CAJ | 학술논문

目的:建立毕赤酵母重组小鼠血管内皮生长因子(mVEGF)的制备方法,为研究 mVEGF 的生物活性、抗原性等提供基础.方法:通过全基因合成方法获得编码 mVEGF 的基因片段,将其克隆至表达载体 pPICZαA 上,电转化整合到毕赤酵母 GS115基因组中,用甲醇诱导表达目的蛋白,表达上清经硫酸铵沉淀、Sephadex G25柱脱盐、阳离子交换层析三步纯化获得目的蛋白;用还原型和非还原型 SDS-PAGE 检测目的蛋白的聚体状态,用 Western 印迹验证纯化蛋白;通过 PNGase F 酶切分析目的蛋白的 N-糖基化修饰;通过人脐静脉内皮细胞(HUVEC)增殖实验检测目的蛋白的生物活性.结果:获得 mVEGF 的重组毕赤酵母表达菌株,SDS-PAGE 分析可见 GS115表达的重组 mVEGF 在还原状态下表观相对分子质量约为20×103,在非还原状态下约为40×103;经 Western 印迹检测,这些条带均为目的蛋白条带,能被兔抗 mVEGF 抗体特异性结合,PNGase F 酶切后相对分子质量降至18×103左右,证明目的蛋白发生了 N-糖基化修饰;细胞测活实验表明,mVEGF 具有刺激 HUVEC 增殖的生物活性.结论:利用毕赤酵母菌制备了具有生物活性的重组 mVEGF.
목적:건립필적효모중조소서혈관내피생장인자(mVEGF)적제비방법,위연구 mVEGF 적생물활성、항원성등제공기출.방법:통과전기인합성방법획득편마 mVEGF 적기인편단,장기극륭지표체재체 pPICZαA 상,전전화정합도필적효모 GS115기인조중,용갑순유도표체목적단백,표체상청경류산안침정、Sephadex G25주탈염、양리자교환층석삼보순화획득목적단백;용환원형화비환원형 SDS-PAGE 검측목적단백적취체상태,용 Western 인적험증순화단백;통과 PNGase F 매절분석목적단백적 N-당기화수식;통과인제정맥내피세포(HUVEC)증식실험검측목적단백적생물활성.결과:획득 mVEGF 적중조필적효모표체균주,SDS-PAGE 분석가견 GS115표체적중조 mVEGF 재환원상태하표관상대분자질량약위20×103,재비환원상태하약위40×103;경 Western 인적검측,저사조대균위목적단백조대,능피토항 mVEGF 항체특이성결합,PNGase F 매절후상대분자질량강지18×103좌우,증명목적단백발생료 N-당기화수식;세포측활실험표명,mVEGF 구유자격 HUVEC 증식적생물활성.결론:이용필적효모균제비료구유생물활성적중조 mVEGF.
Objective: To express and purify Mus musculus vascular endothelial growth factor(mVEGF) in yeast Pichia pastoris, preparing for further study on its bioactivity and antigen characteristic. Methods: The mVEGF gene was got by PCR and cloned into the expression vector pPICZαA. The plasmid pPICZαA-mvegf was trans?formed into P.pastoris GS115. And the mVEGF gene was expressed in P.pastoris after induced by methanol. The protein was purified by three steps in succession: ammonium sulfate precipitation, G25 desalination and cation ex?change chromatograph. The molecular weight was analyzed by reducing and non-reducing SDS-PAGE. The protein was identified by Western blotting. The N-glycosylation was detected by the PNGase F digesting, and bioactivity of the recombinant mVEGF was analyzed by the human umbilical vein endothelial cells(HUVEC) proliferation ex?periment. Results: The mVEGF forms dimers with 40 kD under non-reducing SDS-PAGE, while with 20 kD monomer under reducing conditions. The mVEGF protein can be recognized by the commercial anti-mVEGF anti?body. The molecular weight of mVEGF decreased to 18 kD after PNGase F digesting. The HUVEC could be prolif?erated by recombinant mVEGF. Conclusion: The purified mVEGF expressed in P.pastoris with the proliferation in?duce HUVEC bioactivity was finally obtained.