CN112538494A - 一种tmprss2突变体蛋白及其表达载体、表达工程菌和制备方法 - Google Patents
一种tmprss2突变体蛋白及其表达载体、表达工程菌和制备方法 Download PDFInfo
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- CN112538494A CN112538494A CN202011440699.5A CN202011440699A CN112538494A CN 112538494 A CN112538494 A CN 112538494A CN 202011440699 A CN202011440699 A CN 202011440699A CN 112538494 A CN112538494 A CN 112538494A
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Abstract
本发明实施例提供了一种TMPRSS2突变体蛋白及其表达载体、表达工程菌和制备方法，所述TMPRSS2突变体蛋白的氨基酸序列如SEQ ID NO.7所示；所述表达载体的表达区的核苷酸序列包括如SEQ ID NO.3所示的核苷酸序列；所述工程菌包含所述的TMPRSS2突变体蛋白表达载体。野生型TMPRSS2蛋白由于本身可在Arg255和Ile256间进行自我切割进行活性形式的转化，故野生型蛋白TMPRSS2存在自我剪切，蛋白纯度不高。本发明实施例通过点突变实现哺乳细胞表达的TMPRSS2蛋白突变体，并成功表达纯化，获得了TMPRSS2高纯度高活性蛋白。
Description
技术领域
本发明实施例属于生物技术领域，涉及一种TMPRSS2突变体蛋白及其表达载体、表达工程菌和制备方法。
背景技术
TMPRSS2是II型跨膜丝氨酸蛋白酶的成员之一，是一类定位于细胞膜上具有保守丝氨酸蛋白酶结构域的蛋白家族。该家族基本结构却高度相似，均含有四部分，从N端到C端依次为短细胞质结构域、跨膜结构域、主干区和丝氨酸蛋白酶结构域，其蛋白酶催化结构域包含His296、Asp345和Ser441氨基酸残基。其拥有单跨膜结构域，其中主干区和丝氨酸蛋白酶结构域位于胞外，不同成员区别主要集中于主干区。C端蛋白酶结构域在胞外，N端位于胞内。由于蛋白酶可降解细胞外基质，增加细胞在组织内迁移和扩散，TMPRSS2参与了癌细胞的增殖、侵袭和转移。目前发现TMPRSS2与前列腺癌的发生密切相关。
此外，目前也发现TMPRSS2在2019年爆发的SARS-COV-2病毒入侵宿主机制中，起到重要作用。SARS-CoV-2进入细胞依赖于与宿主细胞ACE2受体的结合，而TMPRSS2会对SARS-CoV-2的刺突蛋白S进行剪切活化，启动S蛋白，促进SARS-CoV-2的入侵，促进SARS-CoV-2和ACE2的膜融合过程。同时，人们也发现TMPRSS2蛋白在H7N9流感病毒和几种H1N1亚型流感病毒，以及SARS和MERS冠状病毒感染中起着关键作用，提示TMPRSS2可作为抗病毒的一个潜在靶标，可能是治疗冠状病毒和一些低致病性流感病毒感染的新的抗病毒策略。虽然TMPRSS2发现时间不长，且作用机制和生理功能还未完全阐明，但使用遗传学及相关方法已发现多个成员功能异常可导致疾病的发生，突出了它的重要性。
随着新冠状病毒在全球愈演愈烈，制备具有活性的TMPRSS2蛋白，并研究其酶活作用对于我们理解新冠病毒感染机制以及开展药物筛选和疫苗研究都具有现实意义。但目前市面上可选择的TMPRSS2的相关产品并不多见，由于TMPRSS2本身可在Arg255和Ile256间进行自我切割进行活性形式的转化，大部分成熟蛋白是膜结合的，有部分会释放在细胞外环境。所以获得高纯度高活性的蛋白难度相对较高，故野生型蛋白存在自我剪切，蛋白纯度不高。
因此，如何开发一种高纯度高活性的TMPRSS2突变体蛋白，成为亟待解决的技术问题。
发明内容
为了解决所述技术问题，本发明提供了一种TMPRSS2突变体蛋白及其表达载体、表达工程菌和制备方法，本发明通过点突变实现哺乳细胞表达的TMPRSS2蛋白突变体，并成功表达纯化，获得了TMPRSS2高纯度高活性蛋白。
在本发明的第一方面，提供了一种TMPRSS2突变体蛋白表达载体，所述表达载体的表达区的核苷酸序列包括如SEQ ID NO.3所示的核苷酸序列。
进一步地，所述表达载体的表达区的核苷酸序列如SEQ ID NO.6所示。
