CN109536469A - 突变改造Prx6蛋白及其表达基因、制备方法和应用 - Google Patents
突变改造Prx6蛋白及其表达基因、制备方法和应用 Download PDFInfo
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- CN109536469A CN109536469A CN201811406703.9A CN201811406703A CN109536469A CN 109536469 A CN109536469 A CN 109536469A CN 201811406703 A CN201811406703 A CN 201811406703A CN 109536469 A CN109536469 A CN 109536469A
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Abstract
本发明涉及一种突变改造Prx6蛋白及其表达基因、制备方法和应用,属于生物工程技术领域。该突变改造Prx6蛋白通过定点突变技术对人Prx6蛋白进行以下至少一个位点的突变改造得到:第39位氨基酸由His突变成Asn,第50位氨基酸由Glu突变为His,第78位氨基酸由Asp突变为由Arg,第103位氨基酸由Asp突变为Lys,第132位氨基酸由Arg突变为Gln。该突变改造Prx6蛋白的抗氧化活性提高,能够高效的清除自由基,保护细胞免受氧化损伤,适合作为抗氧化及抗衰老药物进行研发和生产。并且该突变改造Prx6蛋白产量高、性质稳定,由此解决了同类型天然抗氧化酶(如GPx、Grx)活性不稳定及来源不足等问题,在生物制药方面具有广阔前景。
Description
技术领域
本发明涉及生物工程技术领域,特别是涉及一种突变改造Prx6蛋白及其表达基因、制备方法和应用。
背景技术
现如今,随着环境污染、电磁辐射、人们不良的生活习惯、食品不安全以及药物的乱用等问题的增多,致使机体内活性氧(ROS)产生过量,其自身抗氧化体系不能及时清除过量的ROS,使得相关疾病发病率越来越高且病情越来越重。
因此,我国乃至全球对于抗氧化酶的研发及应用都给予了高度的重视,抗氧化酶的生产及应用极大程度上将促进农林医药等各领域的发展。目前对超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)等抗氧化酶的研究较为深入且有相应产品问世,而GPx和Prx仍尚未得到广泛应用,仅有GPx的小分子模拟物依布硒现处于Ⅲ期临床实验阶段。
GPx是一种重要的含硒抗氧化蛋白,其主要功能在于催化GSH还原体内有毒的过氧化物(ROOH),从而阻断细胞信号转导、抑制细胞凋亡、控制细胞感染等,进而保护生物膜及其他生物组织免受氧化损伤。研究发现,GPx与克山病、关节炎、糖尿病、心脑血管疾病及癌症等的防治都具有密切关系。然而由于其催化活性中心为通常被称为第21种氨基酸的硒代半胱氨酸(Sec),因此很难利用传统方法对其进行大量制备,加之天然来源有限及性质不稳定等缺陷,至今未被良好的开发及应用。因此其模拟物及相关人工产物的研究日益增多。
过氧化物氧还蛋白(Peroxiredoxins,Prxs)家族作为一类不断扩展的巯基特异的抗氧化蛋白家族,近年来倍受科研工作者的关注。他们通过其过氧化物酶活性还原或降解氢过氧化物、过氧化亚硝酸盐和多种有机氢过氧化物,进而在拮抗机体产生的活性氧、维持机体的氧化还原平衡上发挥重要作用。越来越多研究还表明,Prxs还有许多其它生物学功能,如参与细胞信号传导、细胞的增殖分化、血红素的代谢以及增强自然杀伤细胞的细胞毒活性等。由此可见,该蛋白家族成员的抗氧化反应的能力在某种程度上与我们熟悉的GPx和CAT交迭。已有研究报道,哺乳动物组织中存在6个Prx家族成员,其中五个是2-Cys Prx,以硫氧化蛋白为供电子体,而Prx6是唯一的1-Cys Prx,同GPx一样以谷胱甘肽(GSH)为供电子体。并且Prx6是一个双功能的蛋白质,除了具有GPx活性外,还具有磷脂酶A2(PLA2)的活性。基于其GPx活性,它能够清除过氧化氢、脂肪氢过氧化物及磷脂氢过氧化物等,在机体抗氧化防御生理功能中具有重要作用。细胞水平研究表明,过表达Prx6能明显减少脂质过氧化作用,降低细胞质膜损伤,而抑制Prx6表达则可观察到明显的脂质过氧化、脂膜损伤及细胞凋亡等现象。
研究数据表明,GPx的催化效率约为108M-1S-1,CAT的催化效率约为106M-1S-1,相比较而言Prx的催化效率则显得很普通,约为105M-1S-1,这使得人们曾质疑Prx作为过氧化物酶的重要性。但由于其在细胞中含量丰富,且在最近的研究中发现某种细菌内的负责清除过量H2O2的是Prx,而非CAT,这些事实证明Prx在拮抗体内ROS和维持机体的氧化还原平衡功能上具有十分重要的作用。与此同时Prx还有许多其它的生物学功能,如参与细胞的增殖与分型、参与血红素的代谢和增强自然杀伤细胞的细胞毒活性等。
发明内容
基于此,有必要针对Prx催化效率不高问题,提供一种突变改造Prx6蛋白及其表达基因、制备方法和应用,该突变改造Prx6蛋白具有抗氧化活性高,产量高、性质稳定的优点。
一种突变改造Prx6蛋白,通过定点突变技术对人Prx6蛋白进行以下至少一个位点的突变改造得到:
第39位氨基酸由His突变成Asn,第50位氨基酸由Glu突变为His,第78位氨基酸由Asp突变为由Arg,第103位氨基酸由Asp突变为Lys,第132位氨基酸由Arg突变为Gln。
