CN114966052A - 基于非洲猪瘟p30和p22两种蛋白的间接ELISA检测试剂盒 - Google Patents
基于非洲猪瘟p30和p22两种蛋白的间接ELISA检测试剂盒 Download PDFInfo
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- CN114966052A CN114966052A CN202210560778.2A CN202210560778A CN114966052A CN 114966052 A CN114966052 A CN 114966052A CN 202210560778 A CN202210560778 A CN 202210560778A CN 114966052 A CN114966052 A CN 114966052A
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了基于非洲猪瘟p30和p22两种蛋白的间接ELISA检测试剂盒,涉及动物用生物制品制造技术领域。本发明提供一种非洲猪瘟的间接ELISA检测试剂盒,包括氨基酸序列如SEQ ID NO:2所示的p30蛋白和氨基酸序列如SEQ ID NO:4所示的p22蛋白。p30蛋白和p22蛋白都具有良好的反应原性;非洲猪瘟病毒(ASFV)阳性血清稀释12800次后,检测结果仍为阳性,灵敏度高;本发明的间接ELISA方法仅与ASF阳性血清反应,与猪圆环病毒病、伪狂犬、猪繁殖与呼吸综合征、猪瘟和猪格拉瑟病的阳性血清无交叉反应,特异性强;并且本发明的试剂盒具有良好的重复性。
Description
技术领域
本发明涉及动物用生物制品制造技术领域,特别是涉及基于非洲猪瘟p30和p22两种蛋白的间接ELISA检测试剂盒。
背景技术
非洲猪瘟(African swine fever,ASF)是由非洲猪瘟病毒(African swine fevervirus,ASFV)感染引起的一种猪烈性传染病,该病发病过程短,各年龄段家猪、野猪都可以感染,感染后可在5-14天内出现死亡,病死率高达100%,给养猪产业带来了毁灭性打击。
现阶段控制ASF的唯一有效策略是隔离和消灭被感染的动物。因此,高灵敏度和特异性诊断分析对于快速检测和发现ASFV感染的猪至关重要。ELISA是一种OIE规定针对国际贸易的指定实验,用于检测ASFV的特异性抗体。ELISA方法敏感性强、操作简便且经济实惠。因此,筛选出反应原性好的ASFV抗原,并建立有效的ELISA检测方法,对ASF的防控和净化至关重要。
目前,ASFV抗原检测方法较多,包括荧光定量PCR、RPA、微流控芯片等快速检测方法,但是抗体检测方法相对较少,主要还是集中于ELISA检测试剂盒和胶体金试纸条。而当前商品化非洲猪瘟ELISA检测试剂盒包被的蛋白主要包括p72、p30、p54、pp62,其中p30和p54蛋白在ASFV感染早期表达与分泌,p72和pp62蛋白在ASFV感染晚期表达。研究结果表明p30,p54和p72三种蛋白中,p30诱导表达抗体水平最高。而p30是一种在感染细胞早期表达的磷酸化蛋白,目前商品化ELISA试剂盒大多是基于p30蛋白开发的,其准确性有待进一步考量。
发明内容
本发明的目的是提供基于非洲猪瘟p30和p22两种蛋白的间接ELISA检测试剂盒,以解决上述现有技术存在的问题,本发明提供的试剂盒可有效检测非洲猪瘟抗体水平。
为实现上述目的,本发明提供了如下方案:
本发明提供p30蛋白和p22蛋白在制备非洲猪瘟的间接ELISA检测试剂盒中的应用,所述p30蛋白的氨基酸序列如SEQ ID NO:2所示;所述p22蛋白的氨基酸序列如SEQ IDNO:4所示。
本发明还提供一种非洲猪瘟的间接ELISA检测试剂盒,包括氨基酸序列如SEQ IDNO:2所示的p30蛋白和氨基酸序列如SEQ ID NO:4所示的p22蛋白。
进一步地,所述p30蛋白的编码基因如SEQ ID NO:1所示,所述p22蛋白的编码基因如SEQ ID NO:3所示。
