CN112250769A - Ftcd和cyp2d6融合蛋白及其构建方法、应用 - Google Patents
Ftcd和cyp2d6融合蛋白及其构建方法、应用 Download PDFInfo
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- CN112250769A CN112250769A CN202011057065.1A CN202011057065A CN112250769A CN 112250769 A CN112250769 A CN 112250769A CN 202011057065 A CN202011057065 A CN 202011057065A CN 112250769 A CN112250769 A CN 112250769A
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- fusion protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
本发明公开了FTCD和CYP2D6融合蛋白及其构建方法、应用,构建方法包括以下步骤:S1、重组全基因合成序列:连接FTCD和CYP2D6蛋白区段的核苷酸序列获得目的基因;S2、构建重组质粒:将步骤S1中获得的重组全基因合成序列构建至表达载体,获得重组质粒;S3、一次转染:将步骤S2中的重组质粒转染宿主细胞,获得重组杆粒;S4、二次转染:将步骤S3中的重组杆粒再次转染宿主细胞,获得第一代的杆状病毒;S5、表达:将步骤S4中的第一代的杆状病毒进行表达;S6、获得融合蛋白:将步骤S5中表达得到的细胞培养物进行筛选、纯化,获得融合蛋白。通过本发明所述方法构建的融合蛋白能提高检测试剂盒的灵敏度和特异性,减少漏检和错检。
Description
技术领域
本发明涉及生物医药工程技术领域,具体涉及FTCD和CYP2D6融合蛋白及其构建方法、应用。
背景技术
自身免疫性肝炎(AIH)是一个以肝实质损伤为主要表现的自身免疫性疾病,女性多见,临床表现多样,有肝酶持续性升高、球蛋白升高、多种自身抗体阳性。病理改变为以汇管区淋巴细胞性碎屑坏死为基本特点的慢性活动性肝炎。在鉴别诊断上常需结合患者的临床症状、自身抗体和肝穿病理进行综合判断。自身免疫性肝炎(autoimmune hepatitis,AIH)与原发性胆汁性肝硬化(primary biliary cirrhosis,PBC),原发性硬化性胆管炎(primary sclerosing cholangitis,PSC)和自身免疫性胆管炎(autoimmunecholangitis,AIC)统称为自身免疫性肝病,原发性胆汁性肝硬化或原发性硬化性胆管炎被称为自身免疫性肝病的局外综合征(outlier syndrome)。当自身免疫性肝炎和原发性胆汁性肝硬化或原发性硬化性胆管炎合并存在时,称为重叠综合征(overlap syndrome)。局外综合征和重叠综合征共占自身免疫性肝病的18%。自身免疫性肝炎也常伴发其他自身免疫性疾病如系统性红斑狼疮、干燥综合征、溃疡性结肠炎、甲状腺炎和硬皮病等。
根据血清中自身抗体的不同,可将AIH分为3种类型:
AIH-1型,较常见,可发生于任何年龄,以血清中存在ANA和ASMA为特征,该型对治疗应答效果良好。
AIH-2型,多发于儿童和青少年,以血清中抗-LKM-1和抗-LC-1为标志,该型起病急,进展快,对免疫抑制应答欠佳者常见,且停药后易复发,一般需要长期维持治疗。
AIH-3型,目前一些国外的学者将抗-SLA/LP阳性者划分为AIH-3型,该型与较严重的肝炎相关。
AIH-2型中涉及到的LC-1的靶抗原为FTCD(亚甲基四氢叶酸环化脱氢酶),存在于细胞质中,参与组氨酸代谢和甲酸及衍生物代谢;LKM-1的靶抗原为CYP2D6(细胞色素P4502D6),主要存在于内质网和线粒体内膜上,作为一种末端加氧酶,参与了生物体内的甾醇类激素合成等过程。
目前检测AIH-2型的抗原均为单一抗原,在体外诊断试剂研发过程中无法实现同时检测,检测灵敏度和特异性较差,导致试剂成本过高、检测过程繁琐、患者疾病漏检、竞争力缺乏等不足。
发明内容
本发明目的在于提供一种FTCD和CYP2D6融合蛋白,该融合蛋白能提高检测试剂盒的灵敏度和特异性,以减少漏检和错检。
此外,本发明还提供上述FTCD和CYP2D6融合蛋白的构建方法、应用。
本发明通过下述技术方案实现:
一种FTCD和CYP2D6融合蛋白,其氨基酸序列如SEQ ID No.1所示。
