CN1861633B - 一种基于重组抗原的弓形虫检测试剂盒 - Google Patents
一种基于重组抗原的弓形虫检测试剂盒 Download PDFInfo
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- CN1861633B CN1861633B CN2005100345864A CN200510034586A CN1861633B CN 1861633 B CN1861633 B CN 1861633B CN 2005100345864 A CN2005100345864 A CN 2005100345864A CN 200510034586 A CN200510034586 A CN 200510034586A CN 1861633 B CN1861633 B CN 1861633B
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Abstract
本发明是从弓形虫主要表面抗原基因SAG1中切取542-1218片段(tSAG1)，将其亚克隆到可溶性表达载体pET32a(+)中，并转化大肠杆菌，构建工程菌pET32a—tSAG1/BL21，经IPTG诱导高效表达，表达菌的超声裂解上清经Ni-NTA和Sephadex-G75纯化，包被ELISA板微孔，构建抗体检测试剂盒，同样地将上述纯化的重组抗原点样NC膜上，构建胶体金检测试纸条。本发明还利用纯化的重组抗原免疫小鼠，制备抗弓形虫SAG1蛋白的单克隆抗体，并利用该单克隆抗体构建如上所述的ELISA和胶体金试剂盒用以检测弓形虫SAG1循环抗原。
Description
技术领域
本发明涉及一种基于重组抗原的弓形虫(Toxoplasma gondii)检测试剂盒，这种试剂盒是利用重组抗原及其相应的单克隆抗体组建的检测和/或诊断样品中弓形虫抗体或相应抗原，这种抗原或单克隆抗体的筛选与鉴定、试剂盒的组成成分、检测步骤及应用情况。
背景技术
弓形虫(Toxoplasma gondii)是一种专性细胞内寄生的医学原虫，能感染几乎所有恒温动物(包括人类)，引起弓形虫病，全世界近1/3的人口受到威胁。弓形虫是一种机会性致病原虫，在免疫功能正常的人内通常形成包囊，造成隐性感染；但当免疫功能下降时(如肿瘤患者、器官移植者及AIDS患者等)，弓形虫可在宿主体内播散引起弓形虫病甚至导致死亡。妇女在妊娠期初次感染弓形虫可引起流产、畸胎、死胎，或引起胎儿先天性弓形虫病，表现为智力低下、视网膜脉络膜炎、失明等，这些症状可在胎儿出生时或是成长过程中出现。弓形虫感染也是家畜/家禽流产、畸胎、死产的主要原因之一，对畜牧业生产危害严重。因此，弓形虫病的诊断，尤其是弓形虫现症感染的及时诊断成为弓形虫病防治工作的重点。
目前，国内外普遍采用免疫学方法检测血清中的弓形虫IgG、IgM及IgG亲和力，所用抗原多是从感染动物中收集或经组织/细胞培养的弓形虫速殖子抗原。但这种制备速殖子抗原的方法昂贵费时，纯度低，试剂盒批间差异大，不易标准化。
在原核表达系统中表达特异的目的蛋白，因蛋白产量高、操作简单、费用低廉而被许多研究者选用。SAG1(P30)蛋白是弓形虫速殖子期特异性抗原，在不同虫株之间具有具有高度的保守性，大量实验证明SAG1蛋白具有良好的免疫原性，是弓形虫病诊断和疫苗研究的重要候选抗原分子。但SAG1蛋白是一个高度构象依赖的蛋白，其空间结构的形成主要依赖二硫键的正确连接。原核表达系统(如大肠杆菌)缺乏蛋白质翻译后的修饰功能，因此以往在大肠杆菌中获得的重组SAG1蛋白大都形成包涵体，表达产物由于错误折叠而不具有特异的免疫反应性，需经复杂的变性、复性过程才能恢复部分活性。另外，SAG1全基因克隆到大肠杆菌中时，由于对宿主菌的毒性较大，表达量不高。