进一步地,所述p30蛋白和所述p22蛋白的包被比例为1:3。
进一步地,所述p30蛋白的封闭液为5wt%脱脂奶粉,封闭时间为90min;所述p22蛋白的封闭液为5wt%脱脂奶粉,封闭时间为60min。
本发明公开了以下技术效果:
有研究发现,ASFV的p22蛋白是由KP177R基因编码的22kD的蛋白,是ASFV感染后早期转录的病毒跨膜结构蛋白,位于病毒颗粒的内膜和感染细胞的表面。p22蛋白在ASFV欧亚株系中高度保守,由于该蛋白含有跨膜结构,故表达相对困难,且关于p22蛋白的作用机制研究尚不透彻,因此关于p22蛋白的研究相对较少。且目前生猪疫病检测行业,比较成熟的商品化试剂盒是基于p30蛋白的ELISA检测试剂盒,由于p22蛋白存在跨膜区,因此实现可溶性表达相对困难,目前没有基于p22蛋白的间接ELISA检测试剂盒,且经过比对,单独的p22蛋白的间接ELISA检测试剂盒与现有的试剂盒符合率低于90%。
我们推测建立一种同时检测p30和p22两种早期蛋白的ASF抗体检测方法有望提高现有方法的准确率。本发明基于p30和p22两种蛋白建立的间接ELISA检测试剂盒,p30和p22两种重组蛋白均可以与ASF阳性血清发生反应,两者都具有良好的反应原性;ASFV阳性血清稀释12800次后,检测结果仍为阳性,具有较高的灵敏度;本发明的ELISA方法仅与ASF阳性血清反应,与猪圆环病毒病、伪狂犬、猪繁殖与呼吸综合征、猪瘟和猪格拉瑟病的阳性血清无交叉反应,特异性强;选择四种阳性血清进行检测,批间变异系数(CV)范围为2.0%至4.5%,批内变异系数(CV)范围为3.0%至6.0%,表明本发明的试剂盒具有良好的重复性。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为p22蛋白诱导条件摸索结果,其中A为IPTG浓度的摸索,M:蛋白质分子质量标准,1:pET-32a空载体,2:pET-32a-p22未诱导,3-6:IPTG浓度0.1mM、0.4mM、0.7mM、1.0mM;B为OD值的摸索,M:蛋白质分子质量标准,1:pET-32a空载体,2:pET-32a-p22未诱导,3-6:OD值0.4、0.6、0.8、1;
图2为p30蛋白诱导条件摸索结果,其中A为IPTG浓度的摸索,M:蛋白质分子质量标准,1:pET-32a空载体,2:pET-32a-p30未诱导,3-6:IPTG浓度0.1mM、0.4mM、0.7mM、1.0mM;B为OD值的摸索,M:蛋白质分子质量标准,1:pET-32a空载体,2:pET-32a-p30未诱导,3-6:OD值0.4、0.6、0.8、1;
图3为重组蛋白p30、p22的反应原性鉴定,其中A为p30蛋白反应原性,M:蛋白质分子质量标准,1:p30蛋白,B为p22蛋白反应原性,M:蛋白质分子质量标准,1:p22蛋白;
图4为最佳蛋白包被浓度和最佳血清稀释度,其中A为p30蛋白,B为p22蛋白;
图5为最佳封闭液和最佳封闭时间,其中A为p30蛋白,B为p22蛋白;
图6为编码p22的基因序列剪接和密码子优化过程示意图;
图7为编码p30的基因序列经密码子优化过程示意图。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值,以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见得的。本发明说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
以下实施例中,pET-32a载体购自武汉戴安生物技术有限公司。
实施例1
1.重组阳性质粒的构建与合成
根据非洲猪瘟病毒Pig/HLJ/2018的编码p30和p22的基因序列,经剪接和密码子优化后,经上海捷瑞生物工程有限公司合成并连接到pEASY-Blunt载体上,经测序鉴定正确的即为p30和p22基因阳性克隆载体。
编码p22的基因序列剪接和密码子优化过程:剪接掉跨膜区域(前29个氨基酸)具体核苷酸优化情况见图6。
