CN110746495A - 一种重组蛋白e2及其应用 - Google Patents
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- CN110746495A CN110746495A CN201911054685.7A CN201911054685A CN110746495A CN 110746495 A CN110746495 A CN 110746495A CN 201911054685 A CN201911054685 A CN 201911054685A CN 110746495 A CN110746495 A CN 110746495A
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Abstract
本发明提供了一种重组蛋白E2,具有序列表SEQ.ID.No.1的氨基酸序列;所述重组蛋白E2具有序列表SEQ.ID.No2碱基序列,还提供了应用,用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒。本发明E2重组蛋白准确率高,既保证了蛋白的中和性,又避免了高突变率的困扰,更利于运用在BVDV抗体检测技术的开发。E2重组蛋白使用传统的蛋白原核表达技术,成本低,表达量大,成功表达出的E2重组蛋白,经尿素梯度复性后,West blot检测反应原性强。该E2重组蛋白用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒,建立的BVDV抗体ELISA检测方法灵敏度高,特异性好,更利于在基层广大农户中推广使用,更利于我国牛群中BVDV的净化。
Description
技术领域
本发明属于基因重组技术领域,具体涉及一种重组蛋白E2及其应用。
背景技术
牛病毒性腹泻/黏膜病(Bovine viral diarrhea/mucosal disease,BVDV/MD)是由牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)引起的猪、牛、羊、骆驼及其它野生动物的重要传染病,发病率高,致死率低。临床类型包括肺炎、腹泻、母畜流产、死胎和畸胎等急性感染及黏膜病和持续性感染,引起免疫抑制造成继发感染和其他疫苗免疫失败等。持续感染(PI)也是BVDV自然环境中维持存在的一种形式,主要在母牛妊娠初期时通过子宫内感染而引起。此时胎儿的免疫***尚未发育成熟,不能对外来的非致细胞病变性型BVDV进行识别,使胎儿在出生后呈持续性感染,处于免疫耐受状态,体内不产生特异性抗体,却终生带毒、排毒。持续性感染牛是牛场BVDV的存储器,病牛每天可通过粪便等***物,向环境中排出100万到1000万个病毒颗粒,是造成BVDV扩散的最主要传染源。伴随规模化养殖的推进,更是加快了BVDV的蔓延,使国内BVDV的感染情况日益加剧。目前BVDV暂无有效的治愈方式,养殖过程中主要依赖牛群的净化防控。因此建立出一种低成本,方便简洁,快速有效利于推广的BVDV抗体检测方法,能及时检测并剔除发病和隐性感染牛,更好的保证牛群健康发展。
E2蛋白位于BVDV病毒粒子囊膜表面,是现阶段国内外瘟病毒的研究热点,位于ORF的2077~3198位核苷酸,由374个氨基酸残基组成,非糖基化蛋白分子量约为42kDa。E2蛋白C端含有一个疏水的膜锚定区,通过这一疏水区锚定在囊膜表面,可诱导被感染动物产生病毒中和抗体,刺激机体发生免疫应答,同时参与细胞的识别和吸附。E2靠近N末端的一半含有多种依赖于构象的抗原结构域,其N端伸出BVDV囊膜表面,是决定BVDV抗原性的主要部位,也是与抗BVDV抗体结合、介导免疫中和反应及与宿主细胞识别和吸附的主要部位。E2蛋白具有中和活性,但存在着高突变的特点。因此,在选用E2蛋白作为诊断抗原时,需要对BVDV不同流行株E2蛋白保守区域进行分析,挑选出E2蛋白中既保守稳定又具有代表性,同时又包含优势抗原表位的区域,***到不同载体上,实现E2重组蛋白的高效表达,对BVDV抗体诊断技术的开发和疾病的防制有着重要作用。
发明内容
本发明所要解决的技术问题在于针对上述现有技术的不足,提供一种重组蛋白E2及其应用,该E2重组蛋白准确率高,既保证了蛋白的中和性,又避免了高突变率的困扰,更利于运用在BVDV抗体检测技术的开发。E2重组蛋白使用传统的蛋白原核表达技术,成本低,表达量大,成功表达出的E2重组蛋白,经尿素梯度复性后,West blot检测反应原性强。该E2重组蛋白用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒,建立的BVDV抗体ELISA检测方法灵敏度高,特异性好,更利于在基层广大农户中推广使用。
为解决上述技术问题,本发明采用的技术方案是:一种重组蛋白E2,所述重组蛋白E2具有序列表SEQ.ID.No.1的氨基酸序列;所述重组蛋白E2具有序列表SEQ.ID.No2的核苷酸序列;
所述重组蛋白E2的制备方法为:对E2蛋白氨基酸序列进行优化,采用全基因合成并通过限制性酶切位点Nde I和Hind III将重组E2蛋白基因***到表达载体pET30a中,并通过酶切法和测序确认最终表达载体的准确性,最终依次转到Top10大肠杆菌克隆菌株感受态细胞和BL21(DE3)大肠杆菌表达菌感受态细胞中,通过IPTG诱导表达重组蛋白E2,之后通过Ni-IDA亲和层析纯化,得到具有生物学活性的重组蛋白E2;