本发明是从弓形虫SAG1全长基因中截取其主要抗原表位的编码区域，使其在大肠杆菌中得到高效表达，并且这种表达是可溶性的，表达的重组蛋白无需复性即能具有良好的免疫活性。本发明还筛选针对该重组蛋白的单克隆抗体，建立一套基于重组抗原及其单克隆抗体的弓形虫感染检测体系(包括ELISA检测弓形虫IgG和IgM抗体、IgG亲和力、SAG1循环抗原，免疫胶体金检测IgG和IgM抗体和SAG1循环抗原等)，该体系由于抗原可批量生产，易于标准化控制。
发明内容
本发明是从弓形虫主要表面抗原基因SAG1中切取542-1218片段(tSAG1)，将其亚克隆到可溶性表达载体pET32a(+)中，并转化大肠杆菌，构建工程菌pET32a-tSAG1/BL21，经IPTG诱导高效表达，表达菌的超声裂解上清经Ni-NTA和Sephadex-G75纯化，包被ELISA板微孔，构建抗体检测试剂盒，同样地将上述纯化的重组抗原点样NC膜上，构建胶体金检测试纸条。本发明还利用纯化的重组抗原免疫小鼠，制备抗弓形虫SAG1蛋白的单克隆抗体，并利用该单克隆抗体构建如上所述的ELISA和胶体金试剂盒用以检测弓形虫SAG1循环抗原。
这样，在第一个方面，本发明提供了经改造的重组弓形虫主要表面抗原蛋白(rSAG1)截短片段的编码基因序列：
ATGGGTTTCA CTCTTAAGTG CCCTAAAACA GCGCTCACAG AGCCTCCCAC TCTTGCGTAC 60
TCACCCAACA GGCAAATCTG CCCAGCGGGT ACTACAAGTA GCTGTACATC AAAGGCTGTA 120
ACATTGAGCT CCTTGATTCC TGAAGCAGAA GATAGCTGGT GGACGGGGGA TTCTGCTAGT 180
CTCGACACGG CAGGCATCAA ACTCACAGTT CCAATCGAGA AGTTCCCCGT GACAACGCAG 240
ACGTTTGTGG TCGGTTGCAT CAAGGGAGAC GACGCACAGA GTTGTATGGT CACAGTGACA 300
GTACAAGCCA GAGCCTCATC GGTCGTCAAT AATGTCGCAA GGTGCTCCTA CGGTGCAGAC 360
AGCACTCTTG GTCCTGTCAA GTTGTCTGCG GAAGGACCCA CTACAATGAC CCTCGTGTGC 420
GGGAAAGATG GAGTCAAAGT TCCTCAAGAC AACAATCAGT ACTGTTCCGG GACGACGCTG 480
ACTGGTTGCA ACGAGAAATC GTTCAAAGAT ATTTTGCCAA AATTAACTGA GAACCCGTGG 540
CAGGGTAACG CTTCGAGTGA TAAGGGTGCC ACGCTAACGA TCAAGAAGGA AGCATTTCCA 600
GCCGAGTCAA AAAGCGTCAT TATTGGATGC ACAGGGGGAT CGCCTGAGAA GCATCACTGT 660
ACCGTGAAAC TGGAGTTTGC CGGGGCTGCA GGTTAA 696
在第二个方面，本发明提供了重组弓形虫主要表面抗原蛋白(rSAG1)在变性条件下测定时，其分子量范围在20-25kDa范围内；其融合蛋白在变性条件下测定时，其分子量范围在39-43kDa范围内。
适合地，所述的重组抗原蛋白质具有以下序列：
1 METGlyPheThrLeuLysCysProLysThrAlaLeuThrGluProProThrLeuAlaTyr
21 SerProAsnArgGlnIleCysProAlaGlyThrThrSerSerCysThrSerLysAlaVal
41 ThrLeuSerSerLeuIleProGluAlaGluAspSerTrpTrpThrGlyAspSerAlaSer
61 LeuAspThrAlaGlyIleLysLeuThrValProIleGluLysPheProValThrThrGln