编码p30的基因序列经密码子优化过程:具体核苷酸优化情况见图7。
利用限制性内切酶BamHI和XhoI分别对p30和p22的阳性克隆质粒进行双酶切,分别与同样经双酶切处理的pET-32a载体连接,将连接产物转化至感受态细胞DH5α。经生工生物工程(上海)股份有限公司测序,将序列鉴定正确的两种重组阳性质粒分别命名为pET32a-p30和pET32a-p22。
分别设计p30和p22基因的特异性引物,引物序列如下:
上述斜体下划线表示酶切位点。
密码子优化后p30核苷酸序列如下(SEQ ID NO:1):
ATGGATTTCATCCTGAATATCAGTATGAAAATGGAAGTGATTTTCAAGACCGATCTGCGCAGTAGTAGCCAGGTGGTTTTTCATGCCGGTAGCCTGTATAATTGGTTTAGTGTTGAAATTATCAACAGCGGTCGCATTGTGACCACCGCAATTAAAACCCTGCTGAGCACCGTTAAATATGATATTGTGAAAAGCGCACGCATTTATGCCGGTCAGGGCTATACCGAACATCAGGCCCAGGAAGAATGGAATATGATTCTGCATGTGCTGTTTGAAGAAGAAACCGAAAGTAGCGCAAGTAGCGAAAATATTCATGAAAAAAACGACAACGAGACCAATGAATGTACCAGCAGCTTTGAAACCCTGTTTGAACAGGAACCGAGCAGCGAAGTTCCGAAAGATAGCAAACTGTATATGCTGGCCCAGAAAACCGTGCAGCATATTGAACAGTATGGTAAAGCCCCGGATTTTAATAAAGTTATTCGCGCACATAACTTCATTCAGACCATTTATGGTACCCCGCTGAAAGAAGAAGAAAAAGAAGTGGTTCGTCTGATGGTGATTAAACTGCTGAAAAAAAAATAA.
p30氨基酸序列如下(SEQ ID NO:2):
MDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFSVEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENIHEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTIYGTPLKEEEKEVVRLMVIKLLKKK.
密码子优化后p22核苷酸序列如下(SEQ ID NO:3):
AAAAAACAGCAGCCGCCGAAAAAAGTCTGCAAAGTCGACAAAGATTGCGGTAGCGGCGAACATTGTGTTCGCGGTAGTTGTAGCAGTCTGAGCTGTCTGGACGCCGTCAAAATGGACAAACGCAACATCAAAATCGACAGCAAAATCAGCAGCTGCGAATTTACCCCGAACTTCTACCGTTTTACCGATACCGCAGCGGACGAACAACAAGAATTCGGCAAAACCCGCCATCCGATCAAAATTACCCCGAGTCCGTCTGAAAGCCATAGTCCGCAGGAAGTCTGCGAAAAATACTGCTCTTGGGGTACCGACGATTGTACCGGTTGGGAATACGTTGGCGACGAAAAAGAAGGCACCTGTTACGTGTACAACAATCCGCATCATCCGGTGCTGAAATACGGCAAAGACCATATCATCGCGCTGCCGCGTAATCATAAACACGCATGA.
p22氨基酸序列如下(SEQ ID NO:4):
KKQQPPKKVCKVDKDCGSGEHCVRGSCSSLSCLDAVKMDKRNIKIDSKISSCEFTPNFYRFTDTAADEQQEFGKTRHPIKITPSPSESHSPQEVCEKYCSWGTDDCTGWEYVGDEKEGTCYVYNNPHHPVLKYGKDHIIALPRNHKHA.
