CAJ | 학술논문

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.
목적 연구재대장간균중고효표체인촉황체격소석방소-혈관생성소(luteinizing hormone releasing hormone-angiogenin,LHRH-Ang)중조독소급기복성적방법.방법 이용분자생물학기술구건pET28a/LHRH-Ang표체재체,서렬측정험증융합기인적정학성.장측서정학적질립전화재대장간균BL21(DE3)중,이병기-β-D-류대반유당감(Isopropyl-β-D-Thiogalactoside,IPTG)유도관찰중조독소적표체.채용제도투석법대이포함체형식표체적중조독소진행복성연구.결과 이용기인공정기술,성공구건료표체LHRH-PⅡ-Ang중조독소적원핵표체재체,전화대장간균BL21(DE3)후경IPTG유도,중조독소이포함체적형식획득료표체,기표체량점균체단백총량적18.43%.통과세포렬해、8 mol/L뇨소적변성용해화이안기을기경지응효(diethyl-aminoethyl DEAE)순화과정,획득순도체85%적목적단백.용제도투석법대변성단백진행복성,복성중조독소적질량농도위1.27 mg/ml.결론 응용묘인공정기술능구성공구건LHRH-Ang중조독소,병재대장간균중획득고효표체,이포함체형식존재적중조독소경제도투석가이획득유효적복성.
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.