CN108355618B - 牛来源透明质酸酶亲和介质及其吸附方法 - Google Patents
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Abstract
本发明提供了一种牛来源透明质酸酶的亲和介质,由亲和配体和层析介质连接而成,较佳地,所述亲和配体是6‑Chloromethyluracil与2‑Aminobenzimi‑dazole的改造分子,所述层析介质是Sepharose;本发明还提供了上述牛来源透明质酸酶的亲和介质的合成方法,以及利用上述的亲和介质吸附牛来源透明质酸酶的方法,利用本发明的亲和介质可以特异性吸附并纯化牛来源透明质酸酶,有较大的应用推广潜力。
Description
技术领域
本发明涉及生物工程技术领域,特别是涉及医疗用酶生产技术领域,具体涉及一组牛来源透明质酸酶亲和介质及其吸附工艺。
背景技术
透明质酸酶(hyaluronidase,HAase;EC 4.2.99.1)为一种能水解透明质酸的酶(透明质酸为组织基质中具有限制水分及其它细胞外物质扩散作用的成分),用于人体能暂时降低细胞间质的粘性,可促使皮下输液、局部积贮的渗出液或血液加快扩散而利于吸收,为一种重要的药物扩散剂。临床用作药物渗透剂,促进药物的吸收,促进手术及创伤后局部水肿或血肿消散。在整形美容行业,透明质酸酶被用于消解微注射后结块的透明质酸凝胶,具有较大的市场需求。
透明质酸酶的原料主要是牛、羊等家畜的睾丸。长期以来,硫酸铵沉淀、三氯乙酸沉淀、透析、凝胶层析等传统方法被用于透明质酸酶的提取与制备。总体而言,透明质酸酶整体制备步骤复杂且回收率较低。目前,也有利用大肠杆菌表达重组透明质酸酶并利用镍柱亲和纯化的报道,但组氨酸标签的加入可能对目标蛋白的结构和功能产生重大影响,且宿主菌的安全性仍存在一定的隐患。
Kaya MO,Arslan O,Guler OO等人(J Enzyme Inhib Med Chem.2015;30(4):524-7.doi:10.3109/14756366.2014.949253.)利用溴化氰活化了Sepharose 4B介质,以L-酪氨酸作为间隔臂,以间茴香胺为亲和配体。通过硫酸铵沉淀和亲和层析两步分离牛睾丸中的透明质酸酶,活性回收率为16.95%。
A.K.Barsukov等人(Applied Biochemistry and Microbiology,Vol.39,No.6,2003,pp.549-552.)利用硫酸铵沉淀、Sepharose Blue、抗体亲和柱、DEAE应离子交换层析以及Sephadex G200分子筛层析五步,分离牛血清中的透明质酸酶,活性回收率为22%。
Luo Feng等人(Comparative Biochemistry and Physiology,Part C 148(2008)250-257)利用Sephadex G200分子筛层析,UNSO1离子交换层析以及C18反相层析三步,分离中国红蝎子Buthus martensi毒液中的透明质酸酶,活性回收率为12.4%。
R.Marta等人(Toxicon 51(2008)1060-1067)利用Sephacryl S-100分子筛层析以及SP-Sepharose离子交换层析两步,分离Potamotrygon motoro毒液中的透明质酸酶,活性回收率为2.9%。
以上研究的缺陷都是回收率过低,成本高、操作复杂,无法进行工业化生产。为此,有必要开拓思路,寻找更为高效稳定的提取方法。
发明内容
针对现有技术存在的上述问题,本申请提供了一组牛来源透明质酸酶亲和介质及其吸附条件。本发明从仿生学的角度,基于透明质酸酶活性位点的结构,筛选设计并优化出能够与酶高效结合与解离的配体分子,并构建亲和基质,建立高效、快速且不影响透明质酸酶天然构像与活性的纯化工艺,以推进医用级透明质酸酶分离纯化的基础研究和技术创新。