所述BL21(DE3)大肠杆菌是一种菌株,该菌株用于T7 RNA聚合酶为表达***的高效外源基因的蛋白表达宿主,T7噬菌体RNA聚合酶基因的表达受控于入噬菌体DE3区的lacUV5启动子,该区整合于BL21的染色体上,该菌株适合于非毒性蛋白的表达。
本发明还提供了上述的重组蛋白E2的应用,所述重组蛋白E2用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒,所述间接ELISA试剂盒包括包被酶标板、阴性对照血清、阳性对照血清、酶标二抗、酶标二抗稀释液、浓缩洗涤液、样品稀释液、显色液和终止液,所述包被酶标板以重组蛋白E2作为包被抗原。
优选地,所述包被酶标板每孔包被0.2μg所述重组蛋白E2;
所述阴性对照血清和阳性对照血清各2mL;
所述酶标二抗是用HRP藕联物稳定/稀释剂I对兔抗牛IgG进行100倍稀释而得,所述酶标二抗为0.6mL;
所述酶标二抗稀释液为含有pH7.2~7.4的0.01M的磷酸缓冲盐溶液b,所述磷酸缓冲盐溶b中马血清的质量分数为5%,所述酶标二抗稀释液为60mL;
所述浓缩洗涤液为20×洗涤液,所述浓缩洗涤液为pH为7.2~7.4的0.1M的磷酸缓冲盐溶液c,所述磷酸缓冲盐溶c中吐温20的质量分数为0.5%,所述浓缩洗涤液为100mL;
所述样品稀释液为pH为7.2~7.4的0.01M的磷酸缓冲盐溶液d,所述磷酸缓冲盐溶液d中牛血清白蛋白的质量分数为0.1%、蔗糖的质量分数为2%,所述样品稀释液为120mL;
所述显色液包括显色液A和显色液B,所述显色液A的制备方法为:将200mg四甲基联苯胺溶于100mL的无水乙醇中,双蒸水定容至1000mL,得到显色液A;所述显色液B的制备方法为:称取柠檬酸21g、无水磷酸钠28.2g,加入0.75%过氧化氢尿素溶液6.4mL,双蒸水定容至1000mL,调节pH值为4.5~5.0,得到显色液B;所述显色液A和所述显色液B均为30mL;
所述终止液为2M的H2SO4溶液,所述终止液为100mL。
本发明与现有技术相比具有以下优点:
BVDV中的E2蛋白具有中和活性强,突变率较高的特点。结合E2蛋白反应原性强,对BVDV流行株E2蛋白保守性部分的碱基序列组成进行分析,选取保守性部分中具有抗原优势表位的部分,通过人工合成的方式得到目的片段。与传统E2蛋白抗原优势表位截短表达相比较,人工合成并表达后的E2重组蛋白准确率高,既保证了蛋白的中和性,又避免了高突变率的困扰,更利于运用在BVDV抗体检测技术的开发。另一方面E2重组蛋白使用传统的蛋白原核表达技术,成本低,表达量大。成功表达出的E2重组蛋白,经尿素梯度复性后,Westblot检测反应原性强。该E2重组蛋白用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒,建立的BVDV抗体ELISA检测方法灵敏度高,特异性好,更利于在基层广大农户中推广使用,更利于我国牛群中BVDV的净化。
下面结合附图和实施例对本发明作进一步详细说明。
附图说明
图1是本发明实施例1的酶切分析鉴定原核表达质粒pET30a-E2的准确性结果图。
图2是本发明实施例1的SDS-PAGE分析鉴定重组蛋白E2表达菌经IPTG诱导表达结果图。
图3是本发明实施例1的SDS-PAGE分析重组蛋白E2表达菌经诱导后破碎除杂的菌液上清液c的结果图。
图4是本发明实施例1的SDS-PAGE分析重组蛋白E2表达菌经诱导、破碎后的包涵体沉淀物b的纯化结果图。
图5是本发明实施例1的纯化后的重组蛋白E2的SDS-PAGE检测结果图。
图6是本发明实施例1的纯化后的重组蛋白E2的Western-blot分析图。
具体实施方式
实施例1
1、E2蛋白序列的扩增及原核表达质粒pET30a-E2的构建:
使用密码子优化软件MaxCodonTM Optimization Program(V13)对E2蛋白的氨基酸序列进行优化,采用全基因合成重组E2蛋白基因,所述E2重组蛋白的氨基酸序列表为SEQ.ID.No.1,所述重组E2蛋白基因的核苷酸序列表为SEQ.ID.No.2,通过限制性酶切位点Nde I和Hind III将重组E2蛋白基因***到表达载体pET30a中,在200μL的离心管中分别加入重组E2蛋白基因片段4.5μL、pET-30a表达载体0.5μL、DNA连接试剂盒5μL充分混匀,置于温度为16℃的低温连接仪中进行连接反应30min,得到连接产物,将连接产物通过限制性酶切位点Nde I和Hind III进行双酶切和测序确认最终表达载体的准确性,得到原核表达质粒pET30a-E2。图1为酶切分析鉴定原核表达质粒pET30a-E2的准确性结果图,图中M为10000bp DNA Marker;泳道1为原核表达质粒pET-30a-E2的Nde I和Hind III的双酶切产物;泳道2为空质粒对照,由图1可知,重组E2蛋白基因片段与预期分子量相符,经测序后,表明重组E2蛋白表达载体构建成功。
2、连接产物的转化:
将Top10大肠杆菌克隆菌株置于冰块上预冷,向10μL的连接产物中加入Top10大肠杆菌克隆菌株,吹打混匀,在冰块上静置30min,后在42℃恒温水浴锅内热击90s,立即转至冰块上静置3min后,加入600~800μL的无抗性的LB液体培养基,在转速为200rmp/min、温度为37℃的摇床中活化60min,然后在转速为500rmp/min、室温条件下离心5min,弃去上清液,再加入100μL的无抗LB培养基混匀,涂板,得到转化后的Top10大肠杆菌克隆菌株;