在本发明的第二方面，提供了所述的TMPRSS2突变体蛋白表达载体的制备方法，所述方法包括：
获得TMPRSS2突变体蛋白的优化密码子片段，所述优化密码子片段的核苷酸序列如SEQ ID NO.3所示；
获得表达载体，将所述TMPRSS2突变体蛋白的优化密码子片段插入到所述表达载体的表达区，获得TMPRSS2突变体蛋白表达载体。
进一步地，所述获得TMPRSS2突变体蛋白的优化密码子片段，具体包括：
合成得到含有所述TMPRSS2突变体蛋白的优化密码子片段的载体，以所述载体为模板，采用如SEQ ID NO.4-5所示的引物对进行PCR，获得TMPRSS2突变体蛋白的优化密码子片段。
进一步地，所述表达载体为pSecTag2A载体。
进一步地，所述将所述TMPRSS2突变体蛋白的优化密码子片段插入到所述表达载体的表达区，获得TMPRSS2突变体蛋白表达载体，具体包括：
将所述pSecTag2A载体采用Hind III/Not I双酶切，获得酶切载体；
将所述TMPRSS2突变体蛋白的优化密码子片段采用Hind III/Not I双酶切，获得酶切片段；
将所述酶切载体与所述酶切片段进行酶连，获得TMPRSS2突变体蛋白表达载体。
在本发明实施例的第三方面，提供了一种TMPRSS2突变体蛋白表达工程菌，所述工程菌包含所述的TMPRSS2突变体蛋白表达载体。
进一步地，所述工程菌的制备方法为：用所述的TMPRSS2突变体蛋白表达载体转化大肠杆菌感受态细胞，筛选转化子，获得所述TMPRSS2突变体蛋白表达工程菌。
在本发明实施例的第四方面，提供了一种TMPRSS2突变体蛋白，所述TMPRSS2突变体蛋白由所述的TMPRSS2突变体蛋白表达工程菌经诱导表达和纯化制备得到。
进一步地，所述TMPRSS2突变体蛋白的氨基酸序列如SEQ ID NO.7所示。
在本发明实施例的第五方面，提供了一种TMPRSS2蛋白快速和高通量的活性检测方法，所述方法包括：
获得检测体系，所述检测体系的配方为：30-50mM Tris-HCl，100-150mM NaCl，pH7.0-9.0；
将TMPRSS2蛋白和底物加入到所述检测体系中反应，检测荧光强度；
根据所述荧光强度与所述底物的浓度，计算TMPRSS2蛋白的活性。
本发明实施例中的一个或多个技术方案，至少具有如下技术效果或优点：
本发明实施例提供的一种TMPRSS2突变体蛋白及其表达载体、表达工程菌和制备方法，本发明实施例对R255Q位点进行位点突变，构建表达TMPRSS2蛋白，并成功表达其突变体，实现了TMPRSS2蛋白稳定高产高纯度表达，获得不自我切割的活性稳定形式的TMPRSS2，同时根据TMPRSS2蛋白快速和高通量的活性筛选，保证实现高纯度和高活性蛋白的生产。
附图说明
为了更清楚地说明本发明实施例中的技术方案，下面将对实施例描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明实施例的一些实施例，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它的附图。
图1为实施例1中PCR产物回收图；其中，泳道1、Marker；泳道2、TMPRSS2-pSecTag2A产物；
图2为实施例1中载体回收图；其中，泳道1、Marker，Marker从上到下依次是5000、3000、2000、1500、1000、750、500、250、100(bp)；泳道2、pSecTag2 A双酶切产物；
图3为实施例1中转化后的挑斑检测图；泳道1为阴性带，泳道2-4为阳性带；
图4为实施例2中表达上清SDS-PAGE检测图；泳道1、分泌上清(上)；泳道2、胞内上清(中)；泳道3、细胞沉淀(下)；泳道4、阳性对照；
图5为实施例2中TMPRSS2哺乳表达蛋白纯化SDS-PAGE图；泳道1-6分别为裂解液上清、流穿液、20mM咪唑洗脱液、60mM咪唑洗脱液、200mM咪唑洗脱液和500mM咪唑洗脱液；
图6为实施例3中不同浓度TMPRSS2蛋白梯度对BOC-Gln-Ala-Arg-AMC底物切割实验；
图7为TMPRSS2蛋白在不同底物BOC-Gln-Ala-Arg-AMC梯度的测试结果；
图8为TMPRSS2蛋白酶Km；
