摘要
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。
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 pPICZaA. The plasmid pPICZaA-mvegf was trans- formed into P.pastoris GSllS. 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.
出处
《生物技术通讯》
CAS
2012年第6期785-789,共5页
Letters in Biotechnology
基金
国家自然科学基金(31200082)
北京市自然科学基金(5102037)
关键词
小鼠血管内皮生长因子
毕赤酵母
重组蛋白
N-糖基化
Mus musculus vascular endothelial growth factor
Pichia pastoris
recombinant protein
N-glycosyl- ation