最终依次转到Top10大肠杆菌克隆菌株感受态细胞和BL21(DE3)大肠杆菌表达菌感受态细胞中,即使用转化后的Top10大肠杆菌克隆菌株提取质粒转化至BL(21)大肠杆菌表达菌株中,转化步骤同上,得到保存菌液;所述BL21(DE3)是一种菌株,该菌株用于T7 RNA聚合酶为表达***的高效外源基因的蛋白表达宿主,T7噬菌体RNA聚合酶基因的表达受控于入噬菌体DE3区的lacUV5启动子,该区整合于BL21的染色体上,该菌株适合于非毒性蛋白的表达。
3、IPTG诱导表达重组E2蛋白:
使用接种环蘸取保存菌液,在含有浓度为50μg/mL的Kan(卡那霉素)的LB平板上划线培养,倒置于温度为37℃的培养箱内培养12h,观察菌落生长情况,挑选单克隆,接种至含有浓度为50μg/mL的Kan的LB液体培养基中,置于温度为37℃的摇床培养至菌液的OD600nm至0.6-0.8时,得到菌液a;吸取1mL的菌液,在转速为12000rpm的条件下离心1min,弃上清液后,加入80μL的去离子水和20μL的5×蛋白上样缓冲液,得到样品a(0h诱导样本);在菌液a中加入加入终浓度为0.1mM的IPTG(异丙基硫代半乳糖苷),充分混匀;25mL/管分装至离心管中,分别在温度为16℃和37℃的条件下诱导12h后取样,分别得到样品b(16℃诱导样本)和样品c(37℃诱导样本)。
4、SDS-PAGE分析鉴定诱导表达结果:
将诱导后得到的样品a、样品b和样品c,分别放入PCR仪内,在温度为100℃的条件下放置10min,然后在转速为12000rpm的条件下离心5min,分别取上清液进行SDS-PAGE(十二烷基磺酸钠-聚丙烯酰胺凝胶)电泳。溴酚蓝指示剂在浓缩胶时进行电压为80V的稳压电泳,待溴酚蓝指示剂进入分离胶后将电压升至120V,直至溴酚蓝指示剂跑至距离凝胶底约1cm处,结束电泳。
电泳结束后,取出凝胶置于染色盘内,用500mL考马斯亮蓝染液染色10~15min,取出凝胶用蒸馏水漂洗3次,随后转入脱色液中,根据情况,每间隔3~4h更换一次脱色液,直至背景清晰。使用清水漂洗凝胶,在蛋白凝胶成像仪中分析蛋白情况,拍照保存;
图2为SDS-PAGE分析鉴定重组蛋白E2表达菌经IPTG诱导表达结果图,图中M为SDS-PAGE蛋白Marker,泳道0为温度为0h收集得到的样品a,泳道1为温度为16℃的条件下诱导诱导后得到的样品b,泳道2为温度为37℃的条件下诱导后得到的样品c,由图可知表达出的蛋白在分子量为39kDa左右均与E2重组蛋白预期大小相符,表明E2重组蛋白成功表达。重组E2蛋白在16℃和37℃诱导时均可表达,且37℃表达效果较好。
5、重组蛋白E2可溶性鉴定及纯化
将20μL的样品a加入至20mL的含有50μg/mL Kan的液体LB培养基中,然后在摇速为200rmp/min、温度为37℃的条件下培养12h,得到扩繁后的菌液,将6mL扩繁后的菌液加入至600mL的含有50μg/mL Kan的液体LB培养基中,然后在摇速为200rmp/min、温度为37℃的条件下培养至OD600nm到0.8时,加入终浓度为0.1mM的IPTG,然后在温度为37℃条件下诱导表达8h后,在转速为10000rpm的条件下离心5min,弃去上清液,保留沉淀物,向沉淀物中加入40倍体积的BufferA,振荡充分混匀后,在温度为4℃的条件下裂解8h,得到裂解菌液,将裂解菌液放入-80℃冰箱中冷冻5min,然后置于温度为42℃的水浴锅融化,重复进行3次冷冻和融化的操作;然后共进行3次超声破碎菌体操作(所述超声破碎菌体的程序为:超声破碎5s,间歇10s,循环进行15min);收集破碎后的菌液,在温度为4℃、转速为10000rpm的条件下离心10min,分离得到破碎后的菌液上清液a与包涵体沉淀物b,将破碎后的菌液上清液a用粒径为0.45μm的滤膜除去杂质,得到除杂后的破碎后的菌液上清液c;在含有包涵体蛋白沉淀物b的离心管内加入15mL的含8mol/L的尿素的BufferA,充分混匀后,共进行3次超声破碎菌体操作(所述超声破碎菌体的程序为:超声破碎5s,间歇10s,循环进行15min);然后在转速为10000rmp/min的条件下离心10min后,将上清液用粒径为0.45μm的滤膜除去杂质,得到除杂后的包涵体上清液d。将Ni-IDA亲和层析柱用去离子水过柱两遍,再加入5倍填料体积的BufferA润洗一遍。将除杂后的破碎后的菌液上清液c和除杂后的包涵体上清液d分别过柱。使用含有50mmol/L咪唑的Buffer B、含有100mmol/L咪唑的BufferB、含有500mmol/L咪唑的Buffer B,分别对Ni-IDA亲和层析柱进行洗脱,依次收集洗脱液,SDS-PAG鉴定表达情况。
所述BufferA的配制方法为:将29.24g的NaCl和3.12g的NaH2PO4·2H2O,用去离子水稀释至1L,用盐酸溶液或氢氧化钠溶液调节pH值至7.4;
所述BufferB的配制方法为:将16.62g的NaCl和1.56g的NH2PO4·2H2O,用去离子水稀释至1L,用盐酸溶液或氢氧化钠溶液调节pH值至7.4;
图3为SDS-PAGE分析重组蛋白E2表达菌经诱导后破碎除杂的菌液上清液c的纯化结果图,图中M为SDS-PAGE蛋白Marker,泳道1:包涵体沉淀物b;泳道2:除杂后的破碎后的菌液上清液c;泳道3:除杂后的破碎后的菌液上清液c通过Ni-IDA亲和层析柱后的流出液;泳道4-5:50mmol/L咪唑的洗脱组分;泳道6-7:100mmol/L咪唑的洗脱组分;泳道8-9:500mmol/L咪唑的洗脱组分,结果可见诱导后的重组蛋白E2表达菌经破碎除杂后的菌液上清液c中无重组E2蛋白表达。
