CN103509809B - 文昌鱼识别几丁质的丝氨酸蛋白酶casp基因及其应用 - Google Patents
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Abstract
本发明涉及一种文昌鱼识别几丁质的丝氨酸蛋白酶CASP基因、该基因编码的蛋白质及该蛋白质的表达方法和应用。所述CASP基因是通过兼并引物扩增和RACE扩增的方法,从文昌鱼总RNA中克隆得到的一个MASP类似基因。由CASP基因编码的蛋白质,通过重组表达载体PET-32a(+)-CASP,在大肠杆菌中以胞内可溶的形式表达。CASP编码的蛋白在对抗外来病原中起着重要作用,具有成为高效天然抗菌药物的开发价值。
Description
技术领域
本发明涉及文昌鱼特有的MASP类似基因(命名为CASP基因)及其编码的CASP蛋白,以及该基因和蛋白在制备治疗感染性疾病的药物上的应用,属于基因工程领域。
背景技术
补体激活的凝集素途径是先天性免疫的一种重要防御措施,在生物体内发挥着重要的生物学功能,如溶胞作用、溶菌作用、调理作用及介导炎症反应。
激活凝集素途径的复合物是由一系列的模式识别分子以及相关的丝氨酸蛋白酶家族决定的。最早报道的甘露糖结合凝集素(Mannose Bindig lectin,MBL)激活补体途径的发现是在20多年前,从此,补体系统在先天性免疫中重要性重新被认识。而目前研究结果表明,MBL相关的丝氨酸蛋白酶(MASPs):MASP1and MASP2,是参与激活补体系统的重要组成部分。
在病原体的感染时期,MASP在凝集素途径的产生和激活中扮演着重要角色,MASP-2是补体凝集素溶血途径中的重要一员,激活补体凝集素溶血途径中发挥着至关重要的作用。已有的研究表明:MASP2基因的突变和血清含量变化的多少与多种疾病密切相关(自身免疫性疾病,感染性疾病,肿瘤等)。MASP2缺陷,会导致凝集素途径的溶血活性缺失,导致化脓性感染;炎症性肺部疾病,也会导致MASP有关的补体途径损伤。同样的,MASP1缺陷,会导致胚胎细胞移行信息的潜在性丧失,表现为发育性综合征,包括面部异常,唇裂或/腭裂,颅骨缝早闭,学习障碍,生殖器,四肢和泌尿系统畸形,即所谓的3MC综合症;MASP1突变,也会导致其有关的补体途径损伤。
文昌鱼(Branchiostoma belcheri),属于脊索动物门(Chordate),头索动物亚门(cephalochordate),是现存和脊椎动物亲缘关系最近的无脊椎动物。本发明的发明人从文昌鱼中克隆得到了全新的分子,将其命名为CASP(chitin-binding associated serineprotease),该分子C端拥有和MASP同源的CCP和Tryp_SPc结构域,与MASP不同的是,其N端是一个几丁质结合结构域(CBD),这类分子在其他物种中从未报道,因此发明人推测其是文昌鱼所特有的MASP家族成员,并且担负着模式识别的功能。本发明的发明人对其蛋白进行了表达并制备了单抗,结果发现CASP内源蛋白能够结合几丁质糖,免疫胶体金的结果显示其能够识别和结合到金黄色葡萄球菌和大肠杆菌上,并且重组表达的该蛋白具有丝氨酸蛋白酶活性,并且拥有调理素的功能,能够显著地促进巨噬细胞对金葡菌的吞噬率。因此以上结果提示CASP在对抗外来病原中起着重要作用,具有成为高效天然抗菌药物的开发价值。
发明内容
本发明的一个目的在于提供一种新的类MASP家族免疫相关的基因,将其命名为CASP基因,同时还提供该基因所编码的蛋白。
本发明的另一个目的在于提供CASP基因所编码蛋白质的表达方法。
本发明再一个目的在于提供该蛋白在制备治疗感染性疾病药物中的应用。
一种文昌鱼识别几丁质的丝氨酸蛋白酶CASP基因,所述CASP基因是通过兼并引物扩增和RACE扩增的方法,从文昌鱼总RNA中克隆得到的一个MASP类似基因,其DNA序列如序列表中序列1所示。
由CASP基因编码的蛋白质,其氨基酸序列如序列表中序列2所示;该蛋白等电点为5.28,分子量为46834.0道尔顿。
所述的蛋白质通过重组表达载体PET-32a(+)-CASP,在大肠杆菌中以胞内可溶的形式表达。表达方法具体包括以下步骤:
(1)构建重组表达载体PET-32a(+)-CASP;
(2)将重组表达载体PET-32a(+)-CASP转化大肠杆菌菌株BL21(DE3),得到工程菌株;
