CAJ | 학술논문

目的：研究人Flt-1胞外区2-3loop cDNA在Pichia.pastoris酵母中的表达，获得高效表达的具有生物学活性的重组Flt-1(2-3)。方法：采用PCR扩增 Flt-1(2-3)基因，经DNA序列分析后，插入含AOX1 启动子和α分泌信号肽序列的Pichia.pastoris酵母表达载体中，构建重组质粒pPIC9K/Flt-1(2-3)，转化酵母宿主菌GS115，筛选His+Muts表型转化子，摇瓶培养，1%甲醇诱导表达。表达产物经CM-Sepharose FF阳离子交换层析和Sephacryl S-100分子筛层析纯化后，测定其生物学活性。结果：SDS-PAGE分析显示，表达产物以可溶性分子形式存在于上清中，诱导4 d的表达量达上清总蛋白的60%以上。ELISA及Western blot实验表明，表达产物具有良好的抗原性和特异性。经CM-Sepharose FF阳离子交换层析和Sephacryl S-1 00 分子筛层析纯化后，Flt-1(2-3)纯度达到90%以上。生物学活性检测证实其具有结合hVEGF 16 5的能力和抑制hVEGF165对HUVEC的促增殖功能。结论：获得了具有生物学活性的可溶性重组F lt-1受体胞外区2-3loop小片段，为进一步的动物实验和临床应用打下了基础。
목적：연구인Flt-1포외구2-3loop cDNA재Pichia.pastoris효모중적표체，획득고 효 표체적구유생물학활성적중조Flt-1(2-3)。방법：채용PCR확증 Flt-1(2-3)기인，경DNA서렬 분석후，삽입함AOX1 계동자화α분비신호태서렬적Pichia.pastoris효모표체재체중，구 건 중조질립pPIC9K/Flt-1(2-3)，전화효모숙주균GS115，사선His+Muts표형전화자，요 병배양 ，1%갑순유도표체。표체산물경CM-Sepharose FF양리자교환층석화Sephacryl S-100분자 사 층석순화후，측정기생물학활성。결과：SDS-PAGE분석현시，표체산물이가용성분자형식존 재우상청중，유도4 d적표체량체상청총단백적60%이상。ELISA급Western blot실험표명， 표 체산물구유량호적항원성화특이성。경CM-Sepharose FF양리자교환층석화Sephacryl S-1 00 분자사층석순화후，Flt-1(2-3)순도체도90%이상。생물학활성검측증실기구유결합hVEGF 16 5적능력화억제hVEGF165대HUVEC적촉증식공능。결론： 획득료구유생물학활성적가용성중조F lt-1수체포외구2-3loop소편단，위진일보적동물실험화림상응용타하료기출。
Objective: To study the expression of human Flt-1 2-3loop cDNA in Pic hi a.pastoris and to obtain high-level expressed recombinant human Flt-1(2-3) with good biological activity.Methods: Amplifying Flt-1(2-3) cDNA by PCR, after con firmed by DNA sequence analysis, the gene was inserted into the Pichia.pastoris expression vector pPIC9K containing AOX1 promoter and α secreting signal peptid e s, the recombinant expression plasmids pPIC9K/Flt-1(2-3) was constructed and t ra nsformed into GS115. The His+Muts phenotype transformants were screened，fe rment ed in flasks and induced by 1% methanol.Results:After 4 days of methanol induc tion, the expressed Flt-1(2-3) comes up to 60% of total proteins in supernatan t by SDS-PAGE. ELISA and Western blot assay proved it having good antigenicity a nd high specificity. The recombinant protein was further purified with CM-Sephar os e Fast Flow and Sephacryl S-100 chromatography, and was proved having good biol o gical activity to bind hVEGF165 and to inhibit hUVEC proliferation stimula ted by hVEGF165.Conclusion: High-level expression of secreted Flt-1(2-3) with good bi ological activity were successfully achieved in Pichia.pastoris expression syst em and can be applied to further animal and clinical test.