一种FTCD和CYP2D6融合蛋白,包括FTCD蛋白区段和CYP2D6蛋白区段,FTCD蛋白区段氨基酸序列如SEQ ID NO.2所示,CYP2D6蛋白区段氨基酸序列如SEQ ID NO.3所示。
一种FTCD和CYP2D6融合蛋白的构建方法,包括如下步骤:
S1、重组全基因合成序列:依次连接CYP2D6和FTCD蛋白区段的核苷酸序列获得目的基因;
S2、构建重组质粒:将步骤S1中获得的重组全基因合成序列构建至表达载体,获得重组质粒;
S3、一次转染:将步骤S2中的重组质粒转染宿主细胞,获得重组杆粒;
S4、二次转染:将步骤S3中的重组杆粒再次转染宿主细胞,获得第一代的杆状病毒;
S5、表达:将步骤S4中的第一代的杆状病毒进行扩增后表达;
S6、获得融合蛋白:将步骤S5中表达得到的细胞培养物进行纯化,获得融合蛋白。
优选地,所述步骤S1中重组全基因合成序列中还包括柔性肽基因序列、真核KOZAK序列、蜂毒信号肽序列和His标签序列。
优选地,所述步骤S1重组全基因合成序列中,真核KOZAK序列、蜂毒信号肽序列、CYP2D6蛋白区段的核苷酸序列、柔性肽基因序列、FTCD蛋白区段的核苷酸序列和His标签序列依次相连。
优选地,所述步骤S2构建重组质粒中的表达载体包括昆虫细胞表达载体和/或杆状病毒表达载体。
优选地,所述步骤S3一次转染时的宿主细胞包括大肠杆菌宿主和/或动物性细胞宿主。
FTCD和CYP2D6融合蛋白在试剂盒、疫苗或诊断抗原中的应用。
本发明的融合蛋白,是通过在融合蛋白基因序列C端加入His标签序列后构建至pFastBacHT B质粒载体中,然后制备一种含有FTCD和CYP2D6基因序列的重组质粒;重组质粒转化大肠杆菌DH10 Bac,获得重组杆粒;将重组杆粒转染昆虫细胞后分泌表达并将His标签序列纯化后即得FTCD和CYP2D6融合蛋白。
本发明与现有技术相比,具有如下的优点和有益效果:
1、本发明只需要一次表达就能制备得到一种FTCD和CYP2D6两种蛋白的融合蛋白,表达过程简单,降低成本与时间。
2、使用本发明所述FTCD和CYP2D6融合蛋白制备检测试剂盒能提高检测试剂盒的灵敏度、正确性和特异性,减少漏检和错检。
3、用本发明所述FTCD和CYP2D6融合蛋白制备检测试剂盒能够降低反复检测成本,有利于病人节省成本。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解,构成本申请的一部分,并不构成对本发明实施例的限定。在附图中:
图1为目的基因阳性克隆鉴定图;
图2为融合蛋白鉴定图;
图3为纯化蛋白电泳图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
实施例1:
融合基因(目的基因)的全基因序列的合成:
S1、依次连接CYP2D6和FTCD蛋白区段的核苷酸序列,根据UniProt所公开的序列信息,在连接两个基因片段之间去掉终止密码子和起始密码子,并加入5个氨基酸残基(GGGGS)的柔性肽基因序列(Linker序列);在N端加入蜂毒信号肽序列,C端加入6*His标签序列;在序列上游加入限制性核酸内切酶位点BamH I,在其下游则加入Xba I酶切位点,全基因合成。全基因合成时各序列之间的排列顺序为:真核KOZAK-信号肽-FTCD-Linker-CYP2D6-6*His。
实施例2:
含有融合基因的重组质粒构建:
2.1将实施例1中获得的全基因序列和质粒载体pFastBacHT B进行BamH I、Xba I双酶切,试剂盒回收酶切产物,将酶切产物通过T4连接酶连接。
2.2连接产物转化感受态大肠杆菌DH5α,体积不超过感受态细胞的5%,轻轻旋转几次混匀内容物,冰浴30分钟;将管放入42℃水浴,定时60秒热休克,快速将管转移到冰浴120秒;每管加入400μL LB培养基,37℃缓摇60分钟,使细菌复苏并表达质粒编码的抗生素抗性标记基因,低速离心2分钟,去上清,留约100μL培养基在离心管内,重悬菌体,用玻璃铺菌器将菌液在琼脂板上铺匀。
2.3将平板倒置于37℃恒温培养箱,12-16小时后可出现菌落。挑取阳性克隆进行鉴定,如图1所示。
实施例3:
融合蛋白的表达
3.1将实施例2中获得的重组质粒转化至大肠杆菌DH10 Bac感受态细胞,体积不超过感受态细胞的5%,轻轻旋转几次混匀内容物,冰浴30分钟。
3.2将管放入42℃水浴,定时90秒热休克,快速将管转移到冰浴120秒;每管加入800μL LB培养基,37℃,缓摇4小时,使细菌复苏并表达质粒编码的抗生素标记基因;用玻璃铺菌器将30μL菌液在抗性Kan+Gent+Tet琼脂板上铺匀;将平板倒置于37℃恒温培养箱中,30-48小时后可出现蓝白斑菌落。
