CN114957415B - 链霉亲和素突变体及其应用和产品、基因、重组质粒和基因工程菌 - Google Patents
链霉亲和素突变体及其应用和产品、基因、重组质粒和基因工程菌 Download PDFInfo
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- CN114957415B CN114957415B CN202210498265.3A CN202210498265A CN114957415B CN 114957415 B CN114957415 B CN 114957415B CN 202210498265 A CN202210498265 A CN 202210498265A CN 114957415 B CN114957415 B CN 114957415B
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- streptavidin
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- streptavidin mutant
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Abstract
本发明提供了一种链霉亲和素突变体及其应用和产品、基因、重组质粒和基因工程菌，涉及生物技术领域。本发明提供的链霉亲和素突变体，将野生型链霉亲和素截短C端部分氨基酸序列，选取1‑131个氨基酸作为合成序列，并在Y44F和S46C两个位点引入突变点。该链霉亲和素突变体对于生物素的亲和力显著提高；稳定性好，对于抵抗蛋白酶的水解能力显著增强，有利于以此为原料的技术开发。
Description
技术领域
本发明涉及生物技术领域，尤其是涉及一种链霉亲和素突变体及其应用和产品、基因、重组质粒和基因工程菌。
背景技术
链霉亲和素(streptavidin。下称SA)是与亲和素((avidin下称AV)有相似生物学特性的一种蛋白质，1963年，Chaiet在筛选抗苗素时首先发现，并予以报道。近年来，用以检测抗原一抗体系统时，显示其非特异性结合远比AV为低，尤其在组化和DNA分子杂交检测中几无背景显色，因此，SA近年来逐渐受到青睐，大有替代AV之势。
链霉亲和素能与4分子的生物素(Biotin)特异性结合，结合常数为1×10-15/M。同时，由于SA不带糖基、等电点低，在检测中具有比AV低的背景而大大提高了检测的灵敏性。Streptavidin－biotin系统可偶联抗原、抗体、酶、寡核苷酸分子以及PE等荧光物质，因此，Streptavidin-biotin被广泛用于生物反应检测系统，用以检测抗原、抗体及核酸分子。目前，国内外大部分基于酶联免疫反应、荧光分子标记免疫反应和分子杂交为原理的各种检测试剂盒均采用SA-biotin系统。但目前，国内所用SA均为价格昂贵的进口产品，因此研制和生产国产化的SA具有重要的现实意义和广阔的应用前景。链霉亲和素与生物素-构成链霉亲和素-生物素放大系统,是二十世纪七十年代后期发展起来的一种生物反应放大系统，通过一分子亲和素和四分子生物素之间特异结合实现信号放大，还可以通过多级级联放大实现信号多级放大，在生物学特别是免疫检测中有越来越广泛的用途。
链霉亲和素是与亲和素具有相似性质的一种蛋白质，都具有与生物素特异结合的能力，结合常数(Ka值)高达1015M。同时，由于链霉亲和素等电点比亲和素低，且不含糖基链，故在检测中的灵敏度和特异性都高于亲和素，建立在此基础上的生物素-链霉亲和素系统的应用比生物素-亲和素系统有更大优势。
然而链霉亲和素的四级结构、稳定性和功能之间的耦合使得设计用于生物技术应用的稳定、高亲和力单体变得困难。例如，链霉亲和素四聚体的结合口袋由来自多个亚基的残基组成，这些亚基不能在单体蛋白中复制。
有鉴于此，特提出本发明。
发明内容
本发明的第一目的在于提供一种链霉亲和素突变体，以解决上述问题中的至少一种。
本发明的第二目的在于提供上述链霉亲和素突变体在制备检测产品中的应用。
本发明的第三目的在于提供一种检测产品。
本发明的第四目的在于提供一种基因，能够编码上述链霉亲和素突变体。
本发明的第五目的在于提供一种重组质粒。
本发明的第六目的在于提供一种基因工程菌。
第一方面，本发明提供了一种链霉亲和素突变体，所述链霉亲和素突变体的氨基酸序列如SEQ ID NO.1所示。
第二方面，本发明提供了上述链霉亲和素突变体在制备检测产品中的应用；
所述检测产品包括蛋白检测产品和核酸检测产品。
作为进一步技术方案，所述蛋白检测产品包括抗原检测产品和抗体检测产品。
第三方面，本发明提供了一种检测产品，包括上述链霉亲和素突变体。
第四方面，本发明提供了一种基因，所述基因编码上述链霉亲和素突变体。
第五方面，本发明提供了一种重组质粒，所述重组质粒包括载体和上述基因。
作为进一步技术方案，所述载体包括pET-32a(+)质粒。
第六方面，本发明提供了一种基因工程菌，所述基因工程菌含有上述重组质粒。
作为进一步技术方案，所述基因工程菌包括大肠杆菌。