2.重组蛋白p30、p22诱导表达及蛋白纯化
诱导表达的具体操作为:
将pET32a-p30和pET32a-p22两种重组质粒分别转化到感受态细胞BL21中。从37℃温箱培养一夜的固态培养基中挑取单个菌落,放入Amp抗性的LB液体培养基中,37℃,200rpm过夜培养。分别取50μLpET32a-p30、pET32a-p22和pET-32a的菌液,分别置于含有5mLLB液体培养基(Amp+)的试管中,另设一管5mL不加菌液的LB液体培养基(Amp+)为阴性对照,在恒温摇床上37℃,200rpm培养3-4h,使得两种菌液0D600在0.6-0.8之间。每管取500μL菌液作为诱导前对照,4℃保存。之后向剩余三管菌液中各加7.5μL 100mM IPTG溶液,37℃,200rpm培养4h,每管取500μL菌液。将诱导前三管菌液和诱导后三管菌液同时离心(9000rpm,4℃)10min,弃掉上清,留下菌泥。每管用100μLPBS重悬菌液后,4℃离心机下9000rpm离心10min,弃掉上清,留下菌泥。重复上述步骤两次,在最后一次重悬的菌液中加25μL 5×SDS-PAGE Sample Buffer,将6个样品沸水中煮10min,-20℃保存。之后SDS-PAGE凝胶电泳检测。
通过摸索诱导表达条件(IPTG浓度,OD值,诱导温度,诱导时间),确定最佳表达条件(图1、2)。经IPTG诱导表达出目的蛋白p30与p22后,经镍柱纯化获得单一条带的目的蛋白,经BCA蛋白定量试剂盒测定纯化后的两种重组蛋白浓度。结果是:pET32a-p30为0.04g/L;pET32a-p22为0.334g/L。
3.重组蛋白p30、p22的反应原性鉴定
使用纯化的p30、p22两种重组蛋白分别作为抗原,ASF阳性血清作为一抗,羊抗猪GoatpAb to pig lgG HRP为二抗,进行western blot分析。结果显示:p30、p22两种蛋白均条带清晰且单一,且与预期大小相符。表明p30、p22两种重组蛋白均可以与ASF阳性血清发生反应,两者都具有良好的反应原性(图3)。
4.ELISA方法反应条件的优化
试剂盒包括:蛋白包被缓冲液,PBST洗液,封闭液,样品稀释液,酶标二抗,TMB单组分显色液,终止液。
4.1最佳蛋白包被浓度和血清稀释度
应用棋盘滴定法确定了p30蛋白最佳包被浓度为0.4mg/L,样品血清最佳稀释倍数为600。p22蛋白最佳包被浓度为0.11mg/L,血清样品最佳稀释倍数为600倍(图4)。
4.2最佳封闭液和封闭时间的确定
在确定了最佳蛋白包被浓度和最佳血清稀释度的情况下,重复上述操作步骤,实验结果表明p30蛋白最佳封闭液为5%脱脂奶粉,最佳封闭时间为90min,p22蛋白包被板最佳封闭液为5%脱脂奶粉,最佳封闭时间为60min(图5)。
4.3p30与p22最佳包被比例的确定
将p30和p22两种蛋白(按最佳包被浓度)分别以1:1、1:2、2:1、1:3和3:1的体积比例(总体积100mL)用酶标板包被。两种蛋白质的最佳包被比为最大P/N值。实验结果表明,当p30与p22的比例为1:3时,P/N最高(表1)。
表1p30与p22最佳包被比例的确定
4.4临界值的确定
根据上述优化条件,即,选择50份阴性血清(成品试剂盒检测)进行ELISA检测,读取OD450,计算平均值和标准偏差s(0.083)。然后为阴性≤0.34;x+3S=0.423,阳性≥0.423;中间的范围是可疑的。
4.5灵敏性试验
采用p30蛋白间接ELISA试剂盒、p22蛋白间接ELISA试剂盒、p30与p22双蛋白间接ELISA试剂盒和2个商品化试剂盒,分别检测不同稀释率的ASF阳性血清,并测定OD450。结果如表2-5。根据临界值,经血清稀释12800次后,p30和p22包被板的检测结果仍为阳性(表3)。
表2p30蛋白间接ELISA试剂盒灵敏度实验结果
表3p22蛋白间接ELISA试剂盒灵敏度实验结果
表4p30与p22双蛋白间接ELISA试剂盒灵敏度实验结果
表5商品化试剂盒灵敏度实验结果
4.6特异性试验
对猪圆环病毒病、伪狂犬、猪繁殖与呼吸综合征、猪瘟和猪格拉瑟病的阳性血清进行ELISA交叉反应试验的特异性评估。试验结果如下:其他五种猪病的OD450均小于0.34。结果表明,本发明建立的ELISA试剂盒仅与ASFV阳性血清反应,与猪圆环病毒病、伪狂犬、猪繁殖与呼吸综合征、猪瘟和猪格拉瑟病的阳性血清无交叉反应。该方法具有良好的特异性(表6)。
表6特异性试验
4.7重复性试验
选择四种阳性血清,批间变异系数(CV)范围为2.0%至4.5%,批内变异系数(CV)范围为3.0%至6.0%。批内和批间变异系数表明,本发明建立的间接ELISA试剂盒具有良好的重复性(表7)。
表7重复性试验
4.8样品检测