本发明的技术方案如下:
一种透明质酸酶亲和介质,由透明质酸酶亲和配体和层析介质连接而成;所述透明质酸酶亲和配体是6-Chloromethyluracil与2-Aminobenzimidazole的改造分子;所述改造分子分别为通式(1)~(4)所示结构:
优选的,所述层析介质是琼脂糖即Sepharose。
将所述透明质酸酶亲和配体和所述层析介质通过Epichlorohydrin、1,3-propanediamine及Cyanuric chloride作为间隔臂进行连接。
本申请还提供了所述透明质酸酶亲和介质吸附牛来源透明质酸酶的方法,将牛睾丸提取液经过透明质酸酶亲和介质进行亲和吸附,然后采用清洗液冲洗所述亲和介质,用洗脱缓冲液洗脱所述透明质酸酶亲和介质上吸附的透明质酸酶,从而达到纯化目的。
优选的,所述亲和吸附的条件为pH值6.5~7.5,电导率为5~10ms/cm。采用清洗液冲洗所述亲和介质的清洗条件为pH值6.5~7.5,电导率5~10ms/cm。用洗脱缓冲液洗脱所述透明质酸酶亲和介质上吸附的透明质酸酶的洗脱条件为pH值2.5~3.0。
本发明有益的技术效果在于:
本发明克服了上述现有技术中的缺点,提供了一组透明质酸酶亲和介质及其特异性吸附牛来源透明质酸酶的方法,利用该亲和介质可以特异性识别目标蛋白,可在仅用一步亲和层析的情况下,得到较纯的透明质酸酶,蛋白和活性回收率较高,活性回收率为90.1%到95.2%。
本发明通过采用6-Chloromethyluracil与2-Aminobenzimidazole改造分子作为透明质酸酶亲和配体,与层析介质连接合成透明质酸酶亲和介质,可特异性识别牛来源透明质酸酶,适于大规模推广应用;本发明通过优化蛋白质吸附条件使得牛来源透明质酸酶的纯化效率显著增加,具有较大工业应用潜力,体现出较大的经济效益。
附图说明
图1为本发明4种亲和介质在处理牛睾丸提取液的特异性吸附结果电泳图。
图1中,泳道M,分子量marker;
泳道1,牛睾丸提取液总蛋白;
泳道2,6-Chloromethyluracil-ECH介质吸附蛋白;
泳道3,6-Chloromethyluracil-1,3-propanediamine-ECH介质吸附蛋白;
泳道4,2-Aminobenzimidazole-ECH介质吸附蛋白;
泳道5,2-Aminobenzimidazole-CYC-ECH介质吸附蛋白。
具体实施方式
为了能够更清楚地理解本发明的技术内容,特举以下实施例详细说明。
实施例1:Sepharose-6-Chloromethyluracil-ECH的合成及应用
Sepharose CL 4B(100g)用10倍体积的去离子水洗涤,抽干成湿饼状;悬浮于50ml活化缓冲液(0.8M NaOH,20%DMSO,10%Epichlorohydrin,0.5mg/ml硼氢化钠)40℃摇床震荡2.5h,然后倒入玻璃磨砂漏斗中,在抽滤下经过每次10倍体积的蒸馏水洗涤,直到洗涤液pH至中性,在抽干成湿饼状。活化的Sepharose CL 4B介质悬浮于500ml 0.1M NaOH溶液,加入20mL氨水,凝胶在搅拌(200rpm)下30℃恒温12h,用去离子水清洗。称取2g6-Chloromethyluracil,与20g清洗后的介质在60℃恒温12h,维持pH8.0,用去离子水清洗。得到介质Sepharose-6-Chloromethyluracil-ECH,其结构如通式(1)所示,简称结构1。
(1)介质最大吸附量的确定
确定介质的最大吸附量。取透明质酸酶标准品,用去离子水稀释至浓度为100,200,300,400,500,600,700,800,900μg/ml,调节pH值为7.0,分别加入0.01g湿介质,4℃充分振荡2h后,测定上清透明质酸酶浓度,计算介质的最大吸附量。吸附量与上清蛋白浓度关系,结果如表1所示。
