CN113185584A - 重组SARS-CoV-2 N蛋白及其制备方法、应用、及动物用新型冠状病毒ELISA抗体检测试剂盒 - Google Patents
重组SARS-CoV-2 N蛋白及其制备方法、应用、及动物用新型冠状病毒ELISA抗体检测试剂盒 Download PDFInfo
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Abstract
本发明涉及重组SARS‑CoV‑2N蛋白及其制备方法、应用、及动物用新型冠状病毒ELISA抗体检测试剂盒。本发明提供了重组SARS‑CoV‑2N蛋白的制备方法,将经过密码子优化的编码SARS‑CoV‑2N蛋白的核苷酸序列构建入昆虫‑杆状病毒Bac‑to‑Bac表达系统的载体中,并转染昆虫细胞,实现病毒N蛋白的真核表达;亲和层析法提取并纯化昆虫细胞中表达的N蛋白。经过条件优化后每升感染重组杆状病毒的昆虫细胞中重组蛋白含量可达到200mg以上。本发明还提供了动物用新型冠状病毒ELISA抗体检测试剂盒,该试剂盒以优化后的SARS‑CoV‑2N蛋白作为包被抗原,能够对小鼠、水貂、家猫、狗等动物的新冠病毒感染后的血清抗体进行特异性检测,在间接ELISA检测中表现出良好的准确性。
Description
技术领域
本发明属于免疫检测技术领域,具体涉及重组SARS-CoV-2 N蛋白及其制备方法、应用、及动物用新型冠状病毒ELISA抗体检测试剂盒。
背景技术
新型冠状病毒主要在人与人之间通过呼吸道飞沫和密切接触传播,但也有人类和动物之间传播的例子。有报道显示,水貂、家猫、狗、狮子和老虎等几种与感染人类接触过的动物被检测出SARS-CoV-2阳性。如果,动物被感染,可能会充当SARS-CoV-2的“储存库”,使病毒在它们之间扩大传播,并存在病毒从动物到人二次传播的风险。更有报道显示,这些感染SARS-CoV-2的动物(如水貂、小鼠等)可能会引起病毒基因序列的突变(Y453F、N501T),这些突变主要集中于病毒的S蛋白,突变可削弱疫苗的保护效力并影响检测结果。
目前SARS-CoV-2的检测方法主要有分子生物学检测和血清学检测。分子生物学检测(核酸检测)虽然检测结果精准,但是对检测设备或平台要求较高,高耗时较长。相比而言,血清学检测方法(如ELISA试剂盒)具有操作简单、灵敏、快速、低成本等特点,更易于基层开展大规模现场筛查工作。但是,目前仍然缺乏专门针对小鼠、水貂、家猫、狗、狮子和老虎等动物的血清学检测试剂。
发明内容
本发明的目的在于提供重组SARS-CoV-2 N蛋白的氨基酸序列及其编码基因的核苷酸序列。
本发明的第二个目的在于提供包含上述重组SARS-CoV-2 N蛋白编码基因的表达盒、重组载体、重组细胞或重组菌。
本发明的第三个目的在于提供上述重组SARS-CoV-2 N蛋白的制备方法。
本发明的第四个目的在于提供上述重组SARS-CoV-2 N蛋白的应用。
本发明的第五个目的在于提供动物用新型冠状病毒ELISA抗体检测试剂盒。
为了实现上述目的,本发明采用以下技术方案:
重组SARS-CoV-2 N蛋白,其氨基酸序列如SEQ ID NO:1所示;其编码基因的核苷酸序列如SEQ ID NO:2所示。
包含上述重组SARS-CoV-2 N蛋白编码基因的表达盒、重组载体、重组细胞或重组菌。
重组SARS-CoV-2 N蛋白的制备方法,包括如下步骤:将经过密码子优化的编码SARS-CoV-2 N蛋白的核苷酸序列(SEQ ID NO:2)构建入昆虫-杆状病毒Bac-to-Bac表达系统的载体中,并转染昆虫细胞,实现病毒N蛋白的真核表达;并用亲和层析法提取并纯化昆虫细胞中表达的N蛋白。