81 ThrPheValValGlyCysIleLysGlyAspAspAlaGlnSerCysMETValThrValThr
101 ValGlnAlaArgAlaSerSerValValAsnAsnValAlaArgCysSerTyrGlyAlaAsp
121 SerThrLeuGlyProValLysLeuSerAlaGluGlyProThrThrMETThrLeuValCys
141 GlyLysAspGlyValLysValProGlnAspAsnAsnGlnTyrCysSerGlyThrThrLeu
161 ThrGlyCysAsnGluLysSerPheLysAspIleLeuProLysLeuThrGluAsnProTrp
181 GlnGlyAsnAlaSerSerAspLysGlyAlaThrLeuThrIleLysLysGluAlaPhePro
201 AlaGluSerLysSerValIleIleGlyCysThrGlyGlySerProGluLysHisHisCys
221 ThrValLysLeuGluPheAlaGlyAlaAlaGly 231
或者与该序列实质上同源的序列。在氨基酸水平上，如果大量重要的氨基酸序列显示同源性，可以看作蛋白质序列和另一个蛋白质序列实质上同源。随着优先等级的增加，至少40％，50％，60％，70％，80％90％，95％，甚至99％的氨基酸可以是同源的。
在第三个方面，一种如权利要求1或2所限定之蛋白质编码基因的表达载体pET32a(+)-tSAG1。
在第四个方面，一种如权利要求3所限定之的表达载体PET32a(+)-tSAG1，的转化菌株，即工程菌PET32a(+)-tSAG1/BL21。
在第五个方面，一种获得如权利要求1、权利要求2和权利要求3所限定之蛋白质的方法，该方法包括：
(a)用PCR等方法从弓形虫cDNA文库或基因组DNA中获取目的蛋白的编码基因tSAG1；
(b)将目的蛋白的编码基因tSAG1克隆至可溶性表达载体pET32a(+)，并转化大肠杆菌如BL21等，构建工程菌pET32a(+)-tSAG1/BL21；
(c)上述工程菌在一定条件下，经IPTG诱导后高效表达目的蛋白rSAG1；
(d)高效表达的目标菌超声裂解上清经Ni-NTA和Sephadex-G75纯化，获得较高纯度的目的蛋白；
(e)对上述(d)所获得的重组蛋白进行聚丙烯酰胺凝胶电泳，确认具有41kDa左右分子量的特定蛋白质的存在；
(f)对上述(e)所获得的蛋白质进行聚丙烯酰胺凝胶电泳后，电转移至NC膜或PVDF膜上，进行western blotting，鉴定所获得蛋白的免疫反应性。
在第六个方面，本发明提供了一种能用于弓形虫或弓形虫病的检测和/或诊断方法。该方法包括：
(a)使如权利要求1或2所限定的重组蛋白质或其抗原片段与待测样本接触，检测针对弓形虫的抗体；
(b)使如权利要求1或2所限定的重组蛋白质或其抗原片段的单克隆抗体与待测样本接触，检测样本中弓形虫循环抗原。
特别是，可以利用本发明的重组蛋白质或其抗原片段、或其抗原片断组合物检测IgG抗体和/或IgM抗体。适合地，待测样本是生物样品，例如人和动物的血清、脑脊液、尿液、唾液、羊水等所有体液样本。
在第七个方面，本发明提供了用于弓形虫检测和/或诊断的如权利要求1或2所限定的蛋白质或其抗原片段的用途。此种检测是在体外进行的。
本发明的重组蛋白抗原或抗原组分可作为弓形虫虫病检测和/或诊断试剂盒的一部分，这样，在第五个方面，一种用于检测和/或诊断弓形虫的试剂盒，该试剂盒包含如权利要求1或2所限定的蛋白质或其抗原片段，其试剂盒可以是ELISA试剂盒和/或胶体金试剂盒或其他任何品种的试剂盒。
此外，还可以用本发明的重组蛋白抗原或其抗原片段诱导弓形虫的免疫反应。这样在另一方面，本发明提供了一种能够利用如权利要求1或2所限定的蛋白质或其抗原片段免疫动物制备单克隆抗体或多克隆抗体，用此单克隆抗体或多克隆抗体组建检测弓形虫病患者体内的针对如权利要求1或2所限定的蛋白质或其抗原片段的抗原表位(循环抗原)。