表1亲和介质对牛源透明质酸酶的吸附能力
(2)特异性吸附的验证
考察该组介质对牛来源透明质酸酶的特异性识别,取20mL牛睾丸提取液与1g该介质进行吸附并用0.1M HAc洗脱,测定酶活、含量并进行电泳分析,结果如表2Sepharose-6-Chloromethyluracil-ECH对牛睾丸提取液中透明质酸酶的纯化和图1所示。
表2
实施例2:Sepharose-6-Chloromethyluracil-1,3-propanediamine-ECH的合成及应用
Sepharose CL 4B(100g)用10倍体积的去离子水洗涤,抽干成湿饼状;悬浮于50ml活化缓冲液(0.8M NaOH,20%DMSO,10%Epichlorohydrin,0.5mg/ml硼氢化钠)40℃摇床震荡2.5h,然后倒入玻璃磨砂漏斗中,在抽滤下经过每次10倍体积的蒸馏水洗涤,直到洗涤液pH至中性,在抽干成湿饼状。活化的Sepharose CL 4B介质悬浮于500ml0.1M NaOH溶液,加入20ml1,3-propanediamine,凝胶在搅拌(200rpm)下30℃恒温12h,用去离子水清洗。称取2g 6-Chloromethyluracil,与20g清洗后的介质在60℃恒温12h,维持pH8.0,用去离子水清洗。
得到介质Sepharose-6-Chloromethyluracil-1,3-propanediamine-ECH,其结构如通式(2)所示,简称结构2。
(1)介质最大吸附量的确定
确定介质的最大吸附量。取透明质酸酶标准品,用去离子水稀释至浓度为100,200,300,400,500,600,700,800,900μg/ml,调节pH值为7.0,分别加入0.01g湿介质,4℃充分振荡2h后,测定上清透明质酸酶浓度,计算介质的最大吸附量。吸附量与上清蛋白浓度关系,结果如表1所示。
(2)特异性吸附的验证
考察该组介质对牛来源透明质酸酶的特异性识别,取20mL牛睾丸提取液与1g该介质进行吸附并用0.1M HAc洗脱,测定酶活、含量并进行电泳分析,结果如表3Sepharose-6-Chloromethyluracil-1,3-propanediamine-ECH对牛睾丸提取液中透明质酸酶的纯化和图1所示。
表3
实施例3:Sepharose-2-Aminobenzimidazole-ECH的合成及应用
Sepharose CL 4B(100g)用10倍体积的去离子水洗涤,抽干成湿饼状;悬浮于50ml活化缓冲液(0.8M NaOH,20%DMSO,10%Epichlorohydrin,0.5mg/ml硼氢化钠)40℃摇床震荡2.5h,然后倒入玻璃磨砂漏斗中,在抽滤下经过每次10倍体积的蒸馏水洗涤,直到洗涤液pH至中性,在抽干成湿饼状。活化的20g Sepharose CL 4B介质悬浮于50ml 0.1M NaOH溶液,称取加入2g2-Aminobenzimidazole,在60℃恒温12h,用去离子水清洗。
得到介质Sepharose-6-Chloromethyluracil-ECH,其结构如通式(3)所示,简称结构3。
(1)介质最大吸附量的确定
确定介质的最大吸附量。取透明质酸酶标准品,用去离子水稀释至浓度为100,200,300,400,500,600,700,800,900μg/ml,调节pH值为7.0,分别加入0.01g湿介质,4℃充分振荡2h后,测定上清透明质酸酶浓度,计算介质的最大吸附量。吸附量与上清蛋白浓度关系,结果如表1所示。
(2)特异性吸附的验证
考察该组介质对牛来源透明质酸酶的特异性识别,取20mL牛睾丸提取液与1g该介质进行吸附并用0.1M HAc洗脱,测定酶活、含量并进行电泳分析,结果如表4Sepharose-6-Chloromethyluracil-ECH对牛睾丸提取液中透明质酸酶的纯化和图1所示。