优选的,所述载体为pFastBacI、pFastBacDual、pFastBacHTA、pFastBacHTB中任一种。
优选的,所述昆虫细胞为sf9、sf21、High Five中任一种。
重组SARS-CoV-2 N蛋白在制备动物用新型冠状病毒抗体检测试剂盒中的应用。
动物用新型冠状病毒ELISA抗体检测试剂盒,所述试剂盒包括包被抗原的固相载体;所述包被抗原为重组SARS-CoV-2 N蛋白,其编码基因的核苷酸序列如SEQ ID NO:1所示。
优选的,所述重组SARS-CoV-2 N蛋白的包被浓度为2μg/mL。
优选的,所述试剂盒还包括:酶标二抗、洗涤液、显色液、封闭液、样品稀释液、阴性对照、阳性对照中的一种或两种以上。
优选的,所述酶标二抗为HRP标记SPA。
优选的,所述洗涤液为PBST缓冲液。
优选的,所述显色液为TMB显色液。
优选的,所述封闭液为5%脱脂奶。
优选的,所述样品稀释液为磷酸盐缓冲液。
优选的,所述阳性对照为N蛋白免疫小鼠阳性血清。
优选的,所述阴性对照为标准小鼠阴性血清。
本发明取得的有益效果:
本发明提供了重组SARS-CoV-2 N蛋白,该蛋白编码基因(SEQ ID NO.1)是在原始N蛋白基因序列(GenBank序列号为QOQ37303.1)的基础上进行了大幅度的改进设计,获得按照杆状病毒编码特征设计了密码子优化的N蛋白基因。在对SARS-CoV-2 N蛋白基因进行密码子优化时,选择昆虫细胞使用频率最高的密码子,对改造前后基因进行同源比对分析发现改造后的核苷酸序列与原始基因序列同源性仅为72.46%。本发明进一步提供了重组SARS-CoV-2 N蛋白的制备方法,与原核表达系统相比,异源基因的杆状病毒表达允许翻译后修饰,如折叠、寡聚、磷酸化、糖基化、酰化、二硫键形成以及蛋白水解切割等,它们与哺乳动物细胞中的修饰相似或相同,且表达量比哺乳动物细胞中的表达量高,克服了传统杆状病毒表达量低的缺点,够作为包被抗原用于新型冠状病毒抗体检测试剂的制备。本发明经过条件优化后每升感染重组杆状病毒的昆虫细胞中重组蛋白含量可达到200mg以上。
本发明提供了动物用新型冠状病毒ELISA抗体检测试剂盒,该试剂盒以优化后的SARS-CoV-2 N蛋白作为包被抗原,能够对小鼠、水貂、家猫、狗等动物的新冠病毒感染后的血清抗体进行特异性检测,在间接ELISA检测中表现出良好的准确性。
附图说明
图1为重组抗原N蛋白的SDS-PAGE测定结果;
图2为重组抗原N蛋白的Western blot测定结果;
图3为重组抗原N蛋白的纯化结果;
图4为绘制标准曲线;
图5为ELISA检测结果。
具体实施方式
下面结合具体实施方式对本发明作进一步描述,但本发明的保护范围并不仅限于此;实施例及试验例中所用的仪器设备如无特别说明,均为市售常规仪器设备;所用试剂材料,如无特别说明,均为市售常规试剂材料;其中,1)菌株与感受态细胞:E.coli DH5α(全式金生物技术有限公司);E.coli DH10Bac(博迈德基因技术有限公司);2)质粒:pFastBacl(Invitrogen公司);昆虫细胞:sf21(Invitrogen公司);3)培养基:Sf-900ⅡSFM(Gibco 公司);4)试剂盒:昆虫细胞转染试剂盒(Gibco公司);内毒素质粒小提试剂盒(康维世纪生物科技有限公司);5)HRP-SPA(Sigma公司)。重组质粒pFastBacI-N基因测序和引物合成(BamHI和XholI)等技术服务由上海生工生物工程有限公司提供。