本发明人在长期研究人Prx6蛋白(hPrx6)工作的基础上,利用计算机辅助模拟蛋白质结构分析,该hPrx6蛋白的氨基酸序列如SEQ ID NO:15所示,核苷酸序列如SEQ IDNO.16所示;对Prx6的蛋白质晶体结构进行分析,发现该hPrx6蛋白具有一个由80个氨基酸组成的硫氧还蛋白折叠,其中包含四个β折叠和两个α螺旋,其催化中心位于一个β折叠和α螺旋形成的凹槽内。本发明人通过分析后对其催化活性中心进行筛选突变,突变后发现其催化中心的主要氨基酸相互作用位置靠近,且凹槽变浅靠近蛋白质结构表面,更有利于与底物结合发生催化反应。据此设计了系列突变位点的重组蛋白进行比对,最终发现,上述5个位点的突变能够提高Prx6蛋白催化活性。
并且,优选的,对人Prx6蛋白进行至少两个位点的突变改造,所得到的突变改造Prx6蛋白具有更高的催化活性。
在其中一个实施例中,其氨基酸序列如SEQ ID NO:1所示。
本发明还公开了一种编码上述的突变改造Prx6蛋白的表达基因,所述表达基因的序列为:
A)编码权利要求1或2的突变改造Prx6蛋白的核苷酸序列;或者
B)与A)的核苷酸序列编码相同序列的蛋白质,但因遗传密码的简并性而与A)的核苷酸序列不同的序列;或者
C)对上述A)或B)所示核苷酸序列进行一个或多个碱基的取代、缺失、添加修饰的核苷酸序列。
在其中一个实施例中,所述A)中的核苷酸序列如SEQ ID NO.2所示。
本发明还公开了一种表达载体,包含上述的表达基因。
本发明还公开了一种重组细胞,包含上述的重组细胞。
本发明还公开了一种上述的突变改造Prx6蛋白的制备方法,包括以下步骤:
(1)获得人Prx6蛋白表达基因,并将上述表达基因插入至载体中,构建重组表达载体;
(2)以突变引物构建突变体质粒;
(3)将上述突变体质粒转入宿主细胞中,通过筛选得到阳性重组细胞,诱导表达,即得。
在其中一个实施例中,步骤(1)中,通过下述方法获得人Prx6蛋白表达基因:以人宫颈癌hela细胞株cDNA文库为PCR反应模板,设计扩增引物进行PCR扩增反应,扩增产物即为人Prx6蛋白表达基因;
步骤(1)中,通过以下方法将上述表达基因插入至载体中,构建重组表达载体:将获得的人Prx6蛋白表达基因及质粒pColdⅠ进行Hind III/Xba I双酶切,获得含有HindIII/Xba I酶切位点的人Prx6基因和pColdⅠ载体,回收酶切的载体片段;将酶处理后的人Prx6基因片段和pColdⅠ载体在T4连接酶作用下进行连接;连接产物加入DH5α感受态菌中,筛选阳性克隆,得到pColdⅠ-hPrx6重组质粒,即为重组表达载体;
步骤(2)中,通过以下方法构建突变质粒:根据预定突变位点设计突变引物,加入上述pColdⅠ-hPrx6重组质粒进行突变PCR反应,于反应产物中加入QuickCut DpnI限制性内切酶,将原始模板序列降解;酶切后合成产物转化DH5α感受态,对转化子进行菌落PCR验证,并提取质粒测序,得到预定序列的突变质粒。
步骤(3)中,通过以下方法诱导表达突变改造Prx6蛋白:将上述突变质粒转化BL21(DE3)感受态细胞,在含Amp和Kana抗性的固体培养基中筛选阳性克隆;挑取阳性单克隆于含Amp、Kana培养基中培养,加IPTG,诱导表达后收菌,即得突变改造Prx6蛋白。
在其中一个实施例中,步骤(1)中:所述扩增引物包括如下引物对:
hPrx6-F:5’-CCCAAGCTTATGCCCGGAGGTC-3’(SEQ ID NO:3);
hPrx6-R:5’-GCTCTAGATTAAGGCTGGGGTG-3’(SEQ ID NO:4);
所述PCR扩增反应条件为:94℃变性2min;94℃变性50s,55℃退火45s,72℃延伸1min,运行35个循环;72℃保温8min;4℃保持;
所述Hind III/Xba I双酶切的酶切体系为:buffer 2μL、Hind III/Xba I各0.5μL、人Prx6蛋白表达基因17μL;
所述载体片段通过以下方法回收:以0.8%的琼脂糖凝胶电泳,利用胶回收试剂盒回收酶切片段;
所述连接具体操作为:将酶处理后的人Prx6基因片段和pColdⅠ载体按照摩尔比为5:1的比例在T4连接酶作用下进行4℃连接过夜;
所述pColdⅠ-hPrx6重组质粒通过以下方法得到:将连接产物加入DH5α感受态菌中,冰浴30min后,42℃热激90s后立即置于冰上,冷却2min;无菌条件下添加700μL LB培养液,混匀后放入培养箱中100rpm振荡培养1h;将菌液均匀涂布于含Amp的LB固体培养基上,37℃倒置过夜培养,筛选阳性克隆,即得;
步骤(2)中,
所述突变引物包括以下引物对中的至少一对:
第39位H-N突变引物对:
5’-GGGCATTCTCTTCTCCAACCCTCGGGACTTTAC-3’(SEQ ID NO:5)
5’-GTAAAGTCCCGAGGGTTGGAGAAGAGAATGCCC-3’(SEQ ID NO:6)
第50位E-H突变引物对:
5’-GCACCACACACCTTGGCAG-3’(SEQ ID NO:7)
5’-CTGCCAAGGTGTGTGGTGC-3’(SEQ ID NO:8)
第78位D-R突变引物对:
5’-GTGTTGAGCGCCATCTTGCC-3’(SEQ ID NO:9)
5’-GGCAAGATGGCGCTCAACAC-3’(SEQ ID NO:10)
第103位D-K突变引物对:
5’-CCCATCATCAAGGATAGGAATCG-3’(SEQ ID NO:11)
5’-CGATTCCTATCCTTGATGATGGG-3’(SEQ ID NO:12)
第132位R-Q突变引物对:
5’-CATGCCTGTGACAGCTCAGGTGGTGTTTGTTTTTGG-3’(SEQ ID NO:13)
5’-CCAAAAACAAACACCACCTGAGCTGTCACAGGCATG-3’(SEQ ID NO:14)
所述突变PCR反应体系为50μL,包括:PCR buffer(10×)5μL,d NTP(2.5mmol/L)4μL,质粒1μL,上述引物对各1.5μL,Pfu DNA聚合酶1μL,dd H2O余量;
所述突变PCR反应条件为:94℃2min;94℃,30s;55℃,90s;68℃,12min进行20个循环,4℃,保持;
所述原始模板序列降解通过以下方法实现:于反应产物中加入2μL的QuickCutDpnI限制性内切酶,于37℃条件下反应10min;
步骤(3)中,通过以下方法诱导表达突变改造Prx6蛋白:将上述突变质粒转化BL21(DE3)感受态细胞,37℃倒置培养过夜,在含100μg/mL Amp和25μg/mL Kana抗性的固体培养基中筛选阳性克隆;挑取阳性单克隆于20mL含Amp、Kana培养基中,37℃震荡培养,当其OD600值为0.6±0.1时,加终浓度为1mmol/L的IPTG,37℃诱导表达4h后收菌;
还包括步骤(4)蛋白纯化步骤,具体如下:将上述收集得到的菌沉用20mmol/L的PBS重悬,在冰水浴中对菌体进行超声破碎,4℃10000g离心10min收集蛋白上清液,上清液用0.45μm的滤膜过滤;用含50mmol/L咪唑的PBS缓冲液平衡Ni离子亲和层析柱料,将超声后的蛋白上清液按1mL/min的流速挂柱,反复上样3次;分别用六倍柱料体积的含50mmol/L、75mmol/L、100mmol/L咪唑的PBS缓冲液洗脱杂蛋白,再用六倍柱料体积的含300mmol/L咪唑的PBS缓冲液洗脱目的蛋白,即得纯化突变改造Prx6蛋白。
本发明还公开了上述的突变改造Prx6蛋白在制备用于抗氧化的药物中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明的一种突变改造Prx6蛋白,是利用计算机辅助模拟蛋白质结构分析,结合定点突变技术对重组蛋白系列突变位点的进行比对筛选后得到的一种人工改造的高活力过氧化物氧还蛋白(突变改造Prx6蛋白),该突变改造Prx6蛋白的抗氧化活性提高,能够高效的清除自由基,保护细胞免受氧化损伤,适合作为抗氧化及抗衰老药物进行研发和生产。
并且该突变改造Prx6蛋白产量高、性质稳定,由此解决了同类型天然抗氧化酶(如GPx、Grx)活性不稳定及来源不足等问题,在生物制药方面具有广阔前景。
附图说明
图1为实施例中h Prx6基因调取(A)及pColdⅠ-hPrx6质粒构建(B)图。
其中:A为h Prx6基因凝胶电泳图,B为pColdⅠ-hPrx6质粒酶切鉴定凝胶电泳图。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
以下实施例所用原料,均为市售购得。
实施例
一种突变改造Prx6蛋白,通过以下方法制备得到:
一、获得人Prx6蛋白表达基因,并将上述表达基因插入至载体中,构建重组表达载体。
1、人Prx6蛋白表达基因(hPrx6基因)的调取。
以人宫颈癌hela细胞株cDNA文库(按常规方法收集生长状态良好的hela细胞,Trizol法抽提总RNA,用TAKARA反转录试剂盒制备得到cDNA文库,现于北华大学生命科学中心保存)为PCR反应的模板,根据已报道的hPrx6基因序列设计以下引物对进行PCR反应,得到人Prx6蛋白表达基因。
hPrx6-F:5’-CCCAAGCTTATGCCCGGAGGTC-3’(SEQ ID NO:3)
hPrx6-R:5’-GCTCTAGATTAAGGCTGGGGTG-3’(SEQ ID NO:4)。
PCR反应条件:94℃变性2min;94℃变性50s,55℃退火45s,72℃延伸1min,运行35个循环;72℃保温8min;4℃保持。
2、构建重组表达载体。
将获得的PCR产物及质粒pColdⅠ(来源:购于生工生物有限公司)进行Hind III/Xba I双酶切。
酶切体系包括:buffer 2μL、Hind III/Xba I(来源:购于生工生物有限公司)各0.5μL、hPrx6基因(浓度:0.3ng/μL)17μL。
以上酶切体系37℃酶切过夜,获得含有Hind III/Xba I酶切位点的hPrx6基因和pColdⅠ载体,然后进行0.8%的琼脂糖凝胶电泳,利用胶回收试剂盒(来源:购于鼎国生物技术有限公司)回收酶切片段。
将酶处理后的hPrx6基因片段和pColdⅠ载体按照摩尔比为5:1的比例在T4连接酶(来源:购于生工生物有限公司)作用下进行4℃连接过夜,得到连接产物。
将连接产物加入DH5α感受态菌(来源:购于鼎国生物技术有限公司)中,冰浴30min后,42℃热激90s后立即置于冰上,冷却2min。无菌条件下添加700μL LB培养液,混匀后放入培养箱中100rpm振荡培养1h。将菌液均匀涂布于含Amp(氨苄青霉素)的LB固体培养基上,37℃倒置过夜培养。筛选阳性克隆,结果如图1所示,图中A表示从人宫颈癌hela细胞株cDNA文库中成功调取hPrx6基因,M为mark,1和2泳道为hPrx6基因,B表示将hPrx6基因连接于pColdⅠ载体后酶切鉴定,M为mark,1泳道为pColdⅠ-hPrx6经Hind III/Xba I酶切后电泳结果,从图中可以看出经Hind III/Xba I酶切后有目的基因大小的条带,说明成功调取了hPrx6基因并构建重组质粒pColdⅠ-hPrx6。