图4为SDS-PAGE分析重组蛋白E2表达菌经诱导、破碎后的包涵体沉淀物b的纯化结果图,图中M为SDS-PAGE蛋白Marker,泳道1:包涵体上清液d;泳道2:包涵体上清液d通过Ni-IDA亲和层析柱后的流出液;泳道3-5:50mmol/L咪唑的洗脱组分;泳道6-9:100mmol/L咪唑的洗脱组分;泳道10-12:500mmol/L咪唑的洗脱组分;由图可知,在不同咪唑浓度的洗脱组分均可见与预期大小一致的E2重组蛋白,表明E2重组蛋白通过包涵体形式进行表达,可通过亲和层析对E2重组蛋白进行纯化。
6、重组蛋白E2蛋白的透析与浓缩:
使用0.0372g的EDTA·2Na和2g的NaHCO3溶于100mL去离子水中,得到煮沸液a,用煮沸液a将透析袋煮沸10min;然后用去离子水清洗透析袋1~2次,得到一次处理的透析袋;再将0.0372g EDTA·2Na溶于100mL去离子水中,得到煮沸液b,用煮沸液b将一次处理的透析袋煮沸10min;然后用去离子水清洗1~2次后,自然冷却,得到二次处理的透析袋;收集包涵体上清过柱后的50mmol/L、100mmol/L和500mmol/L咪唑的洗脱组分,加入至二次处理的透析袋中,封口后查漏,放入容器内,加入透析液(刚好淹没透析袋即可),在温度为4℃的条件下透析2~3d,每隔12h更换一次透析液,使用去离子水清洗超滤管1~2次,将透析后的蛋白加入超滤管内离心10~15min,得到纯化浓缩后的重组蛋白E2。
如图5纯化浓缩后的重组蛋白E2的SDS-PAGE检测结果图所示,图中M为SDS-PAGE蛋白Marker,泳道1为牛血清白蛋白(1.5μg)对照,泳道2为纯化浓缩后的重组的E2蛋白。由图可知,纯化浓缩后的重组蛋白E2大小为39kDa,与预期大小相符,表明E2重组蛋白纯化成功。
7、E2重组蛋白的Western Blot分析
将纯化浓缩后E2重组蛋白,进行SDS-PAGE电泳,根据预期蛋白大小E2重组蛋白所在位置,用小刀切下多余的部分后,将分离胶浸泡于膜转移液中。准备6张与分离胶尺寸相同的滤纸和1张硝酸纤维膜(NC膜),一同浸泡于转膜液内。按照三层滤纸-凝胶-NC膜-三层滤纸的顺序依次摆放到转膜仪上,每一层都要用滚轴小心排出夹层中的气泡,吸去旁边多余的转膜液。盖上石墨电极盖,根据转印槽正负极连接,200mA(恒流),开始转膜;按照目的蛋白大小,判定最佳转膜时间(常用1kDa/min)。转膜结束后,用镊子取出NC膜(切勿用手接触),剪角标记;使用丽春红进行染色。染色5min,放入去离子水中洗去多余染液,再放入Western洗涤缓冲液TBS T中清洗10~15min,每5min更换洗液。封闭:用Western洗涤缓冲液TBS T配置5%脱脂奶粉溶液,将NC膜放入溶液中,室温,摇床封闭2h。封闭结束后,放入5~10mLTBS T中洗净NC膜上残余的溶液,洗涤3次,每次10min。一抗孵育:将NC膜放入杂交袋中,使用5%脱脂奶粉1:50稀释牛病毒性腹泻病毒(BVDV)抗体阳性血清,37℃孵育1h,洗涤步骤同上。二抗孵育:将1:10000倍稀释后的HRP标记(辣根过氧化物酶标记)的兔抗牛IgG加入杂交袋中,37℃孵育1h,洗涤步骤同上。显色:使用ECL化学发光底物进行显色,将ECL试剂中的A液和B液(ECL试剂由Cyanagen公司生产)各取1mL均匀混合,覆盖在NC膜上,使用保鲜膜包裹,避光孵育3min取出NC膜后,将多余的ECL溶液用滤纸吸净,使用化学发光成像仪成像,保存并分析结果。
如图6对纯化后的重组蛋白E2的Western-blot(免疫印迹试验)分析图,图中M为SDS-PAGE蛋白Marker,泳道1为重组的E2蛋白,结果可见在预期大小处可见明显条带,证明纯化后的重组蛋白E2具有良好的反应原性。
BVDV中的E2蛋白具有中和活性强,突变率较高的特点。结合E2蛋白反应原性强,对BVDV流行株E2蛋白保守性部分的碱基序列组成进行分析,选取保守性部分中具有抗原优势表位的部分,通过人工合成的方式得到目的片段。与传统E2蛋白抗原优势表位截短表达相比较,人工合成并表达后的E2重组蛋白准确率高,既保证了蛋白的中和性,又避免了高突变率的困扰,更利于运用在BVDV抗体检测技术的开发。另一方面E2重组蛋白使用传统的蛋白原核表达技术,成本低,表达量大。成功表达出的E2重组蛋白,经尿素梯度复性后,Westblot检测反应原性强。
实施例2
本实施例为实施例1所述的重组蛋白E2的应用,所述重组蛋白E2用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒,所述间接ELISA试剂盒包括包被酶标板、阴性对照血清、阳性对照血清、酶标二抗、酶标二抗稀释液、浓缩洗涤液、样品稀释液、显色液和终止液,所述包被酶标板以重组蛋白E2作为包被抗原。
1、酶标二抗的制备:将兔抗牛IgG(美国Jackson ImmunoResearch公司生产)用HRP藕联物稳定/稀释剂I(湖州英创生物科技有限公司生菜)进行稀释100倍而得。
2、包被缓冲液、封闭液、样品稀释液、酶标二抗稀释液、洗涤液的配制:
包被缓冲液为pH为9.6的0.05M的碳酸盐缓冲液,所述碳酸盐缓冲液的配制方法为:取Na2CO3 1.59g、NaHCO3 2.93g,加灭菌的双蒸水800mL溶解,调pH至9.6后,加灭菌的双蒸水至1000mL;