实施例1抗原制备与纯化鉴定
1.目的基因的优化:
基因表达受到多种因素的调控和影响,如密码子使用偏好、核糖体结合、mRNA结构等。考虑这些因素,得到的基因序列可以达到可能的最高表达。编码同一蛋白质序列的mRNA序列有多种可能。优化序列的密码子使用偏倚根据多组学数据分析建立的专有参考密码子偏倚进行调整,以符合目标宿主中高表达基因的表达谱。
重组N蛋白基因是在原始N蛋白基因序列(GenBank序列号为QOQ37303.1)的基础上进行了大幅度的改进设计,获得按照杆状病毒编码特征设计了密码子优化的N蛋白基因,其核苷酸序列如SEQ ID NO.1所示,其氨基酸序列如SEQ ID NO.2所示。
在对SARS-CoV-2 N蛋白基因进行密码子优化时,选择昆虫细胞使用频率最高的密码子,对改造前后基因进行同源比对分析发现改造后的核苷酸序列与原始基因序列同源性仅为 72.46%。
与原核表达系统相比,异源基因的杆状病毒表达允许翻译后修饰,如折叠、寡聚、磷酸化、糖基化、酰化、二硫键形成以及蛋白水解切割等,它们与哺乳动物细胞中的修饰相似或相同,且表达量比哺乳动物细胞中的表达量高,克服了传统杆状病毒表达量低的缺点。改造合成的N蛋白基因,与原始基因的序列相比较,表达的蛋白表达水平通常更高。本发明经过条件优化后每升感染重组杆状病毒的昆虫细胞中重组蛋白含量可达到200mg以上。
2.载体构建
根据N蛋白编码序列,设计一对扩增引物,具体序列如下:
正向引物:TTAGGATCCATGAGCGACAACGGTCCT
反向引物:ATCTCGAGTTAAGCCTGGGTGGAGTC
以合成基因pUC57-N(委托上海生工生物有限公司合成)为模板进行PCR扩增;
PCR反应体系如下:
PCR反应条件为95℃预变性1min,95℃变性30s,58℃退火90s,72℃延伸1分钟,共进行30个循环;
将得到的扩增产物或载体从BamHI和XholI双酶切,双酶切消化体系为:
总体系为50μl,反应条件为37℃,4h。反应结束后用TianGen DNA回收试剂盒从反应液中直接回收DNA。将回收的空载体和目的基因的双酶切产物用T4 DNA连接酶连接(分子摩尔比为外源基因:载体DNA=3:1),连接反应体系如下:
16℃连接过夜。取上述连接产物10μl转化E.coli感受态细胞DH5α感受态细胞中,涂于含有氨苄抗性(终浓度为100μg/ml)的LB固体平板上(胰蛋白胨10g,琼脂粉15g,酵母提取物5g,NaCl 10g,去离子水定容至1L)过夜培养;次日,于平板上挑取白色克隆菌落并进行基因测序鉴定(委托上海生工生物有限公司),将测序正确的重组质粒pFastBac1-N转入E.coli DH10Bac感受态细胞中,涂板培养;从培养出的蓝白斑菌落中挑选白斑,提取其杆状病毒质粒Bacmid。
3.N蛋白的表达及鉴定
将经过鉴定的杆状病毒质粒Bacmid用Cellreagent转染试剂盒转入sf21昆虫细胞。具体转染包括如下步骤:
1)将sf21细胞以2×106cells/mL的细胞密度,接种于六孔细胞培养板中(每孔1mL),孵育2h;
2)取l-2ng重组杆粒Bacmid用100μL的Sf-900 II SFM稀释;
3)取转染试剂Cellfectin稀释于100μL的Sf-900 II SFM中;
4)混合2、3两份稀释液,室温孵育40min后,加入1mL的Sf-900 II SFM,制成转染混合物;
5)除去1中细胞培养上清,加入4所述转染混合物,28℃孵育4-6h;