图9为体外表达TMPRSS2蛋白的SDS-PAGE检测图；其中，泳道1：Marker；泳道2：裂解液上清；泳道3：上样流穿；泳道4：10mM咪唑洗脱液；泳道5：30mM咪唑洗脱液；泳道6：60mM咪唑洗脱液；泳道7：250mM咪唑洗脱液。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明实施例，本发明实施例的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明实施例，而非限制本发明实施例。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明实施例所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
除非另有特别说明，本发明实施例中用到的各种原材料、试剂、仪器和设备等，均可通过市场购买得到或者可通过现有方法制备得到。
本发明实施例提供的技术方案为解决上述技术问题，总体思路如下：
(1)本发明实施例首先将TMPRSS2密码子优化，使得采用本发明实施例的表达系统进行蛋白表达时，对密码子偏爱性好，优化后基因片段的核苷酸序列如SEQ ID NO.3所示；
(2)获得TMPRSS2突变体蛋白的表达工程菌
合成得到含有所述TMPRSS2突变体蛋白的优化密码子片段的载体，以所述载体为模板，采用如SEQ ID NO.4-5所示的引物对进行PCR，获得TMPRSS2突变体蛋白的优化密码子片段。
将所述pSecTag2A载体采用Hind III/Not I双酶切，获得酶切载体；
将所述TMPRSS2突变体蛋白的优化密码子片段采用Hind III/Not I双酶切，获得酶切片段；
将所述酶切载体与所述酶切片段进行酶连，获得TMPRSS2突变体蛋白表达载体。
用所述的TMPRSS2突变体蛋白表达载体转化大肠杆菌感受态细胞，筛选转化子，获得所述TMPRSS2突变体蛋白表达工程菌。
(3)将所述的TMPRSS2突变体蛋白表达工程菌经诱导表达和纯化制备得到TMPRSS2突变体蛋白。
(4)建立了一种TMPRSS2蛋白快速和高通量的活性检测方法，所述方法包括：
获得检测体系，所述检测体系的配方为：30-50mM Tris-HCl，100-150mM NaCl，pH7.0-9.0；
将TMPRSS2蛋白和底物加入到所述检测体系中反应，检测荧光强度；
根据所述荧光强度与所述底物的浓度，计算TMPRSS2蛋白的活性。
根据所述方法对制备得到TMPRSS2突变体蛋白进行活性筛选，制备得到了高活性高纯度的TMPRSS2突变体蛋白。
本发明实施例提供通过点突变实现哺乳细胞表达的TMPRSS2蛋白突变体，并成功表达纯化，获得了TMPRSS2高纯度高活性蛋白。
为解决上述技术问题，本发明实施例的技术方案如下：
根据本发明实施例的一种典型实施方式，提供了一种TMPRSS2突变体蛋白表达载体，所述表达载体的表达区的核苷酸序列包括如SEQ ID NO.3所示的核苷酸序列。
作为优选的实施方式，所述表达载体的表达区的核苷酸序列如SEQ ID NO.6所示。
根据本发明实施例的另一种典型实施方式，提供了所述的TMPRSS2突变体蛋白表达载体的制备方法，所述方法包括：
获得TMPRSS2突变体蛋白的优化密码子片段(具体包括：合成得到含有所述TMPRSS2突变体蛋白的优化密码子片段的载体，以pSecTag2A载体为模板，采用如SEQ IDNO.4-5所示的引物对进行PCR，获得TMPRSS2突变体蛋白的优化密码子片段)，所述优化密码子片段的核苷酸序列如SEQ ID NO.3所示；
获得表达载体，将所述TMPRSS2突变体蛋白的优化密码子片段插入到所述表达载体的表达区，获得TMPRSS2突变体蛋白表达载体；具体包括：
将所述pSecTag2A载体采用Hind III/Not I双酶切，获得酶切载体；
将所述TMPRSS2突变体蛋白的优化密码子片段采用Hind III/Not I双酶切，获得酶切片段；
将所述酶切载体与所述酶切片段进行酶连，获得TMPRSS2突变体蛋白表达载体。
本实施例中TMPRSS2突变体蛋白表达载体具体为HU-TMPRSS2-pET28a-SUMO的测序结果为：核苷酸序列如SEQ ID NO.6所示。
根据本发明实施例的另一种典型实施方式，提供了一种TMPRSS2突变体蛋白表达工程菌，所述工程菌包含所述的TMPRSS2突变体蛋白表达载体。
所述工程菌的制备方法为：用所述的TMPRSS2突变体蛋白表达载体转化大肠杆菌感受态细胞，筛选转化子，获得所述TMPRSS2突变体蛋白表达工程菌。