封闭液为pH为7.2~7.4的0.01M的磷酸缓冲盐溶液a,所述磷酸缓冲盐溶液a中马血清的质量分数为5%、酪蛋白的质量分数为0.25%;
酶标二抗稀释液为含有pH7.2~7.4的0.01M的磷酸缓冲盐溶液b,所述磷酸缓冲盐溶b中马血清的质量分数为5%;
浓缩洗涤液为20×洗涤液,所述浓缩洗涤液为pH为7.2~7.4的0.1M的磷酸缓冲盐溶液c,所述磷酸缓冲盐溶c中吐温20的质量分数为0.5%;
样品稀释液为pH为7.2~7.4的0.01M的磷酸缓冲盐溶液d,所述磷酸缓冲盐溶液d中牛血清白蛋白的质量分数为0.1%、蔗糖的质量分数为2%。
3、显色液及终止液的配制:
显色液包括显色液A和显色液B;
显色液A的制备方法为:将200mg四甲基联苯胺溶于100mL的无水乙醇中,双蒸水定容至1000mL;
显色液B的制备方法为:称取柠檬酸21g、无水磷酸钠28.2g,加入0.75%过氧化氢尿素溶液6.4mL,双蒸水定容至1000mL,调节pH值为4.5~5.0;
终止液为2M的H2SO4溶液。
4、ELISA反应条件的确定:
最佳包被浓度最佳样品稀释倍数的确定:采用方阵法,分别将抗原(重组蛋白E2)按照(0.0125、0.025、0.05、0.1、0.2和0.4)μg/孔的包被量包被;将阴性对照血清和阳性血清均分别进行1:25、1:50、1:100和1:200倍的样品稀释液稀释,进行间接ELISA。根据P/N值(阳性样品OD值/阴性样品OD值),最终确定最佳抗原包被量为0.2μg/孔,样品的最佳稀释倍数为1:50。
对最佳包被条件、最佳封闭液及封闭条件、血清孵育时间、酶标二抗稀释度、酶标二抗作用时间以及底物显色时间等作了逐一优化,最终确定本实施例的牛病毒性腹泻病毒抗体的间接ELISA的最佳反应条件为:最佳包被抗原量为0.2μg/孔,4℃包被16h,37℃封闭2h;待检血清稀释50倍,37℃孵育0.5h;酶标二抗抗稀释度为1:10000,37℃反应0.5h;底物显色条件为TMB(3,3',5,5'-四甲基联苯胺)底物室温避光显色10min。
5、阴性对照血清和阳性对照血清制备:
阴性对照血清2mL,为健康未免疫犊牛血清,经牛病毒性腹泻病毒(BVDV)总抗体检测试剂盒检测为阴性,用样品稀释液50倍稀释后,作为阴性对照血清,所述阴性对照血清的OD450nm≥0.1;
阳性对照血清2mL,为成年牛免疫E2蛋白后1月采集的血清(血清中E2蛋白抗体效价为大于1:1024),用HRP藕联物稳定/稀释剂I稀释20倍,得到阳性对照血清,所述阳性对照血清的1.8≦OD450nm≦2.3;所述HRP藕联物稳定/稀释剂I由湖州英创生物科技有限公司生产。
6、牛病毒性腹泻病毒抗体间接ELISA阴阳性临界值的确定:
用建立的间接ELISA方法对46份健康牛血清进行检测,计算S/P值(样本OD值/阳性对照血清OD值),及S/P的平均值和标准差SD,当被检样品的时判为阳性,时判为阴性。经计算46份阴性血清的S/P的平均值为0.138,标准差为0.0365,所以故将阴阳性临界值定为0.211,为了判定方便,把临界值定为0.20。即在已优化好的ELISA反应条件下,S/P值≥0.20,即判定为阳性,反之为阴性。
7、牛病毒性腹泻病毒抗体间接ELISA检测试剂盒组成:
(1)包被板包被酶标板(包被重组蛋白E2)5板;
(2)阴性对照血清2mL;
(3)阳性对照血清2mL;
(4)酶标二抗0.6mL;
(5)酶标二抗稀释液60mL;
(6)样品稀释液120mL;
(7)显色液A 30mL;
(8)显色液B 30mL;
(9)终止液30mL;
(10)浓缩洗涤液(20×洗涤液)100mL;
(11)盖板膜5张;
(12)血清稀释板5块;
(13)说明书1份。
8、牛病毒性腹泻病毒抗体间接ELISA试剂盒操作方法:
(1)洗涤液配制
洗涤液:使用前,浓缩洗涤液恢复至室温,摇动使结晶溶解后,用去离子水稀释20倍,得到洗涤液。
(2)样品处理:
取牛全血,待血液凝固后,在转速为4000rpm的条件下离心10min,收集上清液,得到牛血清,在血清稀释板中将牛血清用样品稀释液稀释得到待检血清样品,按牛血清和样品稀释液的体积比为1:50。
(3)操作步骤按以下步骤进行:
①设置阴性对照血清、阳性对照血清各2孔,每孔100μL。
②将待检血清样品加入到包被酶标板中,加入量为100μL/孔,加样结束后,盖上盖板膜,置37℃温育30min。
③取出包被酶标板,弃去反应液,每孔每次加入260μL的洗涤液,洗涤4次后,在吸水纸上拍干洗涤液。
④用酶标二抗稀释液稀释酶标二抗,酶标二抗和酶标二抗稀释液稀释的体积比为1:100,得到稀释后的酶结合物,将稀释后的酶结合物物加入到包被酶标板中,加入量为100μL/孔,加样结束后,盖上盖板膜,37℃温育30min。
⑤取出包被酶标板,弃去反应液,每孔每次加入260μL的洗涤液,洗涤4次后,在吸水纸上拍干洗涤液。
⑥将显色液A和显色液B按等体积混合,混合后立即加入到包被酶标板中,加入量为100μL/孔,加样结束后,盖上盖板膜,37℃避光显色10min。
⑦每个反应孔加入100μL的终止液,避免出现气泡,加入终止液后的10min内完成结果测定。
(4)结果判定:
用酶标仪于在波长为450nm的条件下读取各反应孔OD值。
实验成立条件:
计算公式:
公式中PC表示阳性对照;
公式中NC表示阴性对照;
公式中S/P表示样本判断值。
阴阳性判定:S/P<0.2,判为阴性;S/P≥0.2,判为阳性。
9、牛病毒性腹泻病毒抗体间接ELISA在临床检测中的应用