与现有技术相比，本发明具有如下有益效果：
本发明提供的链霉亲和素突变体，将野生型链霉亲和素截短C端部分氨基酸序列，选取1-131个氨基酸作为合成序列，并在Y44F和S46C两个位点引入突变点。该链霉亲和素突变体对于生物素的亲和力显著提高；稳定性好，对于抵抗蛋白酶的水解能力显著增强，有利于以此为原料的技术开发。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为m2-SA1突变体表达结果；
图2为m2-SA1突变体纯化结果；
图3为m2-SA1突变体对不同蛋白酶的耐受性对比。
具体实施方式
下面将结合实施方式和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施方式和实施例仅用于说明本发明，而不应视为限制本发明的范围。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
第一方面，本发明提供了一种链霉亲和素突变体，所述链霉亲和素突变体的氨基酸序列如SEQ ID NO.1所示：
mdpskdskaqvsaaeagitgtwynqlgstfivtagadgaltgtfecavgnaesryvltgrydsapatdgsgtalgwtvawknnyrnahsattwsgqyvggaearintqwlltsgtteanawkstlvghdtft(SEQ IDNO.1)。
本发明将野生型链霉亲和素截短C端部分氨基酸序列，选取1-131个氨基酸作为合成序列，并在Y44F和S46C两个位点引入突变点，获得链霉亲和素突变体。该链霉亲和素突变体对于生物素的亲和力显著提高；稳定性好，对于抵抗蛋白酶的水解能力显著增强，有利于以此为原料的技术开发。其中野生型链霉亲和素的氨基酸序列如SEQ ID NO.3所示：
mdpskdskaqvsaaeagitgtwynqlgstfivtagadgaltgtyesavgnaesryvltgrydsapatdgsgtalgwtvawknnyrnahsattwsgqyvggaearintqwlltsgtteanawkstlvghdtftkvkpsaasidaakkagvnngnpldavqq(SEQ ID NO.3)。
链霉亲和素的核苷酸序列如SEQ ID NO.2所示：
ATGGACCCTTCTAAAGACTCCAAGGCTCAAGTTTCAGCCGCAGAAGCGGGTATTACTGGCACCTGGTATAATCAGTTAGGATCGACATTTATCGTCACGGCTGGGGCCGATGGTGCATTGACTGGCACCTACGAGAGTGCGGTAGGAAACGCTGAAAGTCGTTACGTGCTTACAGGGCGCTATGACAGCGCCCCCGCAACGGATGGTTCTGGCACTGCGCTCGGATGGACCGTTGCTTGGAAAAATAACTACCGAAATGCCCATTCCGCAACAACGTGGTCAGGGCAATATGTCGGTGGCGCGGAGGCTCGGATAAACACTCAGTGGCTACTGACCTCGGGAACAACGGAAGCCAATGCATGGAAGAGTACTTTAGTAGGGCACGACACCTTTACATAA(SEQ ID NO.2)。
第二方面，本发明提供了上述链霉亲和素突变体在制备检测产品中的应用。所述检测产品包括蛋白检测产品和核酸检测产品，其中，蛋白检测产品包括抗原检测产品和抗体检测产品。
Streptavidin－biotin系统可偶联抗原、抗体、酶、寡核苷酸分子以及PE等荧光物质，被广泛用于生物反应检测系统，用以检测抗原、抗体及核酸分子。目前，国内外大部分基于酶联免疫反应、荧光分子标记免疫反应和分子杂交为原理的各种检测试剂盒均采用SA-biotin系统。而本发明提供的链霉亲和素突变体的生物素亲和力高，稳定性好，具有替代链霉亲和素的潜力，能够作为原料用于检测产品的制备。
第三方面，本发明提供了一种检测产品，包括上述链霉亲和素突变体。该检测产品检测灵敏度高，稳定性好。
第四方面，本发明提供了一种基因，所述基因编码上述链霉亲和素突变体。例如该基因可以具有SEQ ID NO.2所示的核苷酸序列。
第五方面，本发明提供了一种重组质粒，所述重组质粒包括载体和上述基因。其中，载体包括但不限于pET-32a(+)质粒。
第六方面，本发明提供了一种基因工程菌，所述基因工程菌含有上述重组质粒。其中，基因工程菌例如可以为大肠杆菌。
下面通过具体的实施例和对比例进一步说明本发明，但是，应当理解为，这些实施例仅仅是用于更详细地说明之用，而不应理解为用于以任何形式限制本发明。
以下实施例中所用的引物如下表所示：
实施例1链霉亲和素突变体的制备
1)在所获得的突变体氨基酸序列利用UpGene对其进行优化，优化之后获得核酸序列SEQ ID NO.2；
2)在SEQ ID NO.2上加入N端ATG起始密码子和C端TTA终止密码子之后通过人工合成获得pMD18T-SA1(插入有SEQ ID NO.2的PMD-18T)；