用上述建立的3种间接ELISA试剂盒检测184份猪血样本,检测出105份阳性样本和79份阴性样本。p30与p22双蛋白间接ELISA试剂盒与成品试剂盒id.vet的虽然符合率稍低,为94.6%,但检出的阳性样品数量比商品化试剂盒1多2个,可疑样品数量多2个,阴性样品数量少4个。商品化剂盒2与商品化试剂盒1的符合率为96.2%。结果说明本发明建立的p30与p22双蛋白间接ELISA试剂盒的阳性样品和可疑样品检出率要高于市面上2个商品化试剂盒(表8和表9)。
表8不同试剂盒样品检测结果
表9不同试剂盒样品检测率统计结果
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 山东省农业科学院畜牧兽医研究所
<120> 基于非洲猪瘟p30和p22两种蛋白的间接ELISA检测试剂盒
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 585
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggatttca tcctgaatat cagtatgaaa atggaagtga ttttcaagac cgatctgcgc 60
agtagtagcc aggtggtttt tcatgccggt agcctgtata attggtttag tgttgaaatt 120
atcaacagcg gtcgcattgt gaccaccgca attaaaaccc tgctgagcac cgttaaatat 180
gatattgtga aaagcgcacg catttatgcc ggtcagggct ataccgaaca tcaggcccag 240
gaagaatgga atatgattct gcatgtgctg tttgaagaag aaaccgaaag tagcgcaagt 300
agcgaaaata ttcatgaaaa aaacgacaac gagaccaatg aatgtaccag cagctttgaa 360
accctgtttg aacaggaacc gagcagcgaa gttccgaaag atagcaaact gtatatgctg 420
gcccagaaaa ccgtgcagca tattgaacag tatggtaaag ccccggattt taataaagtt 480
attcgcgcac ataacttcat tcagaccatt tatggtaccc cgctgaaaga agaagaaaaa 540
gaagtggttc gtctgatggt gattaaactg ctgaaaaaaa aataa 585
<210> 2
<211> 194
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Asp Phe Ile Leu Asn Ile Ser Met Lys Met Glu Val Ile Phe Lys
1 5 10 15
Thr Asp Leu Arg Ser Ser Ser Gln Val Val Phe His Ala Gly Ser Leu
20 25 30
Tyr Asn Trp Phe Ser Val Glu Ile Ile Asn Ser Gly Arg Ile Val Thr
35 40 45
Thr Ala Ile Lys Thr Leu Leu Ser Thr Val Lys Tyr Asp Ile Val Lys
50 55 60
Ser Ala Arg Ile Tyr Ala Gly Gln Gly Tyr Thr Glu His Gln Ala Gln
65 70 75 80
Glu Glu Trp Asn Met Ile Leu His Val Leu Phe Glu Glu Glu Thr Glu
85 90 95
Ser Ser Ala Ser Ser Glu Asn Ile His Glu Lys Asn Asp Asn Glu Thr
100 105 110
Asn Glu Cys Thr Ser Ser Phe Glu Thr Leu Phe Glu Gln Glu Pro Ser
115 120 125
Ser Glu Val Pro Lys Asp Ser Lys Leu Tyr Met Leu Ala Gln Lys Thr
130 135 140
Val Gln His Ile Glu Gln Tyr Gly Lys Ala Pro Asp Phe Asn Lys Val
145 150 155 160
Ile Arg Ala His Asn Phe Ile Gln Thr Ile Tyr Gly Thr Pro Leu Lys
165 170 175
Glu Glu Glu Lys Glu Val Val Arg Leu Met Val Ile Lys Leu Leu Lys
180 185 190
Lys Lys
<210> 3
<211> 447