应用本实施例的牛病毒性腹泻病毒抗体间接ELISA试剂盒,检测新疆石河子、沙湾、玛纳斯、塔城等地区部分奶牛场642份临床血清样品,结果如表1所示:
表1 临床血清样品检测结果
来源 | A | B | C | D | E | F | G | H | 总计 |
血清样本数 | 153 | 46 | 39 | 103 | 96 | 73 | 26 | 106 | 642 |
阳性样本数 | 116 | 8 | 3 | 85 | 73 | 53 | 5 | 81 | 424 |
阳性率 | 75.82% | 17.39% | 7.69% | 82.52% | 76.04% | 72.60% | 19.23% | 76.42% | 66.04% |
结果表明,当前石河子、沙湾、玛纳斯、塔城等地区牛场都面临着不同程度的BVDV(牛病毒性腹泻病毒)感染,其中规模化牛场的BVDV抗体阳性率普遍高于散户牛场。
本实施例的检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒在新疆部分地区牛病毒性腹泻病毒抗体的检测实践中得到进一步验证,具有特异性强、敏感度高、操作简单等特点,易于大范围推广使用,具有广阔的市场前景,可用于流行性腹泻感染情况的血清学调查、抗体监测及疫苗免疫效果的评估等方面。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何限制。凡是根据发明技术实质对以上实施例所作的任何简单修改、变更以及等效变化,均仍属于本发明技术方案的保护范围内。
序列表
<110> 石河子大学
<120> 一种重组蛋白E2及其应用
<130> 2019.7.26
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 349
<212> PRT
<213> 人工合成(Artificial synthesis)
<400> 1
Met His His His His His His Ile Pro Glu Cys Arg Ser Asp Trp Ser
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Tyr Ala Ile Ala Arg Ser Glu Lys Ile Gly Pro Leu Gly Ala Glu Glu
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Leu Thr Thr Gln Trp Tyr Asp Tyr Ser Glu Gly Met Arg Leu Lys Asp
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Ala Met Val Glu Val Trp Cys Thr Asn Gly Lys Val Lys Phe Leu Lys
50 55 60
Lys Cys Ala Arg Glu Ala Arg Tyr Leu Ala Ala Leu His Thr Arg Ala
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Leu Pro Thr Ser Val Val Phe Lys Gln Leu Leu Ser Gly Gln Lys Leu
85 90 95
Ala Asp Asn Ile Asp Met Glu Asp Asn Phe Glu Phe Gly Leu Cys Pro
100 105 110
Cys Asp Ala Arg Pro Val Val Lys Gly Lys Phe Asn Thr Thr Leu Leu
115 120 125
Asn Gly Pro Ala Phe Gln Met Val Cys Pro Ile Gly Trp Thr Gly Ser
130 135 140
Val Glu Cys Thr Leu Val Asn Lys Asp Thr Leu Arg Thr Ala Val Val
145 150 155 160
Arg Thr Tyr Lys Arg Ala Lys Pro Phe Pro Arg Arg Gln Gly Cys Ala
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Thr Gln Lys Leu Val Gly Glu Asp Leu Tyr Asp Cys Met Leu Gly Gly
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Asn Trp Thr Cys Ile Thr Gly Asp Gln Val Lys Tyr Ala Gly Gly Glu
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Val Lys Thr Cys Lys Trp Cys Gly Tyr Asn Phe Gln Gly Ser Lys Gly
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Leu Pro His Tyr Pro Ile Gly Lys Cys Lys Leu Gly Asn Glu Thr Gly
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Tyr Arg Leu Val Asp Glu Thr Pro Cys Asn Arg Asp Gly Val Ala Ile