二、以突变引物构建突变体质粒。
1、突变引物设计。
以pColdⅠ-hPrx6为扩增模板进行突变,设计突变引物对如下所示:
Primer 39H-N
5’-GGGCATTCTCTTCTCCAACCCTCGGGACTTTAC-3’(SEQ ID NO:5)
5’-GTAAAGTCCCGAGGGTTGGAGAAGAGAATGCCC-3’(SEQ ID NO:6)
Primer 50E-H
5’-GCACCACACACCTTGGCAG-3’(SEQ ID NO:7)
5’-CTGCCAAGGTGTGTGGTGC-3’(SEQ ID NO:8)
Primer 78D-R
5’-GTGTTGAGCGCCATCTTGCC-3’(SEQ ID NO:9)
5’-GGCAAGATGGCGCTCAACAC-3’(SEQ ID NO:10)
Primer 103D-K
5’-CCCATCATCAAGGATAGGAATCG-3’(SEQ ID NO:11)
5’-CGATTCCTATCCTTGATGATGGG-3’(SEQ ID NO:12)
Primer 132R-Q
5’-CATGCCTGTGACAGCTCAGGTGGTGTTTGTTTTTGG-3’(SEQ ID NO:13)
5’-CCAAAAACAAACACCACCTGAGCTGTCACAGGCATG-3’(SEQ ID NO:14)
2、突变PCR反应。
突变PCR反应体系为50μL:PCR buffer(10×)5μL,d NTP(2.5mmol/L)4μL,pColdⅠ-hPrx6重组质粒(4ng/μL)1μL,上述引物对(浓度5μmol/L,每次突变PCR加入一对突变引物)各1.5μL,Pfu DNA聚合酶(2.5U/μL)1μL,dd H2O 36μL。
突变PCR反应条件:94℃2min;94℃,30s;55℃,90s;68℃,12min进行20个循环,4℃,保持。
3、降解原始模板序列。
在上述合成产物中加入2μL的QuickCut DpnI限制性内切酶(来源:购于生工生物有限公司),于37℃条件下反应10min,将原始模板序列彻底降解。
4、构建突变质粒。
将酶切后合成产物转化DH5α感受态,对转化子进行菌落PCR验证,并提取质粒测序,对测序正确的质粒保藏备用。
以每次突变成功的质粒为模板继续突变,在此突变过程中,我们得到了Prx6-39N(第39位氨基酸由His突变成Asn)、Prx6-78R(第78位氨基酸由Asp突变为由Arg)、Prx6-39N78R(第39位氨基酸由His突变成Asn、第78位氨基酸由Asp突变为由Arg)、Prx6-39N50H78R(第39位氨基酸由His突变成Asn、第50位氨基酸由Glu突变为His、第78位氨基酸由Asp突变为由Arg)、Prx6-39N50H78R103K(第39位氨基酸由His突变成Asn、第50位氨基酸由Glu突变为His、第78位氨基酸由Asp突变为由Arg、第132位氨基酸由Arg突变为Gln)、Prx6-39N50H78R103K132Q(第39位氨基酸由His突变成Asn,第50位氨基酸由Glu突变为His,第78位氨基酸由Asp突变为由Arg,第103位氨基酸由Asp突变为Lys,第132位氨基酸由Arg突变为Gln)等几个突变体。
三、将上述突变体质粒转入宿主细胞中,通过筛选得到阳性重组细胞,诱导表达。
将上述构建好的质粒转化BL21(DE3)感受态细胞,37℃倒置培养过夜,在含Amp(氨苄青霉素100μg/mL)和Kana(卡那霉素,25μg/mL)抗性的固体培养基中筛选阳性克隆。挑取阳性单克隆于20mL含Amp、Kana培养基中,37℃震荡培养,当其OD600值约为0.6时,加终浓度为1mmol/L的IPTG(异丙基硫代半乳糖苷),37℃诱导表达4h后收菌。
四、蛋白纯化。
将收集的菌沉用20mmol/L的PBS重悬,在冰水浴中对菌体进行超声破碎,4℃10000g离心10min收集蛋白上清液,上清液用0.45μm的滤膜过滤。
用含50mmol/L咪唑的PBS缓冲液平衡Ni离子亲和层析柱料,将超声后的蛋白上清液按1mL/min的流速挂柱,反复上样3次。分别用六倍柱料体积的含50mmol/L、75mmol/L、100mmol/L咪唑的PBS缓冲液洗脱杂蛋白,用六倍柱料体积的含300mmol/L咪唑的PBS缓冲液洗脱目的蛋白,即得突变改造Prx6蛋白。
实验例
考察上述制备得到的突变改造Prx6蛋白的抗氧化活性。
一、Prx6突变体清除DPPH自由基能力的测定。
1、方法。
取纯化后的Prx6蛋白(各约2μg,精密称定),以Vc(抗坏血酸)为对照组,测定其清除DPPH的能力,以95%乙醇为溶液配制0.2mmol/L DPPH备用。分别吸取100μL样品溶液于96孔酶标板中,再分别加入100μL 0.2mmol/L DPPH溶液,恒温振荡器迅速混匀,室温避光反应10min,于517nm读取吸光度值。同时用水或95%乙醇做空白。
以对DPPH·清除率为纵坐标,溶液浓度为横坐标绘制清除率曲线,用Vc做标准对照,按照下式计算清除率:
清除率(%)K=[A0–(Ai–Aj)]/A0×100%。
