醫(yī)學(xué)免費(fèi)論文:pET15bYARAEGFP原核表達(dá)質(zhì)粒的構(gòu)建及融合蛋白YARAEGFP的表達(dá)與純化
【摘要】 目的:構(gòu)建原核表達(dá)載體pET15bYARAEGFP,并進(jìn)行YARAEGFP融合蛋白的表達(dá)和純化。方法:用分子克隆技術(shù)構(gòu)建出表達(dá)型載體pET15bYARAEGFP,在E.coli BL21(DE3)中表達(dá)融合蛋白YARAEGFP,并進(jìn)行Ni2+NTA樹脂柱親和層析以純化蛋白。結(jié)果:經(jīng)測(cè)序證實(shí)成功構(gòu)建了表達(dá)型載體pET15bYARAEGFP,YARAEGFP融合蛋白在E.coli BL21(DE3)中得到表達(dá),純化后的蛋白濃度為1.098 mg/mL。SDSPAGE和Western blot分析表明純化蛋白為目的蛋白YARAEGFP。結(jié)論:已成功制備出YARAEGFP融合蛋白。
【關(guān)鍵詞】 細(xì)胞穿透肽 增強(qiáng)型綠色熒光蛋白 原核表達(dá) 融合蛋白
Construction of Prokaryotic Expression Plasmid pET15bYARAEGFP and Expression and Purification of YARAEGFP Fusion Protein CHEN Sisi1,2,WANG Jianing2^,HUANG Yongzhang2,GUO Lingyun2,ZHENG Fei2 (1Cardiovascular Research Institute,Renmin Hospital,Wuhan University,Wuhan,Hubei 430060;2Institute of Clinical Medicine,Renmin Hospital,Yunyang Medical College,Shiyan,Hubei 442000,China)
Abstract:Objective To construct the expression vector pET15bYARAEGFP and express and purify the fusion protein YARAEGFP.Methods Two oligonucleotides encoding YARA were synthesized and annealed to generate YARAencoding DNA. The recombinant plasmid pET15bYARAEGFP was constructed by inserting YARAencoding DNA into pET15bEGFP. The fusion protein YARAEGFP induced with IPTG in E.coli BL21(DE3)was purified with Ni2+resin affinity chromatography and confirmed with SDSPAGE and western blot.Results Sequence analysis confirmed successful construction of the expression vector pET15bYARAEGFP,the fusion protein YARAEGFP was expressed and purified and its concentration was 1.098 mg/mL.SDSPAGE and Western blot demonstrated the fusion protein was YARAEGFP.Conclusion YARAEGFP fusion protein is successfully prepared醫(yī).學(xué).全.在.線payment-defi.com.
Key words:Cell penetrating peptides;Enhanced green fluorescent protein;Prokaryotic expression;Fusion protein
自人類基因組計(jì)劃的完成和蛋白組計(jì)劃的實(shí)施以來,人們發(fā)現(xiàn)了許多生物大分子具有潛在的治療價(jià)值。但細(xì)胞膜是脂質(zhì)雙分子層,具有選擇通透性,這種天然屏障作用在保護(hù)細(xì)胞的同時(shí)也限制了生物大分子自由進(jìn)入細(xì)胞內(nèi)發(fā)揮作用。傳統(tǒng)的將大分子物質(zhì)轉(zhuǎn)導(dǎo)入細(xì)胞內(nèi)的常用方法有電穿孔、脂質(zhì)體轉(zhuǎn)染等,這些方法不但轉(zhuǎn)導(dǎo)效率低,而且對(duì)轉(zhuǎn)導(dǎo)的細(xì)胞均有毒副作用,且僅適用于體外實(shí)驗(yàn)。最近研究發(fā)現(xiàn),某些蛋白質(zhì)的結(jié)構(gòu)區(qū)域具有將外源性蛋白轉(zhuǎn)導(dǎo)入細(xì)胞內(nèi)的特性,這些區(qū)域被稱為蛋白轉(zhuǎn)導(dǎo)結(jié)構(gòu)域或細(xì)胞穿透肽[1],它能攜帶有治療作用的蛋白質(zhì)轉(zhuǎn)導(dǎo)入細(xì)胞內(nèi)并發(fā)揮相應(yīng)的生物學(xué)效應(yīng),這使它有可能成為一種理想的蛋白轉(zhuǎn)導(dǎo)載體。目前研究最為深入的是來源于人類免疫缺陷病毒反式激活因子的TAT蛋白轉(zhuǎn)導(dǎo)結(jié)構(gòu)域,其氨基酸序列為YGRKKRRQRRR。Choi領(lǐng)導(dǎo)的研究小組[2-8]已經(jīng)證實(shí)了TATGFP、TATGDH、TATSOD和TATCAT等融合蛋白能轉(zhuǎn)導(dǎo)入哺乳動(dòng)物細(xì)胞內(nèi)和皮膚組織。為了進(jìn)一步提高細(xì)胞穿透肽的轉(zhuǎn)導(dǎo)效率,Ho等[9]將TAT的氨基酸序列進(jìn)行優(yōu)化,合成了一種新的蛋白轉(zhuǎn)導(dǎo)結(jié)構(gòu)域即YARA,其氨基酸序列為YARAAARQARA,發(fā)現(xiàn)其轉(zhuǎn)導(dǎo)效率為TAT的33倍。為了探討YARA介導(dǎo)的融合蛋白的離體和在體轉(zhuǎn)導(dǎo)能力,本研究設(shè)計(jì)合成了編碼YARA的DNA序列,通過基因重組技術(shù)構(gòu)建了原核表達(dá)質(zhì)粒pET15bYARAEGFP,表達(dá)并純化出YARAEGFP融合蛋白,為YARA介導(dǎo)的生物大分子EGFP穿透細(xì)胞能力的研究奠定了基礎(chǔ)。