(3)对大肠杆菌BL21(DE3)工程菌株进行培养;
(4)纯化重组的CASP融合蛋白,获得CASP基因编码的蛋白质。
所述步骤(1)中重组表达载体PET-32a(+)-CASP的构建包括以下步骤:
a)依据权利要求1所述的CASP基因的两端序列合成一对引物,其中,上游引物P1为5’-ccgGAATTCAAGCCCGTCCAAAAAACCC-3’,含有EcoRI切割位点;下游引物P2为5’-ccgCTCGAGCACGTACGAA CCAATGGC-3’,含有XhoI切割位点;
b)以CASP基因的全长质粒为模板,以P1、P2为引物,进行PCR扩增;
c)将PCR扩增产物克隆到表达载体PET-32a(+)上,得到重组表达载体PET-32a(+)-CASP。
所述的表达载体PET-32a(+)以TRX为融合伴体,该系统使蛋白在大肠杆菌中以胞内可溶的形式表达。
所述步骤(3)的培养条件为:将单菌落接种于30ml含有100mg/L的氨卞2×YT液体培养基中,37℃、220rpm培养12小时,取培养物按1:100的比例接种于含有100mg/L氨卞的2×YT培养基中,37℃、220rpm培养至OD600=0.6,加入终浓度为0.5mM的IPTG,18℃、180rpm诱导培养18小时后离心收集菌体。
所述步骤(4)为,将总菌体用TBS缓冲液洗涤后,再悬浮,超声破碎处理后,高速离心,收集上清液,将上清液经Ni-NTA Agarose亲和色谱层析纯化,收集目的蛋白的洗脱峰。
本发明所选择的白氏文昌鱼(Branchiostoma belcheri)采集于广东省湛江东海岛与硇洲岛航线海域。
本发明通过兼并引物扩增从文昌鱼的cDNA一链中克隆得到了CASP基因的部分目标片段,并对这个片段进行了扩增,得到目标基因序列的全长克隆,命名为CASP(其DNA序列如序列表中序列1所示)。该基因编码433个氨基酸(其氨基酸序列如序列表中序列2所示),含有12个氨基酸的信号肽。去除信号肽的成熟蛋白等电点为5.22,分子量为45559.3道尔顿。
本发明通过设计一对引物,将编码成熟蛋白序列的新基因CASP克隆到原核融合表达载体pET32a(+)上,连接转化后得到大肠杆菌工程菌株BL21(DE3)-pET32a(+)-CASP。此工程菌株中的重组表达载体pET32a(+)-CASP以T7为启动子,trx为分子融合伴体,帮助重组蛋白正确折叠,以可溶的形式表达,N端和C端各有6×His结构作为标签,便于利用固定化金属螯合亲和层析进行纯化。然后再通过对培养时间,诱导时间,温度等条件的摸索和优化,重组CASP融合蛋白的表达量得到提高。
本发明还摸索和优化了重组CASP蛋白的纯化条件,表达产物的超声破碎裂解液经Ni-NTA Agarose亲和色谱层析,得到纯度较高的融合蛋白。
本发明的表达质粒复制方法:参照Sambrook(Sambrook,et al.1989,Molecular cloing.Cold Spring Harbor Labroratory Press.USA)方法,用CaCl2的方法将质粒转化E.coli.DH5α或BL21(DE3)菌株,用含氨苄青霉素(100μg/mL)的LB培养基培养转化菌株,用Omega公司试剂盒提取质粒。
本发明对其CASP编码的蛋白进行了表达,制备并筛选了蛋白的单克隆抗体,结果发现内源CASP蛋白能够结合几丁质糖,免疫胶体金的结果显示其能够识别和结合到金黄色葡萄球菌和大肠杆菌上,并且重组表达的该蛋白具有丝氨酸蛋白酶活性,并且拥有调理素的功能,能够显著地促进巨噬细胞对金葡菌的吞噬率。结果提示CASP编码的蛋白在对抗外来病原中起着重要作用,具有成为高效天然抗菌药物的开发价值。
附图说明
图1为本发明文昌鱼CASP蛋白与其它MASP家族成员蛋白的Tryp_SPc结构域序列同源性比较结果。以下是图1所列到的其他物种名英文简称,全称及拉丁文命名全称(如斜体):
Bbe,Branchiostoma belcheri Sh,Shark(Triakis scyllium);
Hu,human; Lam,Lamprey(Lethenteron camtschaticum);
Xe,Xenopus As,Ascidian(Halocynthia roretzi);
Ca,Carp; Nv,Nematostella vectensis.