6)弃去转染混合物,加入2mL的Sf-900 II SFM,28℃恒温培养箱培养3至4天;
7)收集细胞培养上清,作为P1代细胞按2%的体积接种到新的sf21细胞中,连续培养两代,从而扩增病毒的毒力;
8)至P3代细胞时,观察记录P3代细胞形态变化,并对P3代细胞进行表达鉴定;鉴定方法采用进行SDS-PAGE鉴定及Western Blot验证。
SDS-PAGE及Western Blot鉴定结果如下:
将样品进行12%SDS-PAGE电泳(260V,40min),考马斯亮蓝凝胶染色1.5小时,经纯水脱色后扫描观察电泳图像;具体结果见图所示,从图1中可以看出,约50kDa处有条带出现,其位置与预期一致,初步说明重组蛋白正确表达;
SDS-PAGE电泳样品转至PVDF膜进行western Blot鉴定,包括如下步骤:
1)用甲醇溶液浸泡PVDF膜5秒,用转膜缓冲液浸泡凝胶和PVDF膜,将PVDF膜铺在凝胶上;
2)在凝胶/滤膜外再包一张3mm滤纸(预先用转膜缓冲液浸湿),将凝胶夹在中间,保持湿润和没有气泡;
3)将此滤纸/凝胶/薄膜滤按照厂家建议方法放入电泳装置中,凝胶面向阴极;
4)15V电压,转膜1h;
5)PVDF膜置于5%脱脂奶的封闭液中于37℃封闭1小时;
6)室温下,用PBS-Tween缓冲液洗涤薄膜;
7)用HRP标记的His单抗室温孵育1h;
8)用PBST充分洗涤后,利用化学发光(ECL)显色,发现在约50kDa有目的条带,表明sf21细胞中N蛋白表达成功(图2)。
4.抗原纯化及鉴定
用上述P3代细胞以感染复数为1感染sf21悬浮细胞,接种病毒时的上清细胞密度为 0.8-1.0×106个/ml;接毒后培养3d,收获细胞上清利用Ni亲和层析柱(His Trap TMexcel, GE)对N蛋白进行纯化,结果如图3所示,获得N蛋白纯度高达95%。
实施例2抗体检测试剂盒的制备
本发明的动物用新型冠状病毒ELISA抗体检测试剂盒包括包被抗原的固相载体;所述包被抗原为重组SARS-CoV-2 N蛋白,其编码基因的核苷酸序列如SEQ ID NO:1所示。该试剂盒可用于间接ELISA检测小鼠、宠物(猫或水貂)等动物血清中的新冠病毒N蛋白抗体。
1.重组N蛋白包被浓度及二抗HRP标记SPA浓度的确定
用碳酸盐缓冲液将高纯度N蛋白进行稀释,分别用0.1μg/mL、0.5μg/mL、1μg/mL、2μg/mL、 4μg/mL的N蛋白包被酶标板,37℃包被2小时;5%脱脂奶37℃封闭1h;一抗选用1:5000 稀释的抗SARS-CoV-2 N蛋白的单克隆抗体,37℃孵育1小时;二抗选用经PBST稀释的HRP-SPA,稀释倍数分别为1:500、1:1000……1:128000,37℃孵育30min;加酶底物TMB 溶液,100μL/孔,室温条件下显色10min,加入2mol/L的硫酸终止反应。根据棋盘法测定结果,以OD450nm为1.0左右的组合为最佳模式,选择N蛋白最佳包被浓度和HRP-SPA最佳反应浓度。根据棋盘法测定结果,以OD450nm为1.0左右的组合为最佳模式(表1)。考虑到节约包被原和适量使用酶标抗体原则,最后确定包被原的浓度为2μg/mL,酶结合物HRP-SPA 稀释4000倍,此时为最佳工作浓度。
表1包被原和HRP-SPA最佳工作浓度的确定
2.ELISA检测方法标准曲线的建立:
按照上述确立的实验条件,进行接ELISA检测:用碳酸盐缓冲液将高纯度N蛋白稀释至 2μg/mL,100μl/孔,包被ELISA,37℃包被2小时;PBST洗涤3次;5%脱脂奶37℃封闭1h;PBST洗涤3次;一抗选用抗SARS-CoV-2 N蛋白的单克隆抗体,稀释浓度分别为1:1000、1:2000……1:64000,每孔对应的蛋白含量分别为:100ng、50ng、25ng、12.5ng、6.25ng、3.125ng和1.56ng;37℃孵育1小时;PBST洗涤6次;加入经PBST1:4000稀释的HRP-SPA, 37℃孵育30min;PBST洗涤6次;加酶底物TMB溶液,100μL/孔,室温条件下显色10min,加入2mol/L的硫酸终止反应。测定OD450nm值(表2)。横坐标为抗SARS-CoV-2 N蛋白的单克隆抗体浓度,纵坐标为OD450nm平均值,建立标准曲线,根据结果绘制直线回归和二次曲线回归(图4),得到线性回归方程y=-0.2346x+1.6589(R2=0.9489)及二次曲线回归方程y=0.0304x2-0.4781x+2.0241(R2=0.9968)。
表2抗SARS-CoV-2 N蛋白的单克隆抗体倍比稀释浓度与OD值
3.重复性试验
1)批内重复性试验
在相同的试验条件下(试验条件参见ELISA检测方法标准曲线的建立),选取1:200稀释的小鼠阴、阳性血清,每份血清样本平行做10个复孔,结果进行统计学分析,如表3所示,阴、阳性样品批内变异系数范围是3.16%和4.13%。
表3批内重复性实验结果
2)批间重复性试验
在相同的试验条件下(试验条件参见ELISA检测方法标准曲线的建立),上述阴阳性血清,在6个不同的时间,每份血清样本平行做3个复孔,结果进行统计学分析,如表4所示,阴、阳性样品批内变异系数范围是9.57%和7.12%。由此说明本研究建立的ELISA试剂盒的检测方法,准确性较高,能够用于检测动物血清样本中的N蛋白抗体。
表4批间重复性实验结果
4.ELISA检测试剂盒的制备
1)包被重组SARS-CoV-2 N蛋白的酶标板的制备
将实施例1获得的重组SARS-CoV-2 N蛋白用包被液稀释至2μg/mL,每孔100μl包被96 孔酶标板,37℃孵育1小时后弃去包被液;PBST洗液洗涤3次;每孔加入300μl的5%脱脂奶的封闭液4℃过夜封闭;PBST洗液洗涤3次,晾干。
其中,包被液为碳酸盐缓冲液(carbonate buffer,CBS):分别称0.159g Na2CO3和0.293 g NaHCO3充分溶于80ml蒸馏水中,定容至100ml,4℃保存。
2)动物用新型冠状病毒ELISA抗体检测试剂盒的组装
该试剂盒中含有包被重组SARS-CoV-2 N蛋白的酶标板、洗涤液、样品稀释液、HRP标记的金黄色葡萄球菌A蛋白(HRP-SPA)、显色液、终止液、阳性对照血清、阴性对照血清。
其中,所述洗涤液为PBST缓冲液;所述显色液为TMB显色液;所述封闭液为5%脱脂奶;所述样品稀释液为磷酸盐缓冲液;所述阳性对照为N蛋白免疫小鼠阳性血清;所述阴性对照为标准小鼠阴性血清。
试验例1试剂盒的应用及结果判定
本试验例采用实施例制备的动物用新型冠状病毒ELISA抗体检测试剂盒对小鼠、雪貂血清样本进行检测,检测方法为间接ELISA法,包括如下步骤:
1)待检血清:用磷酸盐缓冲液(PBS)1:200倍稀释血清样本,酶标板每孔加样100μL, 37℃孵育30分钟;设置N蛋白免疫小鼠阳性血清为阳性对照;标准小鼠阴性血清为阴性对照;
2)洗涤:PBST洗液洗涤6次;
3)二抗:用稀释液1:4000倍稀释的辣根过氧化物酶标记的SPA(HRP-SPA),每次100μL, 37℃孵育30min;
4)洗涤:PBST洗液洗涤6次;
5)显色:加入100μl TMB显色液避光反应10min;