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaaaaacagc agccgccgaa aaaagtctgc aaagtcgaca aagattgcgg tagcggcgaa 60
cattgtgttc gcggtagttg tagcagtctg agctgtctgg acgccgtcaa aatggacaaa 120
cgcaacatca aaatcgacag caaaatcagc agctgcgaat ttaccccgaa cttctaccgt 180
tttaccgata ccgcagcgga cgaacaacaa gaattcggca aaacccgcca tccgatcaaa 240
attaccccga gtccgtctga aagccatagt ccgcaggaag tctgcgaaaa atactgctct 300
tggggtaccg acgattgtac cggttgggaa tacgttggcg acgaaaaaga aggcacctgt 360
tacgtgtaca acaatccgca tcatccggtg ctgaaatacg gcaaagacca tatcatcgcg 420
ctgccgcgta atcataaaca cgcatga 447
<210> 4
<211> 148
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Lys Lys Gln Gln Pro Pro Lys Lys Val Cys Lys Val Asp Lys Asp Cys
1 5 10 15
Gly Ser Gly Glu His Cys Val Arg Gly Ser Cys Ser Ser Leu Ser Cys
20 25 30
Leu Asp Ala Val Lys Met Asp Lys Arg Asn Ile Lys Ile Asp Ser Lys
35 40 45
Ile Ser Ser Cys Glu Phe Thr Pro Asn Phe Tyr Arg Phe Thr Asp Thr
50 55 60
Ala Ala Asp Glu Gln Gln Glu Phe Gly Lys Thr Arg His Pro Ile Lys
65 70 75 80
Ile Thr Pro Ser Pro Ser Glu Ser His Ser Pro Gln Glu Val Cys Glu
85 90 95
Lys Tyr Cys Ser Trp Gly Thr Asp Asp Cys Thr Gly Trp Glu Tyr Val
100 105 110
Gly Asp Glu Lys Glu Gly Thr Cys Tyr Val Tyr Asn Asn Pro His His
115 120 125
Pro Val Leu Lys Tyr Gly Lys Asp His Ile Ile Ala Leu Pro Arg Asn
130 135 140
His Lys His Ala
145
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggatccatgg atttcatcct gaatatc 27
<210> 6
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ctcgagtttt tttttcagca gtttaa 26
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggatccaaaa aacagcagcc gccga 25
<210> 8
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctcgagttat gcgtgtttat gattac 26
Claims (5)
1.p30蛋白和p22蛋白在制备非洲猪瘟的间接ELISA检测试剂盒中的应用,其特征在于,所述p30蛋白的氨基酸序列如SEQ ID NO:2所示;所述p22蛋白的氨基酸序列如SEQ ID NO:4所示。
2.一种非洲猪瘟的间接ELISA检测试剂盒,其特征在于,包括氨基酸序列如SEQ ID NO:2所示的p30蛋白和氨基酸序列如SEQ ID NO:4所示的p22蛋白。
3.根据权利要求2所述的试剂盒,其特征在于,所述p30蛋白的编码基因如SEQ ID NO:1所示,所述p22蛋白的编码基因如SEQ ID NO:3所示。
4.根据权利要求2所述的试剂盒,其特征在于,所述p30蛋白和所述p22蛋白的包被比例为1:3。
5.根据权利要求2所述的试剂盒,其特征在于,所述p30蛋白的封闭液为5wt%脱脂奶粉,封闭时间为90min;所述p22蛋白的封闭液为5wt%脱脂奶粉,封闭时间为60min。
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