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目的:研究病毒基因来源的人趋化因子同源物vMIP-Ⅱ的活性单链重组物在原核细胞的表达及制备方法,为大量获取单链vMIP-Ⅱ和其用于趋化因子受体封闭因子的临床应用打下基础。方法:采用双酶切定点克隆、渗透法破碎菌体,并用MBP亲和层析及重组肽段自脱离方式对表达产物进行纯化。纯化产物用SDS-PAGE蛋白印迹及抑制粘附反应等方法进行鉴定。结果:融合蛋白MBP-vMIP在原核细胞中高效分泌型表达,MBP-vMIP在体外自断裂分离出vMIP-Ⅱ,终产物单链vMIP-Ⅱ具有结合趋化因子受体CCR5活性。结论:vMIP-Ⅱ在原核细胞中的融合表达及终产物自脱离法可用于大量获取重组单链vMIP-Ⅱ。单链vMIP-Ⅱ对人趋化因子受体的结合活性有可能对与此受体有关的疾病如HIV感染、慢性炎症过程有作用。
OBJECTIVE: To study the expression and preparation of active single chain recombinant vMIP-Ⅱ, a homolog of human chemokine homolog of viral gene in prokaryotic cells, and to obtain a large amount of single chain vMIP-Ⅱ and its application in chemokine receptor blockade The clinical application of factor lay the foundation. Methods: The double enzyme digestion of point cloning, infiltration method to break the bacteria, and MBP affinity chromatography and recombinant peptide self-release of the expression product was purified. The purified product was identified by SDS-PAGE Western blot and inhibition of adhesion reaction. Results: The fusion protein MBP-vMIP was highly secreted in prokaryotic cells. MBP-vMIP was self-cleaved in vitro and vMIP-Ⅱ was isolated. The final product of single chain vMIP-Ⅱ had chemokine receptor CCR5 activity. Conclusion: The fusion expression of vMIP-Ⅱ in prokaryotic cells and the self-desorption of final products can be used to obtain recombinant single-chain vMIP-Ⅱ in large quantities. The binding activity of single chain vMIP-II to human chemokine receptors may have an effect on diseases associated with this receptor, such as HIV infection and chronic inflammatory processes.