3.3挑取阳性白斑单菌落接入5mL抗性LB,缓摇12-16h,取菌液进行PCR鉴定,结果显示杆粒重组基因正确。将重组杆粒经转染试剂转染昆虫细胞,3-5天后收取细胞上清液作为第一代杆状病毒。用第一代杆状病毒感染昆虫细胞48-96小时后,获得第二代病毒,用于融合蛋白的表达。FTCD和CYP2D6融合蛋白的氨基酸序列如SEQ ID No.1所示。
3.4第二代病毒以MOI=10感染昆虫细胞SF9,继续培养72小时后,收集细胞培养物。
实施例4:
融合蛋白鉴定:
4.1分别将实施例3中的上清与细胞进行非还原性SDS-PAGE电泳,使用12%分离胶浓度,电泳约90分钟;
4.2将SDS-PAGE电泳凝胶进行转膜(PVDF),湿转100V,60分钟;收集PVDF,使用5%脱脂奶粉封闭;
4.3将4.2封闭后的PVDF膜加入anti-His mAb,再洗净后加入鼠二抗孵育1h,ECL显色;确定蛋白表达于细胞上清中,如图2所示。
实施例5:
融合蛋白纯化
5.1取实施例3中的细胞培养物在5000g离心力下离心20分钟收集细胞培养上清。
5.2细胞培养上清与1mL用平衡缓冲液平衡好的Ni-NTA RESIN孵育过夜,次日,弃上清,加入适量平衡缓冲液重悬填料,将填料转移到自流柱里。
5.3依次用冲洗缓冲液1(50mM Tris-HCl pH8.0;500mM NaCl)洗10个柱体积;冲洗缓冲液2(50mM Tris-HCl pH8.0;500mM NaCl;20mM咪唑)洗10个柱体积;冲洗缓冲液3(50mMTris-HCl pH8.0;500mM NaCl;250mM咪唑)洗5个柱体积。
5.4洗脱缓冲液洗脱目的蛋白,1×PBS透析纯化后目的蛋白保存在-80℃。
5.5 SDS-PAGE电泳检测表达蛋白纯度及浓度,如图1所示,M:Protein Marker;1:纯化后目的蛋白。
实施例6:
试剂盒特异性和灵敏度实验
6.1采用实施例5中的优化条件进行ELISA实验,选择含有ANA、ASMA、LKM-1、LC-1和SLA/LP的样本作为灵敏度和特异性实验的参考样本,FTCD和CYP2D6融合蛋白ELISA检测方法特异性和灵敏度实验结果如表1所示,本发明的FTCD和CYP2D6融合蛋白检测试剂盒具有良好的特异性并提高了灵敏度。
表1
项目 | 结果 |
ANA、ASMA | - |
LKM-1、LC-1 | + |
SLA/LP | - |
ANA、ASMA、LKM-1、LC-1 | + |
LKM-1、LC-1、SLA/LP | + |
ANA、ASMA、LKM-1、LC-1、SLA/LP | + |
以上所述的具体实施方式,对本发明的目的、技术方案和有益效果进行了进一步详细说明,所应理解的是,以上所述仅为本发明的具体实施方式而已,并不用于限定本发明的保护范围,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 四川携光生物技术有限公司
<120> FTCD和CYP2D6融合蛋白及其构建方法、应用
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Ala Thr Met Leu Pro Leu Val Ala Val Ala Leu Val Pro Met Leu Gly
1 5 10 15
Ser Leu Ser Leu Ala Ala Pro Val Gly Gly Val Gly Ala Ala Ser Ala
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Ala Pro Gly Gly Gly Ser Val Ala Ala Ala Ala Ala Ala Met Gly Ala
35 40 45
Ala Leu Gly Ser Met Val Gly Leu Met Thr Thr Gly Ala Ala Gly Pro
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Gly Ser Leu Ala Thr Thr Met Ala Ala Leu Ile Pro Pro Pro Ala Gly
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Ala Ser Ala Leu Leu Thr Thr Leu Val Ala Ala Ala Ala Gly Ala Pro