式中,Ai:样品溶液+DPPH溶液的吸光度值;
Aj:样品溶液+95%乙醇的吸光度值;
A0:水/无水乙醇+DPPH溶液的吸光度值。
2、结果。
上述实验结果如下:
表1各样品对DPPH·自由基清除率
从上述结果中可以看出,当对hPrx6蛋白进行定点突变改造后,特别是至少对其中两个位点进行突变改造,其DPPH·自由基清除率明显提高;尤其是Prx6-39N50H78R103K132Q蛋白其DPPH·自由基清除率可达到85%。
二、GPx活性测定。
1、方法。
根据Wilson酶偶联法检测二者的GPx活力。
检测体系为500μL,其中含50mM pH 7.4的PBS、1mM的EDTA、1mM的GSH、0.5mM的HADPH、1U谷胱甘肽还原酶和50nM的突变改造Prx6蛋白样品。
具体方法未在37℃孵育5min后加入0.5mM的H2O2,在340nm处检测吸光度。
酶活力定义为:37℃下,该蛋白质每分钟氧化1μmol NADPH所需酶的量为一个活力单位。
2、结果。
上述实验结果如下:
表2各样品的GPx活力
通过上述结果可以看出,突变改造Prx6-39N50H78R103K132Q蛋白的GPx活力为1150U/μmol,较未突变前提高了一个数量级,且可以同天然Gpx相媲美。
三、硫氰酸铁法检测Prx6催化H2O2的反应活性
1、方法。
将重组Prx6蛋白(hPrx6蛋白)及其突变体蛋白(5μL)加入含1mmol/L DTT,50mmol/L PBS和0.5%甘油的缓冲液(85μL)中室温孵育2min,然后置于37℃环境中,加入10μL1mmol/L的H2O2使反应开始,反应3min后加入40μL 26%的三氯乙酸终止反应。最后加入40μL硫酸亚铁铵和20μL硫氰酸钾,测定A475nm处吸光度值,计算反应速度。改变H2O2的浓度,通过该方法测定Prx6催化H2O2反应的动力学。
2、结果。
上述实验结果如下:
表3各样品的催化H2O2反应活性
上述结果表明,Prx6-39N50H78R103K132Q突变改造Prx6蛋白的Km值较重组hPrx6蛋白低一个数量级,说明改造后Prx6突变体对H2O2的亲和能力较改造前提高。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 北华大学
<120> 突变改造Prx6蛋白及其表达基因、制备方法和应用
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 224
<212> PRT
<213> Prx6-39N50H78R103K132Q(Prx6-39N50H78R103K132Q)
<400> 1
Met Pro Gly Gly Leu Leu Leu Gly Asp Val Ala Pro Asn Phe Glu Ala
1 5 10 15
Asn Thr Thr Val Gly Arg Ile Arg Phe His Asp Phe Leu Gly Asp Ser
20 25 30
Trp Gly Ile Leu Phe Ser Asn Pro Arg Asp Phe Thr Pro Val Cys Thr
35 40 45
Thr His Leu Gly Arg Ala Ala Lys Leu Ala Pro Glu Phe Ala Lys Arg
50 55 60
Asn Val Lys Leu Ile Ala Leu Ser Ile Asp Ser Val Glu Arg His Leu
65 70 75 80
Ala Trp Ser Lys Asp Ile Asn Ala Tyr Asn Cys Glu Glu Pro Thr Glu
85 90 95
Lys Leu Pro Phe Pro Ile Ile Lys Asp Arg Asn Arg Glu Leu Ala Ile
100 105 110
Leu Leu Gly Met Leu Asp Pro Ala Glu Lys Asp Glu Lys Gly Met Pro
115 120 125
Val Thr Ala Gln Val Val Phe Val Phe Gly Pro Asp Lys Lys Leu Lys
130 135 140
Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Arg Asn Phe Asp Glu Ile
145 150 155 160
Leu Arg Val Val Ile Ser Leu Gln Leu Thr Ala Glu Lys Arg Val Ala
165 170 175
Thr Pro Val Asp Trp Lys Asp Gly Asp Ser Val Met Val Leu Pro Thr
180 185 190
Ile Pro Glu Glu Glu Ala Lys Lys Leu Phe Pro Lys Gly Val Phe Thr
195 200 205
Lys Glu Leu Pro Ser Gly Lys Lys Tyr Leu Arg Tyr Thr Pro Gln Pro
210 215 220
<210> 2
<211> 675
<212> DNA
<213> Prx6-39N50H78R103K132Q(Prx6-39N50H78R103K132Q)
<400> 2
atgcccggag gtctgcttct cggggacgtg gctcccaact ttgaggccaa taccaccgtc 60