以下是图1所列到的其他物种MASP全称以及在NCBI等数据库里的序号(如括号内标注):
HuMASP1(NP_001870); LamMASP1(AB089265.1);
HuMASP2(O00187); LamMASPA(BAC41492.1);
HuMASP3(AAK84071); LamMASPB(AB089266.1);
XeMASP1(NP_001082340.1); AsMASPa(BAC41341.1);
XeMASP2(BAA86865.1); AsMASPb(BAC41342.1);
XeMASP3a(AB078636.1); BbeMASP1(BAC75888.1);
XeMASP3b(AB078637.1); BbeMASP3(BAC75889.1);
CaMASP2(BAE44364.1); NvMASP(AB450044.1)
ShMASP(BAA86867.1);
图2为扩增得到的CASP基因全长片段的电泳图,其中M:DNA DL2000marker;1:CASP基因全长片段。
图3为CASP基因的重组表达质粒的构建图。
图4为重组蛋白CASP经诱导表达、亲和层析纯化后的SDS-PAGE和Western Blot检测显影图。
图5重组蛋白酶切小肽活性结果图。
图6A-图6C为重组蛋白CASP的调理素活性实验结果图,其中,图6A为:流式细胞分析结果图;图6B为:吞噬率统计柱状图;图6C为:激光共聚焦结果图。
图7为文昌鱼体液内源CASP蛋白结合几丁质实验结果。
图8A-图8E为内源CASP识别病原菌的免疫胶体金实验结果,其中图8A为:阴性对照;图8B为:阳性对照;图8C-E为:CASP阳性信号。
具体实施方式
下面通过实施例,对本发明的技术方案作进一步的说明。
实施例1:文昌鱼总RNA的提取和CASP部分片段扩增。
总RNA的提取和RACE cDNA合成:取文昌鱼全鱼,采用Trizol试剂法提取总RNA,酚/氯仿抽提去除蛋白质,获得文昌鱼总RNA。取1μg总RNA,按照TOYOBO公司的First StrandcDNA Synthesis Kit ReverTra Ace-α-TM(code No.FSK-100)说明书操作进行逆转录合成第一链。取2μl cDNA一链产物,以根据佛罗里达文昌鱼测序数据库CASP基因预测序列设计的兼并引物5′PCR Primer:5′-ACCCATCCCTCCCAGTCAC-3′和3′PCR Primer:5′-GTAGACACTCGGCTTGGCG-3′为引物,进行RT-PCR扩增CASP的部分片段899bp。将扩增得到的目的片段连接到pGEX T easy vector(promega)后转化DH5α大肠杆菌,挑选重组克隆测序。Blast同源分析表明,获得的这个片段含有CCP和SP结构域,类似MASP家族成员。
实施例2:RACE扩增CASP基因5′和3′末端。
按照Invitrogen的GeneRacerTM Kit进行RNA的去磷酸化RACE、脱帽反应、RNA oligo的连接及mRNA的逆转录,从而合成cDNA一链,采用Takara的LA Taq聚合酶,按照GeneRacerTMKit的反应体系进行PCR扩增。其中,根据上面扩增得到的CASP部分片段的序列,设计进行5′RACE扩增的基因特异的外套引物为5′-gene-specific primer:5′-GACCATCCAAGGCATCACCAGTT-3′;内巢引物为5′-nested primer:5′-GGACACTGACATGGACTGAAGGAGTA-3′;CASP的3′RACE扩增的基因特异的外套引物为5′-gene-specific primer:5′-GGAAGGAGACCACCCACGGCT-3′;内巢引物为5′-nestedprimer:5′-CGCTACGTAACGGCATGACAGTG-3′。将扩增得到的目的片段连接到pGEX T easyvector(promega)后,转化DH5α大肠杆菌,挑选重组克隆测序。并与实施例1得到的目的片段进行拼接,得到CASP基因的全长EST序列,长度是1976bp,编码434个氨基酸的CASP蛋白。
