【作者】 陈忠广；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 犬干扰素是治疗犬病毒性疾病的一种有效手段，但以原核细胞表达的干扰素多以包涵体形成存在，为无生物学活性的蛋白质，需要经过体外重薪折叠复性后才能恢复为具有生物活性的干扰素。稀释复性是一种简单快捷的蛋白质复性方法，也是研究其他复性方法的基础。本实验对表达犬干扰素—γ的pJLA605-CaIFN-γ重组质粒原核表达系统进行诱导表达，对表达后的菌体经溶菌酶作用后，超声波破碎，获得富含重组犬干扰素-γ的包涵体，洗涤后再通过8mol／L尿素溶解，将溶解后的包涵体通过稀释复性法进行复性；探讨了不同折叠促进剂、加样方式、温度、pH值在稀释法复性条件下对重组犬γ-干扰素体外折叠的复性影响；采用MDCK—VSV系统，通过细胞病变（CPE）效应为基础的结晶紫染色法测定复性后重组犬干扰素-γ的活性，用考马斯亮蓝法测定复性后的犬γ-干扰素的浓度。结果表明，每升诱导后的菌体经8mol／L尿素溶解后，得到的包涵体总蛋白为285mg；利用0.5M精氨酸、0.5M盐酸胍、2.0M尿素均能有效抑制复性过程中蛋白质的聚集，其中0.5M精氨酸的效果最好。在4℃、pH值8.0、0.5M精氨酸复性液，蛋白浓度0.15mg·mL^-1及脉冲加样操作方式下，复性后重组犬γ-干扰素的活性为1.35×10⁴U·mL^-1，比活达3.74×10⁶U·mg^-1。更多还原

【Abstract】 Canine interferon-γ, is a effective treatment of viral infection. Recombinant CaIFN-γwasexpressed as inclusion bodies in Escherichia coli. Inclusion body could recover active protein afterrefolding. Protein refolding by dilution is a simple and quick way of renatueration and is the basisof research on other methods of renaturation.Engineering strain containing pJLA605-CaIFN-γwas highly expressed after inducement andthen Utrasonic discruption adding lysozyme. CaIFN-γinclusion body could be dissolved by8mol/L urea effectively after washing and refolding by dilution. Effects of folding enhancer, waysof adding sample, temperature, pH was researched in the renatutation process of recombinantcanine interferon-gamma by dilution. CaIFN-γ, activity could be assayed in Madin-Darby caninekidney （MDCK） cells using the New Jersey strain of vesicular stomatitis virus （VSV） by cytopathiceffect（CPE） inhibition. Application of coomassic brilliant blue method determinates proteinconcentration after refolding. Engineering strain after inducement obtain inclusion body 285mg/Ldissolved by 8mol/L urea effectively. 0.5M L-arginine、0.5M Guanidine-Hcl、2.0M Urea couldsuppress the aggregation of protein during the renaturation process effectively, and 0.5M L-arginineis the best of them. The activity and specific bioactivity was up to 1.35×10⁴U·ml^-1 and3.47×10⁶U·mg^-1 respectively at 4℃, pH 8.0, 0.5 mol·L^-1 L-arginine, 0.15 mg·mL^-1 proein andpulse renturation operation mode.更多还原