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Thr Ala Thr Leu Gly Ala Met Ala Leu Pro Leu Ala Thr Pro Gly Gly
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Leu Ala Ala Ala Thr Ala Ala Leu Gly Gly Gly Leu Ala Ala Ala Val
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Leu Gly Gly Leu Ala Ala Cys Gly Ala Leu Ala Cys Ala Ser Ala Leu
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Gly Val Ala Ala Leu Ala Leu Gly Met Gly Val Pro Gly Ala Thr Pro
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Ala Val Leu Ile Ala Leu Ala Ala Ile Thr Ala Gly Ala Pro Leu Ala
180 185 190
Gly Ile His His Ala Val Ser Ser Leu Leu Gly Gly Ala Leu Thr Gly
195 200 205
Ala Ala Leu Val Leu Ala Cys Leu Gly Thr Ala Gly Gly Gly Gly Gly
210 215 220
Gly Ser Gly His Ala Met Thr Thr Ala Pro Ala Gly Pro Pro Ala Ala
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Leu Thr Gly Ala Pro Leu Ala Gly Met Gly Leu Ala Leu Gly Ala Pro
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Gly Ser Ser Pro Ala Ala Gly Ala Leu Ala Ile Val Val Ala Ala Leu
260 265 270
Pro Ser Ala Gly Met Val Thr Thr Ser Thr Thr Leu Ala Thr Gly Leu
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Leu Leu Met Ile Leu His Pro Ala Val Gly Ala Ala Val Gly Gly Gly
290 295 300
Ile Ala Ala Val Ile Gly Gly Val Ala Ala Pro Gly Met Gly Ala Gly
305 310 315 320
Ala His Met Pro Thr Thr Thr Ala Val Ile His Gly Val Gly Ala Pro
325 330 335
Gly Ala Ile Val Pro Leu Gly Val Thr His Met Thr Ser Ala Ala Ile
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Gly Val Gly Gly Pro Ala Ile His His His His His His
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Gly Pro Gly Ser Leu Ala Thr Thr Met Ala Ala Leu Ile Pro Pro Pro
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Ala Gly Ala Ser Ala Leu Leu Thr Thr Leu Val Ala Ala Ala Ala Gly
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Ala Pro Thr Ala Thr Leu Gly Ala Met Ala Leu Pro Leu Ala Thr Pro