实施例3:CASP全长的扩增及序列分析。
根据实施例2拼接得到的CASP基因的序列,设计引物5′-TCTGCATTTCACATCATCAAACG-3′和5′-TTGGGTGAGGTTTCTAAGGCTAA-3′,采用Takara的LATaq聚合酶扩增CASP的全基因,扩增得到的1976片段的电泳图谱如图2所示。将扩增得到的目的片段连接到pGEX T easy vector(promega)后转化DH5α大肠杆菌,挑选重组克隆测序。Blast同源分析表明,获得了编码CASP全基因的EST序列。
实施例4:重组CASP表达质粒的构建
依据CASP基因全长序列,设计用于重组表达的引物P1、P2,其中,
上游引物(P1)含有EcoRI切割位点,下游引物(P2)含有XhoI切割位点。
上游引物(P1):5’-ccg GAATTC AAGC CCGTCCAAAA AACCC-3’
保护碱基EcoRI CASP基因序列
下游引物(P2):5’-ccg CTCGAG CACGTACGAA CCAATGGC-3’
保护碱基XhoI CASP基因序列
利用含有CASP全长基因的pGEX T质粒为模板,P1、P2为引物,进行PCR扩增,得到特异扩增的条带,其大小为1302bp。将该PCR扩增产物切胶回收,用Xho I和EcoR I对目的片段和质粒pET32a(+)同时进行双酶切,然后用T4DNA连接酶连接(构建过程见图3),挑选阳性克隆测序,进行BLAST分析,确认目的片段重组连接成功。
实施例5:文昌鱼CASP融合蛋白的表达
将重组表达载体pET32a(+)-CASP转化大肠杆菌BL21(DE3),获得工程菌株BL21(DE3)-pET32a(+)-CASP。基因工程菌超声破碎得到的裂解上清液,经SDS-PAGE电泳分析表明,菌株经诱导后有明显的特异表达产物带,分子量与用软件seqtools84预测的理论值64kD相符。对培养时间,诱导浓度,温度等条件的摸索得出,工程菌株BL21(DE3)-pET32a(+)-CASP的培养条件为:将单菌落接种于30ml氨苄抗性2×YT液体培养基中,37℃、250rpm培养过夜;(扩大培养)取过夜培养物按1:100的比例接种于含有100mg/L的氨卞的2×YT培养基中,37℃、250rpm培养至OD600=0.6;(诱导目的蛋白大量表达)加入终浓度为0.5mM的IPTG,18℃、250rpm诱导培养20hr。离心,收集菌体,分析目的蛋白是否表达,经SDS-PAGE电泳分析表明,在该培养条件下,CASP融合蛋白的表达量占菌体总蛋白的一半左右,处于可溶状态。
实施例6:重组文昌鱼CASP融合蛋白的纯化
将总菌体用TBS缓冲液(50mMTris.Cl,150mMNaCl,pH7.5)洗涤,再用适量的TBS缓冲液悬浮,超声处理120min后,离心获得上清液,进行亲和色谱层析纯化,SDS-PAGE分析融合蛋白的表达和层析的结果。从SDS-PAGE结果可以得出:CASP蛋白能被Ni-NTA Agarose亲和柱所吸附,用100mM咪唑洗柱时,能把重组的CASP目的蛋白洗下。收集得到重组蛋白的洗脱峰后,对应于目的蛋白洗脱峰处的洗脱液经凝胶柱除去蛋白中的咪唑,并用30KD的蛋白超滤柱(Millipore)浓缩。SDS-PAGE分析表明:浓缩后得到成份单一浓度较高的重组的CASP目的蛋白(如图4所示)。
实施例7:CASP重组蛋白酶切小肽活性分析
