CN115725551B - MmBBK2在制备胰蛋白酶和胰凝乳蛋白酶抑制剂中的应用 - Google Patents
MmBBK2在制备胰蛋白酶和胰凝乳蛋白酶抑制剂中的应用 Download PDFInfo
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Abstract
本发明属于基因工程和酶工程技术领域,具体涉及MmBBK2在制备胰蛋白酶和胰凝乳蛋白酶抑制剂中的应用。所述MmBBK2具有SEQ ID NO.1或SEQID NO.2所示的氨基酸序列。本发明首次明确了桑叶中的MmBBK2同时具有胰蛋白酶和胰凝乳蛋白酶抑制活性,并揭示了其理化特性,所述MmBBK2在制备胰蛋白酶和胰凝乳蛋白酶抑制剂方面具有良好的应用前景,在明确其理化性质的基础上,可以针对性消除其活性,推动桑叶资源在动物饲粮中的开发利用,为桑叶在动物饲料、保健食品方面的开发利用提供新的视角和思路,提升桑资源的经济效益。
Description
技术领域
本发明属于基因工程和酶工程技术领域,具体涉及MmBBK2在制备胰蛋白酶和胰凝乳蛋白酶抑制剂中的应用。
背景技术
桑叶作为一种优质、新型的动物饲料添加剂,不仅能提高动物生长性能、提升禽畜产品品质,也可避免桑叶资源浪费,提高蚕桑产业的综合经济效益。然而,由于桑叶中存在抗营养因子如单宁、蛋白酶抑制剂和植物凝集素,动物大量添食桑叶会严重干扰其对饲料营养的代谢和吸收,进而影响畜禽健康和畜禽产品的产量与质量,这也极大限制了桑叶资源在动物饲粮中的开发和应用。丝氨酸蛋白酶抑制剂(serine protease inhibitor,SPI)是蛋白酶抑制剂中数目最多,研究最为深入的一类,包括胰蛋白酶抑制剂(trypsininhibitor,TI)、胰凝乳蛋白酶抑制剂(chymotrypsin inhibitor,CI)、弹性蛋白酶抑制剂(elastase inhibitor,EI)和枯草杆菌蛋白酶抑制剂(subtilisin inhibitor,SI)等。
依据SPI的活性位点、作用机制及在植物中的分布状况,植物SPI可分为8个家族,其中研究较为深入的有Kunitz、Serpin、Bowman-Birk、PI-I和PI-II家族。川桑基因组中共鉴定出79个蛋白酶抑制剂(protease inhibitor,PI),其中SPI有35个,分别为Kunitz、Serpin和PI-I家族。分析不同SPI家族基因在各组织中的表达,发现8个Kunitz和1个Serpin家族SPI基因主要在桑叶中表达。Western Blot检测发现丝氨酸蛋白酶抑制剂MmKPI-9在桑叶中有表达,而胶内活性染色未检测到TI活性条带。王丹丹利用胶内活性染色技术从桑叶叶柄部流出的白色乳汁中检测到多个CI活性条带,而在桑树叶片中未检测到CI活性。目前,桑叶中抗营养因子类SPI的研究报道较少,且这些SPI的序列信息、活性、理化特性尚不清楚。
发明内容
为了解决上述技术问题,MmBBK2在制备胰蛋白酶和胰凝乳蛋白酶抑制剂中的应用,所述MmBBK2具有SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列。
基于同一个发明构思,本发明还提供了编码所述MmBBK2的基因,SEQ ID NO.1所示的氨基酸序列的编码基因的核苷酸序列如SEQ ID NO.3所示,SEQ ID NO.2所示的氨基酸序列的编码基因的核苷酸序列如SEQ ID NO.4所示。
基于同一个发明构思,本发明还提供了携带所述基因的质粒。
基于同一个发明构思,本发明还提供了携带所述质粒的宿主表达菌株。
基于同一个发明构思,本发明还提供了所述菌株的表达产物在制备胰蛋白酶和胰凝乳蛋白酶抑制剂中的应用。
基于同一个发明构思,本发明还提供了所述MmBBK2或所述表达产物的活性消除方法,包括:将MmBBK2置于pH 3~4的环境中;或,置于121℃,0.21MPa环境中处理20min;或,利用还原剂β-巯基乙醇于100℃处理10min;或,利用由还原糖介导的美拉德反应消除。
本发明具有如下有益效果:
本发明首次明确了桑叶中的MmBBK2同时具有胰蛋白酶和胰凝乳蛋白酶抑制活性,并揭示了其理化特性,所述MmBBK2在制备胰蛋白酶抑制剂方面具有良好的应用前景,在明确其理化性质的基础上,可以针对性消除其活性,推动桑叶资源在动物饲粮中的开发利用,为桑叶在动物饲料、保健食品方面的开发利用提供新的视角和思路,提升桑资源的经济效益。
附图说明
图1为MmBBK2(Ma)的PCR扩增产物电泳检测(A),以及菌液PCR检测(B)。
图2为MmBBK2-X1和MmBBK2-X2的PCR扩增产物电泳检测(A),以及的菌液PCR检测(B)。
图3为MmBBK2-X1(A)和MmBBK2-X2(B)核苷酸序列及其衍生的氨基酸序列,灰色背景部分为信号肽序列,下划线部分为BowB结构域,六边形框指示同一蛋白酶抑制剂不同亚型间的差异氨基酸,起始密码子(ATG)和终止密码子(TGA)用方框标出。
图4为BL21(DE3)细胞中表达的MmBBK2-X1和MmBBK2-X2的SDS-PAGE分析。“S”表示可溶性蛋白。“U”表示不可溶蛋白。“Control”,转入p28空载体的BL21(DE3)菌株的细胞裂解物。
图5为Origami 2(DE3)细胞中表达的MmBBK2-X1和MmBBK2-X2的SDS-PAGE分析。“S”表示可溶性蛋白。“U”表示不可溶蛋白。“Control”,转入p28空载体的Origami 2(DE3)菌株的细胞裂解物。
图6为在BL21(DE3)细胞中表达的MmBBK2-X1和MmBBK2-X2的TI(A)和CI(B)活性分析。“TI”和“CI”分别代表胰蛋白酶抑制剂和胰凝乳蛋白酶抑制剂。家蚕5龄第5天血液作为阳性对照。“Control”,转入p28空载体的BL21(DE3)菌株的细胞裂解物。箭头表示蛋白酶抑制剂活性条带。
图7为在Origami 2(DE3)细胞中表达的MmBBK2-X1和MmBBK2-X2的TI(A)和CI(B)活性分析。“TI”和“CI”分别代表胰蛋白酶抑制剂和胰凝乳蛋白酶抑制剂。家蚕5龄第5天血液作为阳性对照。“Control”,转入p28空载体的Origami 2(DE3)菌株的细胞裂解物。箭头表示蛋白酶抑制剂活性条带。
图8为不同pH对MmBBK2-X2活性的影响。
图9为高温高压联用对MmBBK2-X2活性的影响。
图10为β-巯基乙醇对MmBBK2-X2活性的影响。
图11为美拉德反应对对MmBBK2-X2活性的影响。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。如未特殊说明,下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
以下实施例涉及p28表达载体由陕西理工大学维生素D生理与应用研究所保存。
实施例1
1实验方法
1.1“金十”叶片中RNA提取和cDNA第一链的合成
1.1.1桑叶RNA的提取采用Eastep Super总RNA提取试剂盒(Promega公司)按照说明书操作。
1.1.2 cDNA第一链的合成
总RNA变性解链,取总RNA 4μg,加Oligo(dT)1μL,用RNase-free water补足至10μL。置于PCR仪42℃30min,再85℃5s,反应结束后迅速取出置于冰中,2.5倍稀释于-20℃保存。
RT-PCR反应体系:
1.2表达载体构建
1.2.1桑叶中抑制剂目的片段PCR扩增
基于白桑中蛋白酶抑制剂的CDS序列,对MmBBK2基因(名称为申请人团队自己设计、命名)设计引物。其引物序列见表1。
表1蛋白酶抑制剂MmBBK2 TA克隆所用引物
注:下标为“Ma”表示基于白桑数据库所设计的引物。
以金十叶片的cDNA为模板进行PCR扩增,PCR体系如下:
PCR扩增程序为:94℃预变性5min;94℃变性30s,53℃退火30s,72℃延伸1min或1min15 s,30个循环;72℃再延伸10min。PCR产物经过1.5%琼脂糖凝胶电泳分离,并参照EasyPure PCR Purification Kit进行纯化。参照pEASY-T1 Simple Cloning Vector使用说明书,将回收的目的片段连接到pEASY-T1 Simple载体。反应体系为:4μL目的片段加入1μL pEASY-T1 Simple载体,混匀后瞬时离心,PCR仪中25℃连接10min。连接产物转化入大肠杆菌DH5α感受态细胞,选择大且圆润的白色单菌落进行摇菌,通过菌液PCR筛选阳性克隆,测序验证,并提取质粒。