根据本发明实施例的另一种典型实施方式，提供了一种TMPRSS2突变体蛋白，所述TMPRSS2突变体蛋白由所述的TMPRSS2突变体蛋白表达工程菌经诱导表达和纯化制备得到。
本实施例中，所述TMPRSS2突变体蛋白的氨基酸序列如SEQ ID NO.7所示。
根据本发明实施例的另一种典型实施方式，提供了一种TMPRSS2蛋白快速和高通量的活性检测方法，所述方法包括：
获得检测体系，所述检测体系的配方为：30-50mM Tris-HCl，100-150mM NaCl，pH7.0-9.0；
通过设置酶浓度反应梯度2μM，1μM，0.5μM，0.25μM，0.1μM和0.05μM，同时设置阳性对照为同等浓度的AMC底物，阴性对照为缓冲buffer，确定最适的酶反应浓度为0.4μM，然后将0.4μM的TMPRSS2蛋白和浓度梯度为38μM，35μM,30μM,25μM,20μM,15μM,10μM,5μM的底物分别加入到100ul所述检测体系中，检测440nm处的荧光强度。根据荧光信号随时间的变化，计算曲线斜率，即为反应速度。通过各底物梯度先的反应处速度和底物浓度梯度，利用prism 5软件，米氏方程(Michaelis-Menten，
)测算得到哺乳表达系统表达TMPRSS2蛋白的Km)。
下面将结合实施例及实验数据对本申请的效果进行详细说明。
实施例1TMPRSS2质粒制备
1、TMPRSS2密码子优化
根据NCBI编号(O15393)，选择TMPRSS2核酸序列如SEQ ID NO.1所示，其编码的氨基酸为如SEQ ID NO.2所示。
考虑到不同表达系统进行蛋白表达时，对密码子偏爱性不同，本申请人对上述密码子进行优化，优化后基因片段的核苷酸序列如SEQ ID NO.3所示。优化后基因片段的核苷酸序列所编码的氨基酸序列如SEQ ID NO.2所示。
2、TMPRSS2基因片段的获得
所述优化后的基因片段由武汉金开瑞生物工程有限公司进行基因合成。根据TMPRSS2优化后的核酸序列设计引物，引物如表1所示。
表1
以合成的所述优化后的基因片段为模板，采用如SEQ ID NO.4-5所示的引物对进行PCR反应，反应体系如表2所示。
表2
| cDNA(VAA-top1-pUC57) | 4ul |
| TMPRSS2-F(50μM) | 2ul |
| TMPRSS2-R(50μM) | 2ul |
| 10×Pfu PCR BufferⅡ | 20ul |
| 2.5mM dNTP Mixture | 16ul |
| Pfu DNA polymerase | 2ul |
| dH<sub>2</sub>O | 154ul |
PCR反应程序为：94℃预变性4min；94℃变性45sec，52℃退火45sec，72℃延伸90sec，共30个循环；72℃延伸10min；15℃保温5min。反应结束后，以1％琼脂糖凝胶电泳检测扩增产物。结果如图1所示，目的片段长度为1161bp，与预期的一致。
根据目的片段的大小切胶，放置干净离心管。离心管放置-80℃冰箱，15min后取出室温溶解，用1mL蓝色枪头将胶捣碎，12000r/min，2min。离心后上清移至的新空白EP管，备用。
3、载体的酶切Hind III/Not I
选择优化的pSecTag2A载体使用的双酶切体系(150μl)如表3所示(单位：μL)
表3
用枪轻轻吹吸混合均匀，37℃水浴酶切20h以上为佳
4、载体回收
采用常规方法进行载体pSecTag2A(约5.6Kb)回收后，1％琼脂糖凝胶电泳检测回收产物。目的载体长度为5633bp，与预期的一致，见图2。Marker从大到小依次为：5000、3000、2000、1500、1000、750、500、250、100(bp)。
5、连接反应
目的片段TMPRSS2和载体pSecTag2A的酶切产物分别经过回收后做连接反应，反应体系(10μL)如表4所示，用枪轻轻吹吸混匀后，22℃连接2小时以上得到连接产物。
表4
| 项目 | 体积 |
| 目的片段TMPRSS2 | 4μL |
| 载体pSecTag2A | 2μL |
| Buffer | 1μL |
| 连接酶 | 1μL |
| 加水至 | 10μL |
将所述连接产物进行双酶切验证，经过载体双酶切后，图2为哺乳载体pSecTag2 A酶切结果显示与预期一致，实际大小为5.1Kb。
6、转化和阳性克隆筛选