将浓度为400μg/ml的CASP重组蛋白与不同浓度的底物小肽Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin(Sigma)37℃避光孵育30min,每组样品重复三次,用酶标仪检测A360nm/460nm(激发波长360nm,吸收波长460nm)变化,Origin7.5软件统计并拟合酶饱和曲线。结果如图5所示,与阴性对照TRX相比,CASP随着底物量的增加,其产物释放吸光值也相应呈明显增高趋势,证明CASP重组蛋白具有丝氨酸蛋白酶活性。
实施例8:CASP重组蛋白的调理素活性分析
将状态良好的Raw264.7细胞系铺至6孔细胞培养板内,过夜培养。FITC标记过的金黄色葡萄球菌(5×108cells ml-1in PBS)与等体积的CASP重组蛋白于室温避光孵育2hr,之后将该混合样品加入到6孔细胞板中。37℃摇床轻晃1hr,保证吞噬细胞发挥吞噬作用。之后,用5%PFA固定15min,用预冷的PBS洗三次。加入0.2mg/ml的台盼蓝以猝灭细胞表面及背景荧光,再用冷的PBS洗两遍。最后重悬至500μlPBS中,用细胞流式分析技术进行检测。同时处理并固定后的Raw264.7细胞系,通过蛋白单抗的处理,及免疫荧光的后续步骤,利用激光共聚焦的手段对吞噬过程进行捕捉。流式细胞术的原始数据如图6A所示,将三组实验中吞噬细胞对细菌的吞噬率利用GraphPad Prism5软件进行统计比较,得到如图6B的柱状图,可以显示,CASP处理组吞噬率较对照组有明显的提高,并且具有显著性统计意义(P=0.0040)。而加入CASP单抗将目的蛋白屏蔽后,该吞噬率又明显下降,这进一步表明CASP能特异地促进吞噬作用,具有调理素的活性。将上述吞噬作用用激光共聚焦显微镜观察,红色荧光标记为CASP,绿色荧光标记为金黄色葡萄球菌,蓝色为吞噬细胞细胞核。结果发现CASP能够识别金黄色葡萄球菌并帮助吞噬细胞吞噬病原菌(见图6C)。
实施例9:CASP内源蛋白结合几丁质的活性分析
将几丁质珠(chitin beads,NEB)用PBS平衡后,与300μl文昌鱼体液室温摇床孵育3hr。用PBS洗涤三次后,将几丁质珠子煮样,利用CASP单抗及western blot去检测,结果表明文昌鱼体液中CASP内源蛋白(图7Lane3)能特异地结合几丁质,而对照组为未处理的体液中CASP内源蛋白,表明了其模式识别的特性。
实施例10:CASP内源蛋白结合病原菌的活性分析
将活化后的大肠杆菌和金黄色葡萄球菌加入到培养有文昌鱼的海水中,4℃过夜后,将文昌鱼的鳃、肠、肝盲囊解剖提取,用于免疫胶体金的后续处理。用配好的固定液(即:包含有多聚甲醛、苦味酸和戊二醛的NaPB溶液)固定3hr。液氮冻融一次后,山羊血清封闭0.5hr,4℃下用CASP单抗作为一抗孵育24hr,TBS洗涤三次后,在4℃下,用金颗粒标记的羊抗鼠二抗(Nanoprobe)孵育过夜。经NaPB洗涤后,用银增强试剂盒处理,放大胶体金信号。将处理好的样品送至中山大学医学院电镜教研室做进一步的包埋,脱水,切片处理,最后置于电镜下观察照相。结果如图8A-8E,图8A为阴性对照,即鼠源IgG作为一抗的处理组;图8B为阳性对照,即文昌鱼细胞核抗组蛋白的单抗处理组,黑色颗粒为阳性信号,如黑色箭头所示,N为细胞核;图8C-E为阳性结果,即CASP单抗处理组,CASP在消化道上皮细胞表达(表皮细胞黑色箭头a所示),并能分泌至外,与病原菌结合(消化道肠腔病原菌表面,黑色箭头b所示),并且如图8D呈吞噬中间状态,表明内源的CASP蛋白能够结合到病原菌上,并介导吞噬过程,提示了该分子在对抗病原菌时的重要作用。
Claims (5)
1.一种文昌鱼识别几丁质的丝氨酸蛋白酶CASP基因,其特征在于,所述CASP基因是通过兼并引物扩增和RACE扩增的方法,从文昌鱼总RNA中克隆得到的一个MASP类似基因,其DNA序列如序列表中序列1所示。
2.由权利要求1所述的CASP基因编码的蛋白质,其氨基酸序列如序列表中序列2所示。