3)转接pMD18T-SA1/DH5a菌株至LB中培养过夜，取1ml菌液提取质粒DNA模板；
4)依据SEQ ID NO.2设计Y44F定点突变引物Y44F-F与Y44F-R，序列如下，以上一步提取的pMD18T-SA1质粒为模板，以MutUFOTM Fast Mutagenesis Kit快速构建获得Y44F突变体；
5)将上述突变体转化进入DH5ɑ感受态细胞，于37℃培养箱培养12h；
6)以M13F和M13R为引物对转化平板单菌落进行PCR验证，将验证成功的阳性克隆子送至测序；
7)将测序成功的pMD18T-m1-SA1重组质粒进行第二点S46C的突变，设计引物S46C-F与S46C-R，以pMD18T-m1-SA1重组质粒为模板，以MutUFOTM Fast Mutagenesis Kit快速构建获得S46C突变体；
8)将上述突变体转化进入DH5ɑ感受态细胞，于37℃培养箱培养12h；
9)以M13F和M13R为引物对转化平板单菌落进行PCR验证，将验证成功的阳性克隆子送至测序；
10)测序验证获得Y44F和S46C双突变SA蛋白重组质粒pMD18T-m2-SA1，将pMD18T-m2-SA1通过BamH I与Hind III进行双酶切获得Y44F和S46C双突变的SA基因片段；
11)通过BamH I与Hind III对pET32a(+)质粒进行双酶切线性化，通过琼脂糖凝胶电泳获得高纯度的线性SA突变基因片段与pET32a(+)质粒线性片段；
12)将上述基因片段与载体片段用南京巨匠生物科技有限公司的M101试剂盒T4DNALigase在16℃条件下过夜连接。
13)将上述连接产物转接至BL21感受态中，涂布在氨苄青霉素抗性平板中筛选阳性克隆子；
14)用T7以及T7 terminator引物进行菌落PCR验证，将验证成功的pET32-m2-SA/BL21菌株转接至3ml LB培养基中培养8h；
15)转接至500ml LB培养基中培养至OD600＝0.6-0.8左右，加入0.2mM IPTG于25摄氏度条件下诱导12h；
16)将上述发酵获得的发酵液收集菌体，用20mM Tris-HCl、500mM NaCl、PH＝8.0的悬菌Buffer重悬菌体，破碎细胞。取1ml破悬离心收集破细胞上清与破细胞沉淀，以及破细胞全液一起上样SDS-PAGE，跑胶观察表达结果，如图1所示；
17)将上述粗酶液进行进一步纯化，通过饱和硫酸铵(加至75％)共沉淀，Ni柱亲和层析、Sephadex G50分级分离和脱盐，H阳离子交换，Q阴离子交换，Sephadex G-200脱盐分离，超滤浓缩，获取得到高纯度m2-SA1蛋白，纯化蛋白的结果如图2所示。
实施例2突变体SA对生物素亲和力测试
取上述纯化得到的m2-SA1突变体蛋白和野生型链霉亲和素，通过Biacore X100生物传感器分析其与生物素的亲和力。测试得到结果如下：
实施例3不同蛋白酶对野生型SA和m2-SA1突变体的水解
如下表所示，通过选取1％浓度的胰蛋白酶(trypsin)、蛋白酶K(PRK)、胃蛋白酶(pepsin)、嗜热菌蛋白酶(thermolysin)对3μg上述m2-SA1蛋白或者SA在37℃条件下水解1h，处理之后的样品上样SDS-PAGE测试突变体对于不同蛋白酶的耐受性，结果如图3所示(图中CK表示空白对照，即仅添加有m2-SA1蛋白或者SA)。
图3中，CK组、trypsin组、PRK组、pepsin组和thermolysin组中SA或m2-SA1和蛋白酶添加情况如下表所示。
注：蛋白酶指：胰蛋白酶(trypsin)、蛋白酶K(PRK)、胃蛋白酶(pepsin)、嗜热菌蛋白酶(thermolysin)，“+”就表示加了，“－”表示没加。
从图3中可以看出，本发明提供的m2-SA1相较于野生型SA具有更强的蛋白酶耐受性。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
SEQUENCE LISTING
<110> 南京巨匠生物科技有限公司
<120> 链霉亲和素突变体及其应用和产品、基因、重组质粒和基因工程菌
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Claims (8)
1. 一种链霉亲和素突变体，其特征在于，所述链霉亲和素突变体的氨基酸序列如SEQID NO.1所示。
2.权利要求1所述的链霉亲和素突变体在制备检测生物素的产品中的应用。
3.一种检测产品，其特征在于，包括权利要求1所述的链霉亲和素突变体。
4.一种基因，其特征在于，所述基因编码权利要求1所述的链霉亲和素突变体。
5.一种重组质粒，其特征在于，所述重组质粒包括载体和权利要求4所述的基因。
6.根据权利要求5所述的重组质粒，其特征在于，所述载体包括pET-32a(+)质粒。
7.一种基因工程菌，其特征在于，所述基因工程菌含有权利要求5或6所述的重组质粒。
8.根据权利要求7所述的基因工程菌，其特征在于，所述基因工程菌包括大肠杆菌。
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