6)终止:加入100μL浓度为2mol/L的硫酸终止反应;
7)读数:酶标仪读取450nm波长OD值(OD450)。
结果判读:以OD450大于2.1倍的阴性样本OD450且读数大于0.21判读为阳性,OD值小于0.2的为阴性。
采用上述方法,分别选择5只N蛋白免疫小鼠阳性血清样品及5只新型冠状病毒攻毒的实验雪貂灭活阳性血清以及5只小鼠阴性血清和5只雪貂阴性血清用上述ELISA试剂盒进行检测,结果如图5所示:10支阳性血清样品检测结果OD450均大于0.5;10支阴性血清OD450均小于0.2,说明本研究建立的ELISA试剂盒的检测方法,能够用于检测小鼠、雪貂等动物血清样本中的新型冠状病毒N蛋白抗体。
<110> 河南中泽生物工程有限公司
<120> 重组SARS-CoV-2 N蛋白及其制备方法、应用、及动物用新型冠状病毒ELISA抗体检测试剂盒
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<170> PatentIn version 3.5
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<221> 重组SARS-CoV-2 N蛋白
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Claims (10)
1.重组SARS-CoV-2N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所述的重组SARS-CoV-2N蛋白,其特征在于,其编码基因的核苷酸序列如SEQ ID NO:2所示。
3.包含权利要求2所述的重组SARS-CoV-2N蛋白编码基因的表达盒、重组载体、重组细胞或重组菌。
4.如权利要求1所述的重组SARS-CoV-2N蛋白的制备方法,其特征在于,包括如下步骤:将如权利要求2所述的重组SARS-CoV-2N蛋白的编码基因导入昆虫-杆状病毒Bac-to-Bac表达系统的载体中,并转染昆虫细胞,实现病毒N蛋白的真核表达;并用亲和层析法提取并纯化昆虫细胞中表达的N蛋白。
5.根据权利要求4所述的方法,其特征在于,所述载体为pFastBacI、pFastBacDual、pFastBacHTA、pFastBacHTB中任一种。
6.根据权利要求4所述的方法,其特征在于,所述昆虫细胞为sf9、sf21、High Five中任一种。
7.如权利要求1所述的重组SARS-CoV-2N蛋白在制备动物用新型冠状病毒抗体检测试剂盒中的应用。
8.动物用新型冠状病毒ELISA抗体检测试剂盒,其特征在于,所述试剂盒包括包被抗原的固相载体;所述包被抗原为重组SARS-CoV-2N蛋白,其编码基因的核苷酸序列如SEQ IDNO:1所示。
9.根据权利要求8所述的试剂盒,其特征在于,所述重组SARS-CoV-2N蛋白的包被浓度为2μg/mL。
10.根据权利要求8所述的试剂盒,其特征在于,所述试剂盒还包括:酶标二抗、洗涤液、显色液、封闭液、阴性对照、阳性对照中的一种或两种以上。
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CN115261395A (zh) * | 2022-04-26 | 2022-11-01 | 中国疾病预防控制中心传染病预防控制所 | 一种新型冠状病毒n蛋白高效可溶性表达的方法 |
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