ggccgcatcc gtttccacga ctttctggga gactcatggg gcattctctt ctccaaccct 120
cgggacttta ccccagtgtg caccacacac cttggcagag ctgcaaagct ggcaccagaa 180
tttgccaaga ggaatgttaa gttgattgcc ctttcaatag acagtgttga gcgccatctt 240
gcctggagca aggatatcaa tgcttacaat tgtgaagagc ccacagaaaa gttacctttt 300
cccatcatca aggataggaa tcgggagctt gccatcctgt tgggcatgct ggatccagca 360
gagaaggatg aaaagggcat gcctgtgaca gctcaggtgg tgtttgtttt tggtcctgat 420
aagaagctga agctgtctat cctctaccca gctaccactg gcaggaactt tgatgagatt 480
ctcagggtag tcatctctct ccagctgaca gcagaaaaaa gggttgccac cccagttgat 540
tggaaggatg gggatagtgt gatggtcctt ccaaccatcc ctgaagaaga agccaaaaaa 600
cttttcccga aaggagtctt caccaaagag ctcccatctg gcaagaaata cctccgctac 660
acaccccagc cttaa 675
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cccaagctta tgcccggagg tc 22
<210> 4
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gctctagatt aaggctgggg tg 22
<210> 5
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gggcattctc ttctccaacc ctcgggactt tac 33
<210> 6
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gtaaagtccc gagggttgga gaagagaatg ccc 33
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gcaccacaca ccttggcag 19
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctgccaaggt gtgtggtgc 19
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gtgttgagcg ccatcttgcc 20
<210> 10
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggcaagatgg cgctcaacac 20
<210> 11
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cccatcatca aggataggaa tcg 23
<210> 12
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cgattcctat ccttgatgat ggg 23
<210> 13
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
catgcctgtg acagctcagg tggtgtttgt ttttgg 36
<210> 14
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ccaaaaacaa acaccacctg agctgtcaca ggcatg 36
<210> 15
<211> 224
<212> PRT
<213> hPrx6蛋白(hPrx6)
<400> 15
Met Pro Gly Gly Leu Leu Leu Gly Asp Val Ala Pro Asn Phe Glu Ala
1 5 10 15
Asn Thr Thr Val Gly Arg Ile Arg Phe His Asp Phe Leu Gly Asp Ser
20 25 30
Trp Gly Ile Leu Phe Ser His Pro Arg Asp Phe Thr Pro Val Cys Thr
35 40 45
Thr Glu Leu Gly Arg Ala Ala Lys Leu Ala Pro Glu Phe Ala Lys Arg
50 55 60
Asn Val Lys Leu Ile Ala Leu Ser Ile Asp Ser Val Glu Asp His Leu
65 70 75 80
Ala Trp Ser Lys Asp Ile Asn Ala Tyr Asn Cys Glu Glu Pro Thr Glu
85 90 95