从-80℃冰箱中取出感受态细胞至冰上融化。轻轻打开盖子，加入所述连接产物(10μL)；轻轻吹吸并旋转枪头，使DNA与感受态细胞充分混匀。冰上30min；42℃热击90s(此处时间一定要准确)；冰上1min；加入800μL预热的LB培养基，置37℃摇床中158r/120min。6000r离心4min(提前15分钟将需要的平板倒置于37度恒温培养箱，核对抗生素是否正确，15分钟的温育足以让平板加热，倒置会更好的挥发游离的水分)；超净台里面吸掉800μL上清，剩余菌液混匀，涂至含有相应抗生素的平板上；倒置平板，于37℃恒温培养箱中培养，12～16小时后可出现菌落。挑斑检测，见下图。Marker从上到下依次是5000、3000、2000、1500、1000、750、500、250、100(bp),检测体系中引物选择的是TMPRSS2的特异性引物(见引物设计)，经过PCR扩增后条带大小约为1161bp。
选择两个呈阳性结果的单克隆菌液接种至3ml加相应抗生素的LB培养基中培养过夜，第二天进行保种，送本集团兄弟公司武汉金开瑞基因工程有限公司进行测序分析，测序无突变。TMPRSS2-pSecTag2A的测序结果为：核苷酸序列如SEQ ID NO.6所示；氨基酸序列如SEQ ID NO.7所示。
同时培养后进行挑斑检测，经过PCR扩增后条带大小约为1161bp，哺乳载体pSecTag2A阳性率别为3/4，见图3，且挑选的阳性克隆，送测序后与预期相符。可用于下步的诱导表达。
实施例2 TMPRSS2突变体蛋白在哺乳细胞中的表达以及蛋白镍柱纯化
1、哺乳细胞表达
(1)将阳性单克隆菌液转接300ml LB培养基过夜培养大抽质粒；
(2)准备两支15ml的无菌离心管，在其中一支中加入5ml转染试剂和100μg无菌质粒DNA，轻轻吹打混匀；
(3)室温下静置10分钟制备出质粒-载体复合物；
(4)从恒温摇床中取出293T细胞，边摇边加入制备好的质粒-载体复合物，放回5％CO2，37℃恒温摇床中震荡培养。
(5)摇瓶培养5～7天，收获上清，分别取分泌上清和胞内上清做WB his tag检测，以确定蛋白是否表达，结果见图4；
由图4可知，制备质粒-载体复合物后，进行293T细胞的转染表达，获取细胞分泌上清和胞内上清，通过检测his tag的表达情况，可以确认TMPRSS2蛋白进行了表达。
2、蛋白镍柱纯化
(1)重生镍柱，依次用H2O洗3柱体积，0.1M EDTA洗2柱体积，用H2O洗4柱体积，用N2SO4缓冲洗5柱体积，用0.5M NaCl,0.02M CH3COONa,pH4.0缓冲洗1柱体积，用H2O洗5柱体积，用NTA-0洗5柱体积。
(2)将样品加入已平衡好的镍柱中，控制流速，让样品缓慢滴下，样品过镍柱至少2遍；
(3)接穿透；
(4)用含20mM咪唑浓度的NTA-0洗杂，接一滴液体用G250检测对比颜色，当颜色变浅与对照一致，表明可以洗脱目的蛋白；
(5)用含60mM咪唑浓度的NTA-0洗脱，接一滴液体用G250检测对比颜色，寻找洗脱峰。洗脱体积根据颜色对比决定；
(6)用含200mM咪唑浓度的NTA-0洗脱，接一滴液体用G250检测对比颜色，寻找洗脱峰。洗脱体积根据颜色对比决定；
(7)用含500mM咪唑浓度的NTA-0洗脱，接一滴液体用G250检测对比颜色，寻找洗脱峰。洗脱体积根据颜色对比决定；
(8)洗脱下来的液体分别超滤后进行SDS-PAGE检测。见图十
(9)柱子继续用500mM咪唑洗2柱体积彻底去杂，水洗4柱，0.1M EDTA洗2柱，用水洗5柱，用20％乙醇封柱。
经镍柱纯化后的蛋白，取裂解液上清、流穿液、20mM咪唑洗脱液、60mM咪唑洗脱液、200mM咪唑洗脱液和500mM咪唑洗脱液同时进行SDS-PAGE电泳，结果如图5所示，表明在60mM咪唑洗脱液中可见明显目的带，且含量最高，纯度达到90％。
实施例3蛋白活性检测
1、通过实验对多个活性荧光底物肽进行活性筛选。通过使用2μM的TMPRSS2酶与25μM的荧光底物进行酶切反应60min,同时设置阳性对照为25μM的AMC，阴性对照为反应缓冲液，同时设置背景对照为不添加TMPRSS2酶的25μM荧光底物。在340nm激发和440nm吸收处持续检测荧光强度的变化情况，选择出可被TMPRSS2识别并切割的的有效荧光底物肽，包括BOC-Gln-Ala-Arg-AMC和Ac-Val-Arg-Pro-Arg-AMC，其中BOC-Gln-Ala-Arg-AMC信号较强，作为后期主要研究的荧光底物肽；
