趋化因子受体CCR5胞外段重组蛋白的克隆、表达、纯化及鉴定_药学论文.docVIP

趋化因子受体CCR5胞外段重组蛋白的克隆、表达、纯化及鉴定_药学论文 趋化因子受体CCR5胞外段重组蛋白的克隆、表达、纯化及鉴定_药学论文 【关键词】 因子 Cloning, expression, purification and identification of CCR5 extracellular domain recombinant protein 【Abstract】 AIM: To construct a prokaryotic expression vector of CCR5 extracellular domain recombinant protein and to purify and identify it. METHODS: To obtain amino acid sequence of CCR5 extracellular domains, the four peptides of CCR5（Nterm, ECL1, ECL2 and ECL3）were combined using the soft linker and named rCCR5. RCCR5 gene was synthesized according to codon E.coli preferred. The gene was cloned into the vector pET22b（+） and the aim protein was expressed in E.coli. The expression product was purified and then identified with Westernblot. RESULTS: We developed a purification process of rCCR5 from inclusion bodies. The procedure included washing and solubilization of inclusion body, purification by SephacrylS300 and refolding by SephacrylS100. The result of Western blot showed the specific binding of CCR5 mAb with aim protein. CONCLUSION: Purified recombinant protein rCCR5 is obtained successfully, which can be used as a candidate antigen for CCR5 autovaccine to challenge HIV1 infection. 【Keywords】 CCR5; PADRE; inclusion bodies; refolding; autovaccine; HIV1 infection 【摘要】 目的：构建趋化因子受体5(CCR5)胞外段重组蛋白(命名为：rCCR5)原核表达载体，并进行诱导表达. 对表达产物进行纯化和鉴定. 方法：截取CCR5胞外结构4个片段NTerm，ECL1, ECL2和ECL3的氨基酸序列，应用柔性linker分别串联，模拟CCR5胞外结构，依据大肠杆菌偏爱密码子人工合成基因. 运用基因重组方法在大肠杆菌中进行表达,表达产物经纯化、复性后进行Western blot鉴定. 结果：目的蛋白以包涵体形式存在，经包涵体洗涤、变性溶解，SephacrylS300凝胶过滤层析及SephacrylS100凝胶柱复性获得了纯化的蛋白质，目的蛋白能与CCR5 mAb特异性结合. 结论：获得了大量纯化的rCCR5重组蛋白，可作为研制CCR5自体蛋白疫苗以阻断HIV1感染的候选抗原蛋白. 【关键词】 CCR5;人工手动HTL表位；包涵体;复性;自体疫苗;HIV1感染 0引言 趋化因子受体5(CCR5)是HIV1感染初期病毒进入体内最重要的辅助受体［1］，近几年来，以CCR5为靶点的抗HIV1策略倍受关注，通过小分子拮抗剂、单克隆抗体及基因干涉等不同手段减弱或降低细胞表面CCR5的功能达到对抗HIV1感染的目的［2-5］. 其中以CCR5为靶向的自体疫苗开辟了艾滋病疫苗研究的新途径［6］. CCR5自体疫苗通过诱导机体自身产生针对自身分子CCR5的抗体进而与细胞表面的CCR5结合，降低细胞表面的CCR5的数量，减少了HIV1病毒感染的机会，达到预防HIV1感染的效果. 我们构建人CCR5胞外段重组蛋白表达载体，对获得的重组蛋白纯化、复性. 为构建CCR5自体蛋白疫苗提供实验依据. 1材料和方法 1.1材料各种