3.权利要求2所述的蛋白质的表达方法,其特征在于,通过重组表达载体PET-32a(+)-CASP,在大肠杆菌中以胞内可溶的形式表达;
所述重组表达载体PET-32a(+)-CASP为通过包括以下步骤的方法构建获得:
a)依据权利要求1所述的CASP基因的两端序列合成一对引物,其中,上游引物P1为5’-ccgGAATTCAAGCCCGTCCAAAAAACCC-3’,含有EcoRI切割位点;下游引物P2为5’-ccgCTCGAGCACGTACGAA CCAATGGC-3’,含有XhoI切割位点;
b)以含有CASP全长基因的pGEX T easy vector质粒为模板,以P1、P2为引物,进行PCR扩增;
c)将PCR扩增产物克隆到表达载体pET-32a(+)上,得到重组表达载体PET-32a(+)-CASP。
4.根据权利要求3所述的表达方法,其特征在于,所述表达方法包括以下步骤:
(1)构建重组表达载体PET-32a(+)-CASP;
(2)将重组表达载体PET-32a(+)-CASP转化大肠杆菌菌株BL21(DE3),得到工程菌株;
(3)对步骤(2)所述的工程菌株进行培养,培养条件为:将单菌落接种于30ml含有100mg/L氨卞霉素的2×YT液体培养基中,37℃、220rpm培养12小时,取培养物按1:100的体积比接种于含有100mg/L氨卞霉素的2×YT培养基中,37℃、220rpm培养至OD600=0.6,加入终浓度为0.5mM的IPTG,18℃、180rpm诱导培养18小时后离心收集菌体;
(4)纯化重组的融合蛋白,获得CASP基因编码的蛋白质,按如下方法进行:将总菌体用TBS缓冲液洗涤后,再悬浮,超声破碎处理后,高速离心,收集上清液,将上清液用Ni-NTA Agarose亲和柱吸附,然后用100mM咪唑洗柱,收集目的蛋白的洗脱峰;其中,所述TBS缓冲液其Tris-Cl浓度为50mM、NaCl浓度为150mM,pH为7.5。
5.权利要求2所述的蛋白质在制备治疗大肠杆菌和金黄色葡萄球菌感染性疾病的药物中的应用。
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CN1356907A (zh) * | 1999-06-18 | 2002-07-03 | 布亚纳森·乔·布拉吉 | 鱼丝氨酸蛋白酶及其在制药和化妆品上的应用 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1356907A (zh) * | 1999-06-18 | 2002-07-03 | 布亚纳森·乔·布拉吉 | 鱼丝氨酸蛋白酶及其在制药和化妆品上的应用 |
CN101328478A (zh) * | 2008-04-24 | 2008-12-24 | 中山大学 | 一种具有抗血小板性栓塞活性的蛋白质及生产方法和应用 |
CN103007258A (zh) * | 2011-09-22 | 2013-04-03 | 安淇生物控释技术(苏州)有限公司 | 含鱼丝氨酸蛋白酶和抗菌化合物的医用组合物及其用途 |
CN102827784A (zh) * | 2012-09-11 | 2012-12-19 | 集美大学 | 重组鲫鱼mbsp表达菌株及其构建方法 |
Non-Patent Citations (2)
Title |
---|
Putnam,N.H.等."GenBank Accession No:XM_002610326.1 ".《GenBank》.2009,第1-2页. * |
Putnam,N.H.等."GenBank Accession No:XP_002610372.1".《GenBank》.2009,第1-2页. * |
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