Lys Leu Pro Phe Pro Ile Ile Asp Asp Arg Asn Arg Glu Leu Ala Ile
100 105 110
Leu Leu Gly Met Leu Asp Pro Ala Glu Lys Asp Glu Lys Gly Met Pro
115 120 125
Val Thr Ala Arg Val Val Phe Val Phe Gly Pro Asp Lys Lys Leu Lys
130 135 140
Leu Ser Ile Leu Tyr Pro Ala Thr Thr Gly Arg Asn Phe Asp Glu Ile
145 150 155 160
Leu Arg Val Val Ile Ser Leu Gln Leu Thr Ala Glu Lys Arg Val Ala
165 170 175
Thr Pro Val Asp Trp Lys Asp Gly Asp Ser Val Met Val Leu Pro Thr
180 185 190
Ile Pro Glu Glu Glu Ala Lys Lys Leu Phe Pro Lys Gly Val Phe Thr
195 200 205
Lys Glu Leu Pro Ser Gly Lys Lys Tyr Leu Arg Tyr Thr Pro Gln Pro
210 215 220
<210> 16
<211> 675
<212> DNA
<213> hPrx6蛋白(hPrx6)
<400> 16
atgcccggag gtctgcttct cggggacgtg gctcccaact ttgaggccaa taccaccgtc 60
ggccgcatcc gtttccacga ctttctggga gactcatggg gcattctctt ctcccaccct 120
cgggacttta ccccagtgtg caccacagag cttggcagag ctgcaaagct ggcaccagaa 180
tttgccaaga ggaatgttaa gttgattgcc ctttcaatag acagtgttga ggaccatctt 240
gcctggagca aggatatcaa tgcttacaat tgtgaagagc ccacagaaaa gttacctttt 300
cccatcatcg atgataggaa tcgggagctt gccatcctgt tgggcatgct ggatccagca 360
gagaaggatg aaaagggcat gcctgtgaca gctcgtgtgg tgtttgtttt tggtcctgat 420
aagaagctga agctgtctat cctctaccca gctaccactg gcaggaactt tgatgagatt 480
ctcagggtag tcatctctct ccagctgaca gcagaaaaaa gggttgccac cccagttgat 540
tggaaggatg gggatagtgt gatggtcctt ccaaccatcc ctgaagaaga agccaaaaaa 600
cttttcccga aaggagtctt caccaaagag ctcccatctg gcaagaaata cctccgctac 660
acaccccagc cttaa 675
Claims (10)
1.一种突变改造Prx6蛋白,其特征在于,通过定点突变技术对人Prx6蛋白进行以下至少一个位点的突变改造得到:
第39位氨基酸由His突变成Asn,第50位氨基酸由Glu突变为His,第78位氨基酸由Asp突变为由Arg,第103位氨基酸由Asp突变为Lys,第132位氨基酸由Arg突变为Gln。
2.根据权利要求1所述的突变改造Prx6蛋白,其特征在于,其氨基酸序列如SEQ ID NO:1所示。
3.一种编码权利要求1或2所述的突变改造Prx6蛋白的表达基因,其特征在于,所述表达基因的序列为:
A)编码权利要求1或2的突变改造Prx6蛋白的核苷酸序列;或者
B)与A)的核苷酸序列编码相同序列的蛋白质,但因遗传密码的简并性而与A)的核苷酸序列不同的序列;或者
C)对上述A)或B)所示核苷酸序列进行一个或多个碱基的取代、缺失、添加修饰的核苷酸序列。
4.根据权利要求3所述的突变改造Prx6蛋白的表达基因,其特征在于,所述A)中的核苷酸序列如SEQ ID NO.2所示。
5.一种表达载体,其特征在于,包含权利要求3或4所述的表达基因。
6.一种重组细胞,其特征在于,包含权利要求5所述的重组细胞。
7.一种权利要求1或2所述的突变改造Prx6蛋白的制备方法,其特征在于,包括以下步骤:
(1)获得人Prx6蛋白表达基因,并将上述表达基因插入至载体中,构建重组表达载体;
(2)以突变引物构建突变体质粒;
(3)将上述突变体质粒转入宿主细胞中,通过筛选得到阳性重组细胞,诱导表达,即得。
8.根据权利要求7所述的突变改造Prx6蛋白的制备方法,其特征在于,
步骤(1)中,通过下述方法获得人Prx6蛋白表达基因:以人宫颈癌hela细胞株cDNA文库为PCR反应模板,设计扩增引物进行PCR扩增反应,扩增产物即为人Prx6蛋白表达基因;
