence correction of fusion gene was confirmed by DNA sequencing After IPGT induction, recombinant LHRH-Ang protein was expressed in BL21 （ DE3 ） as inclusion body ,it took 18.43% of total protein. Inclusion body was resolved in 8 mol/L urea and purified by DEAE-Sepharose FF column, the purity was 85%. Recombinant LHRH-Ang toxin was refolded and concentrated by gradient dialysis and PEG 20000, respectively. Conclusions Recombinant LHRH-Ang protein was expressed in E. coli and refolded successfully.

关 键 词：促黄体素释放激素 血管生成素类 免疫毒素类 大肠杆菌 

分 类 号：R739.91[医药卫生—肿瘤]