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Ala Leu Gly Val Ala Ala Leu Ala Leu Gly Met Gly Val Pro Gly Ala
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Gly His Ala Met Thr Thr Ala Pro Ala Gly Pro Pro Ala Ala Leu Thr
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Gly Ala Pro Leu Ala Gly Met Gly Leu Ala Leu Gly Ala Pro Gly Ser
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Ser Pro Ala Ala Gly Ala Leu Ala Ile Val Val Ala Ala Leu Pro Ser
35 40 45
Ala Gly Met Val Thr Thr Ser Thr Thr Leu Ala Thr Gly Leu Leu Leu
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Met Ile Leu His Pro Ala Val Gly Ala Ala Val Gly Gly Gly Ile Ala
65 70 75 80
Ala Val Ile Gly Gly Val Ala Ala Pro Gly Met Gly Ala Gly Ala His
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Met Pro Thr Thr Thr Ala Val Ile His Gly Val Gly Ala Pro Gly Ala
100 105 110
Ile Val Pro Leu Gly Val Thr His Met Thr Ser Ala Ala Ile Gly Val
115 120 125
Gly Gly Pro Ala Ile
130
Claims (8)
1.一种FTCD和CYP2D6融合蛋白,其特征在于,其氨基酸序列如SEQ ID No.1所示。
2.根据权利要求1所述的一种FTCD和CYP2D6融合蛋白,其特征在于,包括FTCD蛋白区段和CYP2D6蛋白区段,FTCD蛋白区段氨基酸序列如SEQ ID NO.2所示,CYP2D6蛋白区段氨基酸序列如SEQ ID NO.3所示。
3.一种FTCD和CYP2D6融合蛋白的构建方法,其特征在于,包括如下步骤:
S1、重组全基因合成序列:依次连接CYP2D6和FTCD蛋白区段的核苷酸序列获得目的基因;
S2、构建重组质粒:将步骤S1中获得的重组全基因合成序列构建至表达载体,获得重组质粒;
S3、一次转染:将步骤S2中的重组质粒转染宿主细胞,获得重组杆粒;
S4、二次转染:将步骤S3中的重组杆粒再次转染宿主细胞,获得第一代的杆状病毒;
S5、表达:将步骤S4中的第一代的杆状病毒进行扩增后表达;
S6、获得融合蛋白:将步骤S5中表达得到的细胞培养物进行纯化,获得融合蛋白。
4.根据权利要求3所述的一种FTCD和CYP2D6融合蛋白的构建方法,其特征在于,所述步骤S1中重组全基因合成序列中还包括柔性肽基因序列、真核KOZAK序列、蜂毒信号肽序列和His标签序列。
5.根据权利要求3所述的一种FTCD和CYP2D6融合蛋白的构建方法,其特征在于,所述步骤S1重组全基因合成序列中,真核KOZAK序列、蜂毒信号肽序列、CYP2D6蛋白区段的核苷酸序列、柔性肽基因序列、FTCD蛋白区段的核苷酸序列和His标签序列依次相连。
6.根据权利要求3所述的一种FTCD和CYP2D6融合蛋白的构建方法,其特征在于,所述步骤S2构建重组质粒中的表达载体包括昆虫细胞表达载体和/或杆状病毒表达载体。
7.根据权利要求3所述的一种FTCD和CYP2D6融合蛋白的构建方法,其特征在于,所述步骤S3一次转染时的宿主细胞包括大肠杆菌宿主和/或动物性细胞宿主。
8.如权利要求1-2任一所述的一种FTCD和CYP2D6融合蛋白在试剂盒、疫苗或诊断抗原中的应用。
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