以T克隆得到的质粒(稀释20倍)为模板进行PCR扩增,其引物序列见表2。
表2蛋白酶抑制剂MmBBK2-X1和MmBBK2-X2表达载体构建所用引物
注:下划线表示酶切位点。Nde I位点:CATATG;Ase I位点ATTAAT:Not I位点:GCGGCCGC。下标为“Ma”表示基于白桑数据库所设计的引物。
PCR体系如下:
PCR扩增程序:95℃预变性2min;95℃变性30s,61℃退火30s,72℃延伸30s,30个循环;72℃再延伸10min。PCR产物经过1.5%琼脂糖凝胶电泳分离,并参照EasyPure QuickGel Extraction Kit进行切胶回收纯化,对PCR产物和p28载体进行双酶切,双酶切反应体系见表3。酶切条件:37℃12h,加入10×Loading buffer终止酶切。酶切产物经1.5%琼脂糖凝胶电泳分离,参照EasyPure Quick Gel Extraction Kit切胶回收。
表3双酶切反应体系
37℃过夜酶切后,加入10×Loading buffer终止酶切。酶切产物经1.5%琼脂糖凝胶电泳分离,参照EasyPure Quick Gel Extraction Kit切胶回收。
1.2.2目的片段与p28载体连接
将目的片段16℃2h连接到p28载体上,连接体系如下:
1.2.3转化
将连接产物转化入大肠杆菌DH5ɑ感受态细胞,具体转化步骤如下:
1)将DH5ɑ感受态细胞置于冰上,待其刚融化时,加入连接产物轻轻吹吸混匀,并置于冰上静置30min。
2)42℃热激90s,迅速置于冰上静置5min。
3)加入900μL不含抗生素的2-YT液体培养基,37℃220rpm培养1h。
4)3500rpm离心5min,弃上清800μL,移液枪重悬沉淀,将重悬液加入含卡那霉素抗性的2-YT固体培养基,用灭菌后的涂布棒轻轻涂布于均匀。
5)37℃恒温培养箱中先正置10min,再倒置培养12h。
1.2.4菌液PCR筛选阳性克隆及测序
挑取6个大而圆润的白色单菌落,接种于600μL的含卡那霉素抗性的2-YT液体培养基中,37℃220rpm震荡培养4h。吸取2μL菌液为模板进行菌液PCR。菌液PCR反应体系如下:
PCR程序为:95℃预变性5min;95℃变性30s,61℃退火30s,72℃延伸1min,30个循环;72℃再延伸10min。PCR产物经1.5%琼脂糖凝胶检测,能扩增出目的条带的菌液为阳性克隆。选择3个条带明亮的阳性克隆菌液各吸取200μL送至上海生工进行测序。
1.2.5制备甘油菌及质粒提取
按照1/100~1/1000比例吸取测序正确的菌液于含卡那霉素抗性的2-YT液体培养基,37℃220rpm,震荡培养12h;取300μL菌液中与200μL 50%的甘油混匀制备成甘油菌,-20℃保存。吸取2mL菌液,参考EasyPure Plasmid MiniPrep Kit提取质粒。
1.3 MmBBK2-X1和MmBBK2-X2的原核表达
1.3.1转化入大肠杆菌表达菌株
将质粒转入大肠杆菌BL21(DE3)和Origami 2(DE3)菌株,转化步骤如下:
1)-80℃保存的BL21(DE3)或Origami 2(DE3)感受态细胞置于冰上,待其刚刚融化时,取1μL的质粒轻轻地缓慢加入感受态细胞中轻柔混匀,冰浴30min。
2)42℃热激90s,热激结束快速插入冰中冷却5min。
3)加入不含抗生素的2-YT液体培养基900μL,37℃220rpm孵育1h。
4)3500rpm,离心5min,弃上清液800μL。
5)移液器混匀剩余上清液和沉淀,悬浊液分别置于含有一种抗生素(卡那霉素抗性)或三种抗生素(卡那霉素、链霉素、四环素)的2-YT固体培养基上,用灭菌过的涂布棒轻轻涂布均匀。