2、优化实验反应条件，包括反应时间，温度，pH，缓冲等，确定适宜的反应缓冲buffer体系为：30-50mM Tris-HCl，100-150mM NaCl，pH7.0-9.0；
经过多次条件条调整摸索出TMPRSS2酶反应的最佳反应条件为：30-50mM Tris-HCl，100-150mM NaCl，pH7.0-9.0，并以此条件检测了哺乳表达系统所表达TMPRSS2蛋白酶突变体的Km值，实验过程中，设置对应的阳性对照为与底物同等浓度的AMC，阴性对照为反应缓冲液，同时设置背景对照为不添加TMPRSS2酶的25μM荧光底物。
3、设置酶浓度反应梯度2μM，1μM，0.5μM，0.25μM，0.1μM和0.05μM，底物BOC-Gln-Ala-Arg-AMC浓度为25μM。同时设置阳性对照为与荧光底物同等浓度的AMC，阴性对照为反应缓冲液，同时设置背景对照为不添加TMPRSS2酶的25μM荧光底物，最终确定最适的酶反应浓度为0.4μM；
根据筛选后的有效荧光底物肽，荧光信号的检测情况来看，能够检测到有强的荧光信号，结果如图6所述，说明经过哺乳表达系统表达的TMPRSS2蛋白可对荧光底物进行切割，且检测的荧光信号与底物浓度呈现浓度依赖性关系，选择以0.4μM的底物浓度继续进行后续检测。
4、确定最适酶反应浓度后，设置底物梯度为38μM,35μM,30μM,25μM,20μM,15μM,10μM,5μM对底物浓度进行摸索，设置阳性对照为与底物同等浓度的AMC，阴性对照为反应缓冲液，同时设置背景对照为不添加TMPRSS2酶的25μM荧光底物，根据荧光信号随时间的变化，计算曲线斜率，以确定酶反应初速度；
5、根据酶反应初速度和荧光信号的变化值作图如图8所示，根据米氏方程(Michaelis-Menten，
)测算得到哺乳表达系统表达TMPRSS2蛋白的Km。测算该TMPRSS2蛋白酶的Km为19.13μM。
综上可知，本发明实施例通过哺乳细胞表达系统进行放大表达TMPRSS2，可获得高纯度的TMPRSS2蛋白，其纯度高达90％，并可通过以上摸索的活性检测实验实现对TMPRSS2蛋白进行快速和高通量的活性检测筛选。
对比例1
该对比例没有进行密码子优化，其他方法均同实施例1。
体外表达TMPRSS2蛋白的SDS-PAGE检测结果如图9所示，由图9可知，泳道中杂带较多且目标条带较窄，表明体外表达TMPRSS2蛋白存在表达量低，纯度低的缺点。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已描述了本发明实施例的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明实施例范围的所有变更和修改。
显然，本领域的技术人员可以对本发明实施例进行各种改动和变型而不脱离本发明实施例的精神和范围。这样，倘若本发明实施例的这些修改和变型属于本发明实施例权利要求及其等同技术的范围之内，则本发明实施例也意图包含这些改动和变型在内。
序列表
<110> 武汉华美生物工程有限公司
<120> 一种TMPRSS2突变体蛋白及其表达载体、表达工程菌和制备方法
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aatcggtgtg ttcgcctcta cggaccaaac ttcatccttc aggtgtactc atctcagagg 180
aagtcctggc accctgtgtg ccaagacgac tggaacgaga actacgggcg ggcggcctgc 240
agggacatgg gctataagaa taatttttac tctagccaag gaatagtgga tgacagcgga 300
tccaccagct ttatgaaact gaacacaagt gccggcaatg tcgatatcta taaaaaactg 360
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gcctggccct ggcaggtcag cctgcacgtc cagaacgtcc acgtgtgcgg aggctccatc 540
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tggaagttca tgggcagcaa gtgcagcaac agcggcatcg agtgcgacag cagcggcacc 60
