摘要
目的探索人白介素18(IL-18)成熟蛋白(mhIL-18)在毕赤酵母中高效表达载体的构建,以及多拷贝阳性转化子的筛选。方法采用SOEing及不对称PCR方法扩增出mhIL-18基因并构建融合型表达载体pPIC9IL-18-In-tein,再运用大片段套接的方法避开酶切位点,构建pPIC9KIL-18-Intein。利用G418的加压筛选寻找重组克隆高拷贝菌株。结果成功构建pPIC9KIL-18-Intein载体,并通过双抗筛选、PCR、酶切以及DNA测序证明。寻找到重组克隆高拷贝菌株。结论成功构建mhIL-18在毕赤酵母中的表达载体pPIC9KIL-18-Intein,并筛选到重组克隆高拷贝菌株,为IL-18融合蛋白的高表达及纯化奠定了基础。
Objective To study recombinant vector and screen the highest resistance transforming of mature human IL-18（mhIL-18） which can be expressed in high level and purified effectively in pichia pastoris. Methods The gene of mhIL-18-Intein was amplified by SOEing and asymmetry PCR. And the recombinant expression plasmid pPICgIL-18-Intein and pPICgKIL-18- Intein were constructed. The transform containing highest resistance of pichia pastoris were selected with increasing concentration of G418. Results The recombinant vector was identified by transformation,PCR, restriction enzymes digestion analysis and DNA sequencing. The recombinant pichia pastoris was selected by G418. Conclusion The recombinant vector pPICgKIL-18-Intein is constructed and the highest resistance of pichia pastoris of mhIL-18 is obtained successfully.
出处
《江西医学院学报》
2007年第2期15-19,共5页
Acta Academiae Medicinae Jiangxi