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Val Pro His Gly Lys Val Lys Cys Lys Ile Gly Asp Thr Val Val Gln
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Val Ile Ala Met Asp Thr Lys Leu Gly Pro Met Pro Cys Thr Pro Tyr
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Glu Ile Ile Pro Ser Glu Gly Pro Val Glu Lys Thr Ala Cys Thr Phe
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Asn Tyr Thr Lys Thr Leu Lys Asn Lys His Phe Glu Pro Arg Asp Ser
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Tyr Phe Gln Gln Tyr Met Leu Lys Gly Glu Tyr Gln Tyr Trp Phe Asp
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<210> 2
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<213> 人工合成(Artificial synthesis)
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gtcaaattcc tgaaaaaatg cgcgcgcgaa gcacgttatc tggcagcact gcatacccgc 240
gcactgccga cctctgttgt tttcaaacag ctgctgagcg gtcagaaact ggcggataac 300
atcgatatgg aggacaactt cgaattcggc ctgtgtccgt gtgacgcacg tccggttgtt 360
aaaggtaaat tcaacaccac cctgctgaac ggtccggctt ttcagatggt ttgcccgatt 420
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ctggttggcg aagatctgta cgattgcatg ctgggcggta attggacctg tattacgggc 600
gatcaggtca aatacgcagg cggcgaagtt aaaacctgca aatggtgcgg ctacaacttt 660
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ggctaccgtc tggttgacga aaccccgtgt aatcgcgatg gcgttgcaat tgttccgcac 780
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agctatttcc agcagtacat gctgaaaggc gagtaccagt actggttcga tctggacgcg 1020
attgatcacc ataacgacta cttcgcggaa taatgaaagc tt 1062
Claims (3)
1.一种重组蛋白E2,其特征在于,所述重组蛋白E2具有序列表SEQ.ID.No.1的氨基酸序列;所述重组蛋白E2具有序列表SEQ.ID.No2的核苷酸序列;所述重组蛋白E2的制备方法为:对E2蛋白氨基酸序列进行优化,采用全基因合成并通过限制性酶切位点NdeI和HindIII将重组E2蛋白基因***到表达载体pET30a中,并通过酶切法和测序确认最终表达载体的准确性,最终依次转到Top10大肠杆菌克隆菌株感受态细胞和BL21(DE3)大肠杆菌表达菌感受态细胞中,通过IPTG诱导表达重组蛋白E2,之后通过Ni-IDA亲和层析纯化,得到具有生物学活性的重组蛋白E2。
2.一种如权利要求1所述的重组蛋白E2的应用,其特征在于,所述重组蛋白E2用于检测牛病毒性腹泻病毒抗体的间接ELISA试剂盒,所述间接ELISA试剂盒包括包被酶标板、阴性对照血清、阳性对照血清、酶标二抗、酶标二抗稀释液、浓缩洗涤液、样品稀释液、显色液和终止液,所述包被酶标板以重组蛋白E2作为包被抗原。
3.根据权利要求2所述的应用,其特征在于,所述包被酶标板每孔包被0.2μg所述重组蛋白E2;
所述阴性对照血清和阳性对照血清各2mL;
所述酶标二抗是用HRP藕联物稳定/稀释剂I对兔抗牛IgG进行100倍稀释而得,所述酶标二抗为0.6mL;
所述酶标二抗稀释液为含有pH7.2~7.4的0.01M的磷酸缓冲盐溶液b,所述磷酸缓冲盐溶b中马血清的质量分数为5%,所述酶标二抗稀释液为60mL;
所述浓缩洗涤液为20×洗涤液,所述浓缩洗涤液为pH为7.2~7.4的0.1M的磷酸缓冲盐溶液c,所述磷酸缓冲盐溶c中吐温20的质量分数为0.5%,所述浓缩洗涤液为100mL;