6)将固体平板在37℃培养箱中先正置10min,再倒置培养12h。
1.3.2诱导表达
1)挑取大而圆润的单菌落,分别置于600μL含有一种抗生素(卡那霉素抗性)或三种抗生素(卡那霉素、链霉素、四环素)的2-YT液体培养基,恒温摇床37℃220rpm过夜培养。
2)取过夜摇混的菌液150μL于15mL具有相应抗生素的2-YT液体培养基,37℃220rpm培养至菌液OD600=0.6~1.0时迅速插入冰中延缓生长。
3)按1/500比例加入0.1M IPTG贮藏液(即工作浓度0.2mM),37℃220rpm诱导5h或16℃220rpm诱导20h。
4)诱导结束后,4℃6000rpm离心20min,弃上清液。
5)加入1.5mL的1×结合缓冲液重悬菌体,4℃6000rpm离心10min,弃上清液。
6)再加入1mL的1×结合缓冲液重悬菌体,再次离心,弃上清液。
7)最后加入450μL 1×结合缓冲液重悬菌体,-20℃保存。
1.3.3细胞破碎
将菌体混匀物置于冰水混合物中,使用超声波破碎仪30W破碎至菌体混匀液透亮,4℃16000g,离心30min,分离上清,沉淀中加入250μL 1×结合缓冲液重悬,获得包涵体溶液,-20℃保存上清液及沉淀物。
1.4SDS-PAGE以及Native PAGE和MmBBK2-X1和MmBBK2-X2的活性染色
操作步骤参照申请号为202111086415.1的中国专利。
1.5不同pH、高温高压联用、还原剂和美拉德反应对MmBBK2-X2活性影响
操作步骤参照申请号为202111086415.1的中国专利。
2结果与分析
2.1 MmBBK2(Ma)的表达载体构建
PCR产物检测结果显示,PCR扩增目的基因条带单一,且条带较亮。将PCR产物连接到T载体上转入DH5ɑ感受态细胞,取菌液进行菌液PCR,用1.0%琼脂糖凝胶对菌液PCR产物进行检测,结果发现,MmBBK2(Ma)有阳性克隆(图1)。通过菌液测序后发现MmBBK2(Ma)存在2种形式。我们将成功克隆的MmBBK2的2种形式分别命名为:MmBBK2-X1和MmBBK2-X2。接下来,我们提取质粒,并进行亚克隆。经PCR扩增MmBBK2-X1和MmBBK2-X2得到明亮且单一的条带。之后,将PCR产物连接到p28载体上转入DH5ɑ感受态细胞,进行菌液PCR,并采用1%的琼脂糖凝胶电泳检测,结果如图2所示。挑选阳性克隆的菌液,经过测序证实,MmBBK2-X1-p28和MmBBK2-X2-p28表达载体构建成功。
2.2 MmBBK2-X1和MmBBK2-X2的一级结构分析
MmBBK2-X1和MmBBK2-X2的CDS编码框均由369个核苷酸组成,编码一个有122个氨基酸的蛋白质,氨基酸序列中均有12个半胱氨酸残基,它们的氨基酸序列中有6个氨基酸的替换,但氨基酸性质相似,MmBBK2-X1基因序列如SEQ ID NO.1所示,其编码蛋白的氨基酸序列如SEQ ID NO.3所示;MmBBK2-X2基因序列如SEQ ID NO.2所示,其编码蛋白的氨基酸序列如SEQ ID NO.4所示。MmBBK2-X1和MmBBK2-X2蛋白都具有一个24个氨基酸组成的信号肽(图3),蛋白成熟体分子量为10801.38Da和10815.33Da,等电点(pI)分别为5.08和5.34,都具有一个包含12个半胱氨酸残基的BowB结构域。
2.3 MmBBK2-X1和MmBBK2-X2的原核表达
为了实现MmBBK2-X1和MmBBK2-X2的原核表达,将重组质粒转入大肠杆菌BL21(DE3)(图4)和Origami 2(DE3)(图5)两种表达菌株中,使用工作浓度为0.2mM的IPTG进行诱导表达。用16.5%的SDS-PAGE对目的蛋白进行分离检测,结果显示,在IPTG浓度为0.2mM时,MmBBK2-X1和MmBBK2-X2在Origami 2(DE3)菌株中主要以可溶形式表达,在BL21(DE3)菌株中主要以包涵体形式存在。
2.4 MmBBK2-X1和MmBBK2-X2的活性分析
