犬干扰素-γ的复性及活性测定
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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1.35×10~4U·mL~(-1)，比活达3.74×10~6U·mg~(-1)。
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 1.35×10~4U·ml~(-1) and3.47×10~6U·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.
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1.35×10~4U·mL~(-1)，比活达3.74×10~6U·mg~(-1)。
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 1.35×10~4U·ml~(-1) and3.47×10~6U·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.
引文
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董晓燕等．2000．固定化分子伴侣GroE促进变性溶菌酶复性的研究．生物工程学报．16：169-172
额尔敦，郭美香，韩玲等．1999．γ—干扰素时间分辨免疫荧光分析方法的建立．生物化学与物物理进展．26(4)：388～391
方敏，黄华裸．2001．包涵体蛋白体外复性的研究进展．生物上程学报．17(6)：608～612
杜平．1984．医用干扰素学．解放军出版社．北京．pp231～234
费峥峥等．2004．重组人γ干扰素复性过程中体外活性检测方法研究．微生物学通报．31(3)：65～69
靳挺，关怡新，姚善泾．2006．离子交换层析复性重组人γ-干扰素折叠二聚体的形成．浙江大学学报：农业与生命科学版．32(1)：101～105
费峥峥．罗曼．姚善泾．2004．小分子伴侣协助重组人γ-干扰素体外复性研究．生物化学与生物物理进展关怡新．31(10)907～911
付向卫．2003．细胞病变抑制法在重组人干扰素γ效价测定中的应用。四川食品与发酵．39(3)：22～24
高永贵，关怡新，姚善径．2003．包涵体蛋白的变复性研究[J]．科技通讯．1(19)10～15
郭立安，朱宝全，陈代杰．1999．使用三态模型选择基因重组蛋白质的复性条件．中国医药工业杂志．30(3)：141～144
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靳挺，关怡新等．2004．重组人γ-干扰素包涵体稀释复性[J]．化工学报，(5)：770～774
靳挺，关怡新，林东强，姚善泾．2006．脲梯度扩张床吸附层析复性重组人γ-干扰素．浙江大学学报：工学版．40(7)：1257～1261
李会成，王同昌，李集新．1994．大肠杆菌表达的真核蛋白产物的复性．生物工程进展．14(5)36～39
李伟，欧阳藩．1999．重组链激酶三种复性方式的比较．药物生物技术．6(4)：230～233
缪晓辉，潘卫，吴清漩等．1994．ABC-ELISA检测人天然及重组γ-干扰素．中华微生物学和免疫学杂志．14(1)：58～61
宁云山，李妍，王小宁．2001．包涵体蛋白质的复性研究进展．生物技术通讯．12(3)：237～240
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王骊丽，耿信笃，韩骅．2003．人IFN-γ在大肠杆菌中高效表达和色谱复性研究．西北大学学报：自然科学版．33(5)：540～544
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吴蕾，雷鸣．2002．超声破碎重组大肠杆菌释放包含体的过程研究．化学工业与工程．19(6)：422～425
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杨琪等．2002．犬干扰素吖克隆及其在鼠骨髓瘤细胞中表达．生物工程学报．18(3)365～367
杨晓仪，林键，吴文言．2004．重组蛋白包涵体的复性研究生命科学研究．8(2)100～105
张佳艺．2003．小分子伴侣的制备及其协助基因工程蛋白体外重折叠的初步研究．浙江大学硕士学位论文．pp5
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曹瑞兵等．2003．猪γ干扰素的基因克隆、改造、表达及活性测定．南京农业大学学报．26(2)71-75
董晓燕等．2000．固定化分子伴侣GroE促进变性溶菌酶复性的研究．生物工程学报．16：169-172
额尔敦，郭美香，韩玲等．1999．γ—干扰素时间分辨免疫荧光分析方法的建立．生物化学与物物理进展．26(4)：388～391
方敏，黄华裸．2001．包涵体蛋白体外复性的研究进展．生物上程学报．17(6)：608～612
杜平．1984．医用干扰素学．解放军出版社．北京．pp231～234
费峥峥等．2004．重组人γ干扰素复性过程中体外活性检测方法研究．微生物学通报．31(3)：65～69
靳挺，关怡新，姚善泾．2006．离子交换层析复性重组人γ-干扰素折叠二聚体的形成．浙江大学学报：农业与生命科学版．32(1)：101～105
费峥峥．罗曼．姚善泾．2004．小分子伴侣协助重组人γ-干扰素体外复性研究．生物化学与生物物理进展关怡新．31(10)907～911
付向卫．2003．细胞病变抑制法在重组人干扰素γ效价测定中的应用。四川食品与发酵．39(3)：22～24
高永贵，关怡新，姚善径．2003．包涵体蛋白的变复性研究[J]．科技通讯．1(19)10～15
郭立安，朱宝全，陈代杰．1999．使用三态模型选择基因重组蛋白质的复性条件．中国医药工业杂志．30(3)：141～144
纪剑飞，张成刚．1998．包涵体重组蛋白的纯化及复性[J]．沈阳药科大学学报．10(15)303～307
靳挺，关怡新等．2004．重组人γ-干扰素包涵体稀释复性[J]．化工学报，(5)：770～774
靳挺，关怡新，林东强，姚善泾．2006．脲梯度扩张床吸附层析复性重组人γ-干扰素．浙江大学学报：工学版．40(7)：1257～1261
李会成，王同昌，李集新．1994．大肠杆菌表达的真核蛋白产物的复性．生物工程进展．14(5)36～39
李伟，欧阳藩．1999．重组链激酶三种复性方式的比较．药物生物技术．6(4)：230～233
缪晓辉，潘卫，吴清漩等．1994．ABC-ELISA检测人天然及重组γ-干扰素．中华微生物学和免疫学杂志．14(1)：58～61
宁云山，李妍，王小宁．2001．包涵体蛋白质的复性研究进展．生物技术通讯．12(3)：237～240
沈同，王镜岩．1999．生物化学(第二版上册)，高等教育出版社．pp167～168
唐松山，刘钟滨．1995．大肠杆菌外源基因表达产物的下游技术．国外医学分册．18(3)124-127
王骊丽，耿信笃，韩骅．2003．人IFN-γ在大肠杆菌中高效表达和色谱复性研究．西北大学学报：自然科学版．33(5)：540～544
王艾丽，武建国，王玲等．1994．ELISA检测Y干扰素及临床应用．临床检验杂志．12(4)：197
吴蕾，雷鸣．2002．超声破碎重组大肠杆菌释放包含体的过程研究．化学工业与工程．19(6)：422～425
吴乃虎编著．2001．基因工程原理(下册)[M]．第二版．科学出版社．135
夏春等．1999犬IFN-α基因的克隆与序列分析．农业生物技术学报．7(3)245～247
徐明波，孟文华，马贤凯．1994．分子伴侣(GroE)纯化及其催化的重组蛋白折叠反应．生物化学与生物物理学报．26(4)：365～370
阎哲．1997．原核细胞表达的重组蛋自复性研究进展．《国外医学》预防、诊断、治疗用生物制品分册．20(1)：10～14
杨琪等．2002．犬干扰素吖克隆及其在鼠骨髓瘤细胞中表达．生物工程学报．18(3)365～367
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