表4
实施例4:Sepharose 2-Aminobenzimidazole-CYC-ECH的合成及应用
Sepharose CL 4B(100g)用10倍体积的去离子水洗涤,抽干成湿饼状;悬浮于50ml活化缓冲液(0.8M NaOH,20%DMSO,10%Epichlorohydrin,0.5mg/ml硼氢化钠)40℃摇床震荡2.5h,然后倒入玻璃磨砂漏斗中,在抽滤下经过每次10倍体积的蒸馏水洗涤,直到洗涤液pH至中性,在抽干成湿饼状。活化的Sepharose CL 4B介质悬浮于500ml0.1M NaOH溶液,加入20ml氨水,凝胶在搅拌(200rpm)下30℃恒温12h,用去离子水清洗。称取2gCyanuricchloride,与清洗后的介质在0℃冰水浴恒温12h,维持pH8.0,用去离子水清洗。称取2g 2-Aminobenzimidazole,与清洗后的20g介质在60℃恒温12h,用去离子水清洗。
得到介质Sepharose 2-Aminobenzimidazole-CYC-ECH,其结构如通式(4)所示,简称结构4。
(1)介质最大吸附量的确定
确定介质的最大吸附量。取透明质酸酶标准品,用去离子水稀释至浓度为100,200,300,400,500,600,700,800,900μg/ml,调节pH值为7.0,分别加入0.01g湿介质,4℃充分振荡2h后,测定上清透明质酸酶浓度,计算介质的最大吸附量。吸附量与上清蛋白浓度关系,结果如表1所示。
(2)特异性吸附的验证
考察该组介质对牛来源透明质酸酶的特异性识别,取20mL牛睾丸提取液与1g该介质进行吸附并用0.1M HAc洗脱,测定酶活、含量并进行电泳分析,结果如表5Sepharose 2-Aminobenzimidazole-CYC-ECH对牛睾丸提取液中透明质酸酶的纯化和图1所示。
表5
从图1和表1~表5可以看到,本发明制备得到的四种亲和介质对牛睾丸透明质酸酶均有较好的吸附能力。经过一步亲和层析后,结构1构成的亲和介质对透明质酸酶的活性回收率达到90.1%,纯化倍数达到40.4倍,蛋白纯度为29.2%;结构2构成的亲和介质对透明质酸酶的活性回收率达到92.2%,纯化倍数达到36.0倍,蛋白纯度为28.9%;结构3构成的亲和介质对透明质酸酶的活性回收率达到92.3%,纯化倍数达到173倍,蛋白纯度为90.2%;结构4构成的亲和介质对透明质酸酶的活性回收率达到95.2%,纯化倍数达到198倍,蛋白纯度为91.3%。
Claims (7)
2.根据权利要求1所述的透明质酸酶亲和介质,其特征在于,所述层析介质是琼脂糖即Sepharose。
3.根据权利要求1所述的透明质酸酶亲和介质,其特征在于,将所述透明质酸酶亲和配体和所述层析介质通过Epichlorohydrin、1,3-propanediamine及Cyanuric chloride作为间隔臂进行连接。
4.权利要求1~3任一项所述的透明质酸酶亲和介质吸附牛来源透明质酸酶的方法,其特征在于,将牛睾丸提取液经过透明质酸酶亲和介质进行亲和吸附,然后采用清洗液冲洗所述亲和介质,用洗脱缓冲液洗脱所述透明质酸酶亲和介质上吸附的透明质酸酶,从而达到纯化目的。
5.根据权利要求4所述的方法,其特征在于,所述亲和吸附的条件为pH值6.5~7.5,电导率为5~10ms/cm。
6.根据权利要求4所述的方法,其特征在于,采用清洗液冲洗所述亲和介质的清洗条件为pH值6.5~7.5,电导率5~10ms/cm。
7.根据权利要求4所述的方法,其特征在于,用洗脱缓冲液洗脱所述透明质酸酶亲和介质上吸附的透明质酸酶的洗脱条件为pH值2.5~3.0。
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