与已有技术相比有如下优点：
1.目前弓形虫检测试剂盒的包被抗原都是采用弓形虫速殖子全虫抗原，不仅抗原获得困难，而且批间差异大。利用重组蛋白构建试剂盒不仅抗原容易获得，节约成本，而且可以大批量制备，质量易于控制，故易于试剂盒的标准化生产。目前尚没有用弓形虫重组抗原组建的弓形虫病诊断试剂盒问世。
2.我们在大肠杆菌中进一步以可溶性形式表达成熟SAG1蛋白片段，表达的重组蛋白无需复性即能具有良好的免疫活性；利用高密度发酵生产rSAG1，收获工程菌产量可达26g/L以上，其蛋白表达量达菌体蛋白的30％，经Ni柱、超滤和分子筛纯化后可达90％以上的纯度。经ELISA检测弓形虫感染的动物血清和弓形虫病人血清以及健康动物血清和健康人血清，结果其敏感性和特异性均达90％以上，检测系统的重复性和稳定性均优于目前市售同类产品。
附图说明
图1重组质粒的酶切图谱
1：pET32a(+)Nco I单酶切；
2：pET32a(+)-tSAG1 Nco I单酶切；
3：PET32a(+)-tSAG1 Nco I+HindIII双酶切；
4：DNA标准
图2目的蛋白占菌体蛋白的百分含量分析
图3a Ni柱纯化后的目的蛋白纯度电泳图
1Ni柱纯化的rSAG1
2标准分子量蛋白
3纯化前菌体蛋白超声裂解上清
图3b Ni柱纯化后的目的蛋白含量分析
图4纯化rSAG1蛋白与兔血清的Western-blot结果
1：正常兔血清；
2：弓形虫速殖子免疫兔血清；
3：低分子量标准蛋白
图5rSAG1包被ELISA板的最佳浓度曲线
图6五株单克隆抗体(腹水)的亚类测定
1：Y3A8；2：Y3E10；3：Y5G11；4：K3A3；5：K7H3
图7五株单克隆抗体(腹水)与弓形虫速殖子抗原的Western-blot分析
1：K7H3；2：K3A3；3：Y5G11；4：Y3E10；5：Y3A8；6：正常兔血清；7：弓形虫速殖子免疫兔血清；8：低分子量标准蛋白
具体实施方式
实施例一pET32a(+)-tSAG1的构建
提取转化菌重组质粒DNA，与空载质粒DNA进行1％琼脂糖电泳，而后分别进行单酶切及双酶切鉴定，pET32a(+)-tSAG1阳性重组质粒经Nco I单酶切可见大小约6600bp左右的单条带，经Nco I+HindIII双酶切后可产生大小约5900bp及700bp两条带，与预期结果相符。而pET32a(+)经Nco I单酶切后只有5900bp一条带，以上结果表明已获得了pET32a(+)-tSAG1重组质粒(图1)。
实施例二工程菌pET32a(+)-tSAG1/BL21诱导表达
pET32a(+)-tSAG1/BL21经IPTG诱导后，经SDS-PAGE分析，在约40kDa处出现特异性表达带，与预期分子量基本吻合。空载体在21kDa的位置表达了硫氧还蛋白(Thioredoxin，Trx)，而空载体、重组质粒在诱导前均未出现特异的表达蛋白条带。重组菌经超声波破菌后，14000rpm离心20min，分别取上清和沉淀进行SDS-PAGE，结果显示目的蛋白主要以可溶性形式表达。目的蛋白占菌体蛋白的34.36％。(图2)
实施例三Ni柱纯化后rSAG1的纯度
将重组蛋白在优化条件下进行大量表达，经超声破菌后，重组蛋白大部分以可溶性形式存在于超声上清中，上清中的重组蛋白经Ni-NTA一步纯化后，纯度可达60以上％，该纯化的重组蛋白再经Sephadex-G75纯化后可获得90％以上的纯度(图3)。
实施例四Ni柱纯化后rSAG1的免疫活性
将纯化的rSAG1与弓形虫速殖子免疫兔血清进行Western-blot，结果显示重组蛋白与弓形虫速殖子免疫兔血清在约40kDa处有一特异性反应条带，而与正常兔血清无明显反应条带出现(图4)
实施例五rSAG1检测人血清的ELISA结果
经ELISA测定，弓形虫速殖子免疫兔血清针对rSAG1的最适工作稀释度为1∶300左右，rSAG1的最佳包被浓度约为0.2ug/孔(图5)。