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aaccgctgcg tgcgcctgta cggccccaac ttcatcctgc aggtgtacag cagccagcgc 180
aagagctggc accccgtgtg ccaggacgac tggaacgaga actacggccg cgccgcctgc 240
cgcgacatgg gctacaagaa caacttctac agcagccagg gcatcgtgga cgacagcggc 300
agcaccagct tcatgaagct gaacaccagc gccggcaacg tggacatcta caagaagctg 360
taccacagcg acgcctgcag cagcaaggcc gtggtgagcc tgcgctgcat cgcctgcggc 420
gtgaacctga acagcagccg ccagagccgc atcgtgggcg gcgagagcgc cctgcccggc 480
gcctggccct ggcaggtgag cctgcacgtg cagaacgtgc acgtgtgcgg cggcagcatc 540
atcacccccg agtggatcgt gaccgccgcc cactgcgtgg agaagcccct gaacaacccc 600
tggcactgga ccgccttcgc cggcatcctg cgccagagct tcatgttcta cggcgccggc 660
taccaggtgg agaaggtgat cagccacccc aactacgaca gcaagaccaa gaacaacgac 720
atcgccctga tgaagctgca gaagcccctg accttcaacg acctggtgaa gcccgtgtgc 780
ctgcccaacc ccggcatgat gctgcagccc gagcagctgt gctggatcag cggctggggc 840
gccaccgagg agaagggcaa gaccagcgag gtgctgaacg ccgccaaggt gctgctgatc 900
gagacccagc gctgcaacag ccgctacgtg tacgacaacc tgatcacccc cgccatgatc 960
tgcgccggct tcctgcaggg caacgtggac agctgccagg gcgacagcgg cggccccctg 1020
gtgaccagca agaacaacat ctggtggctg atcggcgaca ccagctgggg cagcggctgc 1080
gccaaggcct accgccccgg cgtgtacggc aacgtgatgg tgttcaccga ctggatctac 1140
cgccagatgc gcgccgacgg c 1161
<210> 4
<211> 45
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<210> 5
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cagagccgca tcgtgggcgg cgagagcgcc ctgcccggcg cctggccctg gcaggtgagc 660
ctgcacgtgc agaacgtgca cgtgtgcggc ggcagcatca tcacccccga gtggatcgtg 720
accgccgccc actgcgtgga gaagcccctg aacaacccct ggcactggac cgccttcgcc 780
ggcatcctgc gccagagctt catgttctac ggcgccggct accaggtgga gaaggtgatc 840
agccacccca actacgacag caagaccaag aacaacgaca tcgccctgat gaagctgcag 900
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ctgcagcccg agcagctgtg ctggatcagc ggctggggcg ccaccgagga gaagggcaag 1020