步骤(1)中,通过以下方法将上述表达基因插入至载体中,构建重组表达载体:将获得的人Prx6蛋白表达基因及质粒pCold Ⅰ进行Hind III/Xba I双酶切,获得含有Hind III/Xba I酶切位点的人Prx6基因和pCold Ⅰ载体,回收酶切的载体片段;将酶处理后的人Prx6基因片段和pCold Ⅰ载体在T4连接酶作用下进行连接;连接产物加入DH5α感受态菌中,筛选阳性克隆,得到pCold Ⅰ-hPrx6重组质粒,即为重组表达载体;
步骤(2)中,通过以下方法构建突变质粒:根据预定突变位点设计突变引物,加入上述pCold Ⅰ-hPrx6重组质粒进行突变PCR反应,于反应产物中加入Quick Cut DpnI限制性内切酶,将原始模板序列降解;酶切后合成产物转化DH5α感受态,对转化子进行菌落PCR验证,并提取质粒测序,得到预定序列的突变质粒。
步骤(3)中,通过以下方法诱导表达突变改造Prx6蛋白:将上述突变质粒转化BL21(DE3)感受态细胞,在含Amp和Kana抗性的固体培养基中筛选阳性克隆;挑取阳性单克隆于含Amp、Kana培养基中培养,加IPTG,诱导表达后收菌,即得突变改造Prx6蛋白。
9.根据权利要求8所述的突变改造Prx6蛋白的制备方法,其特征在于,
步骤(1)中:
所述扩增引物包括如下引物对:
hPrx6-F:5’-CCCAAGCTTATGCCCGGAGGTC-3’(SEQ ID NO:3);
hPrx6-R:5’-GCTCTAGATTAAGGCTGGGGTG-3’(SEQ ID NO:4);
所述PCR扩增反应条件为:94℃变性2min;94℃变性50s,55℃退火45s,72℃延伸1min,运行35个循环;72℃保温8min;4℃保持;
所述Hind III/Xba I双酶切的酶切体系为:buffer 2μL、Hind III/Xba I各0.5μL、人Prx6蛋白表达基因17μL;
所述载体片段通过以下方法回收:以0.8%的琼脂糖凝胶电泳,利用胶回收试剂盒回收酶切片段;
所述连接具体操作为:将酶处理后的人Prx6基因片段和pCold Ⅰ载体按照摩尔比为5:1的比例在T4连接酶作用下进行4℃连接过夜;
所述pCold Ⅰ-hPrx6重组质粒通过以下方法得到:将连接产物加入DH5α感受态菌中,冰浴30min后,42℃热激90s后立即置于冰上,冷却2min;无菌条件下添加700μL LB培养液,混匀后放入培养箱中100rpm振荡培养1h;将菌液均匀涂布于含Amp的LB固体培养基上,37℃倒置过夜培养,筛选阳性克隆,即得;
步骤(2)中,
所述突变引物包括以下引物对中的至少一对:
第39位H-N突变引物对:
5’-GGGCATTCTCTTCTCCAACCCTCGGGACTTTAC-3’(SEQ ID NO:5)
5’-GTAAAGTCCCGAGGGTTGGAGAAGAGAATGCCC-3’(SEQ ID NO:6)
第50位E-H突变引物对:
5’-GCACCACACACCTTGGCAG-3’(SEQ ID NO:7)
5’-CTGCCAAGGTGTGTGGTGC-3’(SEQ ID NO:8)
第78位D-R突变引物对:
5’-GTGTTGAGCGCCATCTTGCC-3’(SEQ ID NO:9)
5’-GGCAAGATGGCGCTCAACAC-3’(SEQ ID NO:10)
第103位D-K突变引物对:
5’-CCCATCATCAAGGATAGGAATCG-3’(SEQ ID NO:11)
5’-CGATTCCTATCCTTGATGATGGG-3’(SEQ ID NO:12)
第132位R-Q突变引物对:
5’-CATGCCTGTGACAGCTCAGGTGGTGTTTGTTTTTGG-3’(SEQ ID NO:13)
5’-CCAAAAACAAACACCACCTGAGCTGTCACAGGCATG-3’(SEQ ID NO:14)
所述突变PCR反应体系为50μL,包括:PCR buffer(10×)5μL,d NTP(2.5mmol/L)4μL,质粒1μL,上述引物对各1.5μL,Pfu DNA聚合酶1μL,dd H2O余量;
所述突变PCR反应条件为:94℃2min;94℃,30s;55℃,90s;68℃,12min进行20个循环,4℃,保持;
所述原始模板序列降解通过以下方法实现:于反应产物中加入2μL的QuickCut DpnI限制性内切酶,于37℃条件下反应10min;
步骤(3)中,通过以下方法诱导表达突变改造Prx6蛋白:将上述突变质粒转化BL21(DE3)感受态细胞,37℃倒置培养过夜,在含100μg/mL Amp和25μg/mL Kana抗性的固体培养基中筛选阳性克隆;挑取阳性单克隆于20mL含Amp、Kana培养基中,37℃震荡培养,当其OD600值为0.6±0.1时,加终浓度为1mmol/L的IPTG,37℃诱导表达4h后收菌;
还包括步骤(4)蛋白纯化步骤,具体如下:将上述收集得到的菌沉用20mmol/L的PBS重悬,在冰水浴中对菌体进行超声破碎,4℃10000g离心10min收集蛋白上清液,上清液用0.45μm的滤膜过滤;用含50mmol/L咪唑的PBS缓冲液平衡Ni离子亲和层析柱料,将超声后的蛋白上清液按1mL/min的流速挂柱,反复上样3次;分别用六倍柱料体积的含50mmol/L、75mmol/L、100mmol/L咪唑的PBS缓冲液洗脱杂蛋白,再用六倍柱料体积的含300mmol/L咪唑的PBS缓冲液洗脱目的蛋白,即得纯化突变改造Prx6蛋白。
10.权利要求1或2所述的突变改造Prx6蛋白在制备用于抗氧化的药物中的应用。
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