为了分析目的蛋白对胰蛋白酶和胰凝乳蛋白酶的抑制活性,我们将BL21(DE3)和Origami 2(DE3)两种菌株中表达的目的蛋白的上清液经10%的Native PAGE分离后进行胶内活性染色。在BL21(DE3)菌株中未检测到MmBBK2-X1和MmBBK2-X2的胰蛋白酶或胰凝乳蛋白酶抑制剂活性(图6);在Origami 2(DE3)菌株中,检测到MmBBK2-X1和MmBBK2-X2同时具备对胰蛋白酶和胰凝乳蛋白酶的抑制活性(图7)。
2.5 pH、温度、还原剂和美拉德反应对MmBBK2-X2活性的影响
(1)不同pH对MmBBK2-X2活性的影响
如图8所示,MmBBK2-X2在碱性环境中的稳定性比在酸性环境中的稳定性更强,在pH 5~6范围内,MmBBK2-X2表现出最强的抑制活性,且相比在pH(7~11)活性条带出现了位置上的改变,在pH 3~4范围内,MmBBK2-X2的抑制活性基本丧失。
(2)高温高压联用对MmBBK2-X2活性的影响
为验证高温或高温高压联用对蛋白酶抑制剂MmBBK2-X2活性的影响,我们将蛋白酶抑制剂置于121℃0.21MPa或100℃处理20min,之后经Native PAGE和胶内活性染色分析其对蛋白酶的抑制活性。胶内活性染色结果显示(图9),相比于对照组,100℃处理20min会使MmBBK2-X2对胰蛋白酶和胰凝乳蛋白酶的抑制活性略有降低,而高温高压(121℃0.21MPa)联用会极大的削弱MmBBK2-X2对胰蛋白酶和胰凝乳蛋白酶的抑制活性。
(3)还原剂对MmBBK2-X2活性的影响
为了探究还原剂对MmBBK2-X2活性的影响,我们采用β-巯基乙醇对蛋白酶抑制剂MmBBK2-X2进行处理,并分析其对胰蛋白酶和胰凝乳蛋白酶的抑制活性。结果如图10所示,无β-巯基乙醇时,加热对蛋白酶抑制剂的活性无明显影响;有β-巯基乙醇不加热时,MmBBK2-X2对蛋白酶的抑制活性有一定程度的降低;有β-巯基乙醇且加热时,MmBBK2-X2对蛋白酶的抑制活性完全丧失。这表说明MmBBK2-X2结构中存在二硫键桥,且二硫键桥是保持其活性的重要结构。
(4)葡萄糖介导的美拉德反应对MmPI活性的影响
如图11所示,无葡萄糖时,100℃加热60min使极大削弱MmBBK2-X2对胰蛋白酶和胰凝乳蛋白酶的抑制活性;有葡萄糖不加热时,MmBBK2-X2对蛋白酶的抑制活性无明显影响。有葡萄糖且加热时MmBBK2-X2完全失活。以上结果表明,由葡萄糖介导的美拉德反应可以完全消除MmBBK2-X2对胰蛋白酶和胰凝乳蛋白酶的抑制活性。
尽管已描述了本发明的优选实施例,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进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (5)
1.MmBBK2在制备胰蛋白酶和胰凝乳蛋白酶抑制剂中的应用,其特征在于,所述MmBBK2的氨基酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
2.编码权利要求1中所述MmBBK2的基因,其特征在于,SEQ ID NO.1所示的氨基酸序列的编码基因的核苷酸序列如SEQ ID NO.3所示,SEQ ID NO.2所示的氨基酸序列的编码基因的核苷酸序列如SEQ ID NO.4所示。
3.携带权利要求2所述基因的质粒,其特征在于,是将SEQ ID NO.3或SEQ ID NO.4所示的编码基因连接至p28表达载体获得,SEQ ID NO.3所示编码基因的连接位点为Nde I和NotI,SEQ ID NO.4所示的编码基因的连接位点为Ase I和Not I。
4.携带权利要求3所述质粒的宿主表达菌株,其特征在于,所述宿主表达菌株为Origami 2(DE3)菌株。
5.权利要求1中所述MmBBK2的活性消除方法,其特征在于,包括:将MmBBK2置于pH 3~4的环境中;或,置于121℃,0.21MPa环境中处理20min;或,利用还原剂β-巯基乙醇于100℃处理10min;或,利用由还原糖介导的美拉德反应消除。
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