accagcgagg tgctgaacgc cgccaaggtg ctgctgatcg agacccagcg ctgcaacagc 1080
cgctacgtgt acgacaacct gatcaccccc gccatgatct gcgccggctt cctgcagggc 1140
aacgtggaca gctgccaggg cgacagcggc ggccccctgg tgaccagcaa gaacaacatc 1200
tggtggctga tcggcgacac cagctggggc agcggctgcg ccaaggccta ccgccccggc 1260
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Claims (10)
1.一种TMPRSS2突变体蛋白表达载体，其特征在于，所述表达载体的表达区的核苷酸序列包括如SEQ ID NO.3所示的核苷酸序列。
2.根据权利要求2所述的一种TMPRSS2突变体蛋白表达载体，其特征在于，所述表达载体的表达区的核苷酸序列如SEQ ID NO.6所示。
3.一种权利要求1-2任一所述的TMPRSS2突变体蛋白表达载体的制备方法，其特征在于，所述方法包括：
获得TMPRSS2突变体蛋白的优化密码子片段，所述优化密码子片段的核苷酸序列如SEQID NO.3所示；
获得表达载体，将所述TMPRSS2突变体蛋白的优化密码子片段插入到所述表达载体的表达区，获得TMPRSS2突变体蛋白表达载体。
4.根据权利要求3所述的TMPRSS2突变体蛋白表达载体的制备方法，其特征在于，所述获得TMPRSS2突变体蛋白的优化密码子片段，具体包括：
合成得到含有所述TMPRSS2突变体蛋白的优化密码子片段的载体，以所述载体为模板，采用如SEQ ID NO.4-5所示的引物对进行PCR，获得TMPRSS2突变体蛋白的优化密码子片段。
5.根据权利要求3所述的TMPRSS2突变体蛋白表达载体的制备方法，其特征在于，所述表达载体为pSecTag2A载体。
6.根据权利要求5所述的TMPRSS2突变体蛋白表达载体的制备方法，其特征在于，所述将所述TMPRSS2突变体蛋白的优化密码子片段插入到所述表达载体的表达区，获得TMPRSS2突变体蛋白表达载体，具体包括：
将所述pSecTag2A载体采用Hind III/Not I双酶切，获得酶切载体；
将所述TMPRSS2突变体蛋白的优化密码子片段采用Hind III/Not I双酶切，获得酶切片段；
将所述酶切载体与所述酶切片段进行酶连，获得TMPRSS2突变体蛋白表达载体。
7.一种TMPRSS2突变体蛋白表达工程菌，其特征在于，所述工程菌包含权利要求1-2任一所述的TMPRSS2突变体蛋白表达载体。
8.一种TMPRSS2突变体蛋白，其特征在于，所述TMPRSS2突变体蛋白由权利要求6-7任一所述的TMPRSS2突变体蛋白表达工程菌经诱导表达和纯化制备得到。
9.如权利要求8所述的一种TMPRSS2突变体蛋白，其特征在于，所述TMPRSS2突变体蛋白的氨基酸序列如SEQ ID NO.7所示。
10.一种TMPRSS2蛋白快速和高通量的活性检测方法，其特征在于，所述方法包括：
获得检测体系，所述检测体系的配方为：30-50mM Tris-HCl，100-150mM NaCl，pH7.0-9.0；
将TMPRSS2蛋白和底物加入到所述检测体系中反应，检测荧光强度；
根据所述荧光强度与所述底物的浓度，计算TMPRSS2蛋白的活性。
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| WO2023164026A3 (en) * | 2022-02-24 | 2023-10-19 | Howard University | Viral receptor-derived peptides against viral diseases |
| CN116003611A (zh) * | 2022-08-17 | 2023-04-25 | 中南大学湘雅医院 | 抗tmprss2抗体及其用途 |
| CN116003611B (zh) * | 2022-08-17 | 2024-02-27 | 中南大学湘雅医院 | 抗tmprss2抗体及其用途 |
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