本文選題：陽離子多肽　切入點(diǎn)：位點(diǎn)特異性重組　出處：《生物工程學(xué)報(bào)》2015年12期

【摘要】：利用Gateway克隆技術(shù)構(gòu)建重組抗瘤肽AIK的原核表達(dá)體系,建立表達(dá)及純化重組AIK的最優(yōu)條件,為深入研究和利用AIK奠定基礎(chǔ)。首先,設(shè)計(jì)含AttB重組位點(diǎn)的引物,通過重疊PCR技術(shù)擴(kuò)增出Att B-TEV-FLAG-AIK序列,利用BP重組反應(yīng)將目的序列TEV-FLAG-AIK克隆到供體載體pDONR223中,構(gòu)建入門載體,再通過LR重組反應(yīng),將目的序列轉(zhuǎn)移到目的載體pDEST15中,構(gòu)建GST-AIK融合蛋白原核表達(dá)質(zhì)粒。隨后,在BL21(DE3)工程菌中優(yōu)化誘導(dǎo)融合蛋白表達(dá)的條件。以谷胱甘肽磁珠純化GST-AIK融合蛋白,再以rTEV酶切除GST,獲得FLAG-AIK重組蛋白。最后以MTS法檢測FLAG-AIK對白血病細(xì)胞HL-60的細(xì)胞毒性。菌液PCR驗(yàn)證和測序分析表明成功構(gòu)建了重組抗瘤肽AIK的入門質(zhì)粒和原核表達(dá)質(zhì)粒。在BL21(DE3)工程菌中實(shí)現(xiàn)了GST-AIK融合蛋白的高效可溶性表達(dá)。并測得在37℃下以0.1 mmol/L IPTG誘導(dǎo)工程菌(OD600=1.0)4 h,重組蛋白表達(dá)量占菌體總蛋白的30%以上。經(jīng)GST親和層析、rTEV酶切除GST標(biāo)簽及二次GST親和層析獲得純度高于95%的FLAG-AIK蛋白。MTS法測得所制備的FLAG-AIK蛋白抑瘤活性與化學(xué)合成的AIK相當(dāng)。總之,本課題應(yīng)用Gateway克隆系統(tǒng)成功構(gòu)建了抗瘤肽AIK的原核表達(dá)質(zhì)粒,實(shí)現(xiàn)了GST-AIK融合蛋白的高效可溶性表達(dá),經(jīng)親和層析獲得了有生物活性的重組AIK多肽,為后續(xù)深入研究和大規(guī)模制備奠定了基礎(chǔ)。
[Abstract]:The prokaryotic expression system of recombinant anti-tumor peptide AIK was constructed by using Gateway cloning technique, and the optimal conditions for expression and purification of recombinant AIK were established, which laid the foundation for further study and utilization of AIK. Firstly, primers containing the recombinant site of AttB were designed. The Att B-TEV-FLAG-AIK sequence was amplified by overlapping PCR technique, the TEV-FLAG-AIK sequence was cloned into donor vector pDONR223 by BP recombination reaction, and the entry vector was constructed. The target sequence was transferred to the target vector pDEST15 by LR recombination reaction. The prokaryotic expression plasmid of GST-AIK fusion protein was constructed. Subsequently, the conditions for inducing the expression of fusion protein were optimized in BL21DDE3 engineering strain. GST-AIK fusion protein was purified by glutathione magnetic beads. Finally, the cytotoxicity of FLAG-AIK to leukemic cell HL-60 was detected by MTS method. The results of PCR verification and sequencing analysis showed that the primer plasmid and prokaryotic expression plasmid of recombinant anti-tumor peptide AIK were successfully constructed. The fusion protein of GST-AIK was expressed in BL21DDE3. The recombinant protein was induced by 0.1 mmol/L IPTG at 37 鈩，

本文編號(hào)：1670925