所述样品稀释液为pH为7.2~7.4的0.01M的磷酸缓冲盐溶液d,所述磷酸缓冲盐溶液d中牛血清白蛋白的质量分数为0.1%、蔗糖的质量分数为2%,所述样品稀释液为120mL;
所述显色液包括显色液A和显色液B,所述显色液A的制备方法为:将200mg四甲基联苯胺溶于100mL的无水乙醇中,双蒸水定容至1000mL,得到显色液A;所述显色液B的制备方法为:称取柠檬酸21g、无水磷酸钠28.2g,加入0.75%过氧化氢尿素溶液6.4mL,双蒸水定容至1000mL,调节pH值为4.5~5.0,得到显色液B;所述显色液A和所述显色液B均为30mL;
所述终止液为2M的H2SO4溶液,所述终止液为100mL。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112034169A (zh) * | 2020-08-27 | 2020-12-04 | 华威特(江苏)生物制药有限公司 | 用于检测牛病毒性腹泻病毒1型的直接免疫荧光试剂及其试剂盒 |
CN112646840A (zh) * | 2020-12-25 | 2021-04-13 | 石河子大学 | 一种杆状病毒表达***制备牛病毒性腹泻病毒e2蛋白的方法及应用 |
CN113512110A (zh) * | 2021-04-13 | 2021-10-19 | 石河子大学 | Bvdv高特异性纳米抗体及其制备方法及应用 |
CN113512098A (zh) * | 2021-07-15 | 2021-10-19 | 武汉大学 | 鉴别猪瘟病毒和牛病毒性腹泻病毒血清抗体间接elisa方法及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120258127A1 (en) * | 2010-09-10 | 2012-10-11 | Graham Simon Paul | Vaccine peptides |
CN108614121A (zh) * | 2018-05-04 | 2018-10-02 | 山东师范大学 | 牛病毒性腹泻病毒e2蛋白抗原多表位融合肽及其制备与应用 |
CN108641993A (zh) * | 2018-05-17 | 2018-10-12 | 新疆农垦科学院 | 一种食品级表达牛病毒性腹泻病毒e2蛋白的重组乳酸菌口服疫苗株 |
CN110004178A (zh) * | 2019-04-02 | 2019-07-12 | 石河子大学 | 一种牛病毒性腹泻病毒样颗粒的制备的制备方法 |
-
2019
- 2019-10-31 CN CN201911054685.7A patent/CN110746495A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120258127A1 (en) * | 2010-09-10 | 2012-10-11 | Graham Simon Paul | Vaccine peptides |
CN108614121A (zh) * | 2018-05-04 | 2018-10-02 | 山东师范大学 | 牛病毒性腹泻病毒e2蛋白抗原多表位融合肽及其制备与应用 |
CN108641993A (zh) * | 2018-05-17 | 2018-10-12 | 新疆农垦科学院 | 一种食品级表达牛病毒性腹泻病毒e2蛋白的重组乳酸菌口服疫苗株 |
CN110004178A (zh) * | 2019-04-02 | 2019-07-12 | 石河子大学 | 一种牛病毒性腹泻病毒样颗粒的制备的制备方法 |
Non-Patent Citations (6)
Title |
---|
GENBANK: "polyprotein [Bovine viral diarrhea virus 1]", 《GENBANK》 * |
TIANSEN LI等: "Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein", 《PLOS ONE》 * |
柴顺秀等: "牛病毒性腹泻病毒重组E2蛋白PPA-ELISA方法的建立及临床应用", 《动物医学进展》 * |
钟发刚等: "牛病毒腹泻病毒(BVDV)E2重组蛋白ELISA检测方法的初步建立", 《新疆农业科学》 * |
马旭升等: "牛病毒性腹泻病毒BVDV-LC株的分离与鉴定", 《中国预防兽医学报》 * |
黄美玲等: "牛病毒性腹泻病毒E2抗原表位表达及验证", 《生物技术》 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112034169A (zh) * | 2020-08-27 | 2020-12-04 | 华威特(江苏)生物制药有限公司 | 用于检测牛病毒性腹泻病毒1型的直接免疫荧光试剂及其试剂盒 |
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CN113512110A (zh) * | 2021-04-13 | 2021-10-19 | 石河子大学 | Bvdv高特异性纳米抗体及其制备方法及应用 |
CN113512110B (zh) * | 2021-04-13 | 2022-10-14 | 石河子大学 | Bvdv纳米抗体及其制备方法及应用 |
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