以rSAG1抗原和弓形虫速殖子超声抗原包被ELISA板微孔，用间接ELISA法检测人血清中IgG抗体，共检测60份人血清。结果20份血清两种抗原检测均为阳性，13份血清两种抗原检测均为阴性；rSAG1抗原检测为阳性而弓形虫速殖子抗原检测为阴性的14份，rSAG1抗原检测为阴性而弓形虫速殖子抗原检测为阳性的13份。经配对计数资料的x检验，应用二项分布原理计算双侧精确概率，P＝1.000，说明两种方法检测的阳性率无显著性差异。
表1-1不同抗原对弓形虫IgG抗体检测结果比较表
实施例六rSAG1单克隆抗体的亚类鉴定
经Mouse monoclonal antibody isotyping kit测定，5株单克隆抗体重链均为IgG1，轻链均为κ链(图6)。
实施例七单克隆抗体的特异性鉴定
5株单克隆抗体均能识别在还原条件下分子量约为30KDa的天然SAG1抗原，与rSAG1在约40KDa处有特异性反应条带，而与pET单体蛋白无反应条带出现(图7)。
实施例七单克隆抗体的效价
五株杂交瘤细胞诱生的腹水经SmartspecTM3000测定，蛋白浓度分别为：Y3A8：42.2mg/ml；Y3E10：33.14mg/ml；Y5G11：29.93mg/ml；K3A3：32.24mg/ml，K7H3：29.44mg/ml。分别用rSAG1(包被浓度为0.2ug/孔)和弓形虫速殖子抗原(包被浓度为2ug/孔)作为包被抗原，用间接ELISA方法测定5株单克隆抗体(腹水)的效价。5株单抗均能与重组和天然SAG1抗原发生特异性反应，P/N均大于2.1。
间接ELISA测定5株单克隆抗体(腹水)效价
直接ELISA测定标记单抗效价
序列表
<110>(陈晓光，李华)
<120>(一种基于重组抗原的弓形虫检测试剂盒)
<140>
<141>
<160>2
<210>1
<211>695
<212>cDNA
<213>刚地弓形虫(Toxoplasma gondii)
<220>
<221>CDS
<222>(1)......(695)
<220>
<221>mutation
<222>(1)......(6)，(15)，(693)......(695)
<400>1
ATGGGTTTCA CTCTTAAGTG CCCTAAAACA GCGCTCACAG AGCCTCCCAC TCTTGCGTAC 60
TCACCCAACA GGCAAATCTG CCCAGCGGGT ACTACAAGTA GCTGTACATC AAAGGCTGTA 120
ACATTGAGCT CCTTGATTCC TGAAGCAGAA GATAGCTGGT GGACGGGGGA TTCTGCTAGT 180
CTCGACACGG CAGGCATCAA ACTCACAGTT CCAATCGAGA AGTTCCCCGT GACAACGCAG 240
ACGTTTGTGG TCGGTTGCAT CAAGGGAGAC GACGCACAGA GTTGTATGGT CACAGTGACA 300
GTACAAGCCA GAGCCTCATC GGTCGTCAAT AATGTCGCAA GGTGCTCCTA CGGTGCAGAC 360
AGCACTCTTG GTCCTGTCAA GTTGTCTGCG GAAGGACCCA CTACAATGAC CCTCGTGTGC 420
GGGAAAGATG GAGTCAAAGT TCCTCAAGAC AACAATCAGT ACTGTTCCGG GACGACGCTG 480
ACTGGTTGCA ACGAGAAATC GTTCAAAGAT ATTTTGCCAA AATTAACTGA GAACCCGTGG 540
CAGGGTAACG CTTCGAGTGA TAAGGGTGCC ACGCTAACGA TCAAGAAGGA AGCATTTCCA 600
GCCGAGTCAA AAAGCGTCAT TATTGGATGC ACAGGGGGAT CGCCTGAGAA GCATCACTGT 660
ACCGTGAAAC TGGAGTTTGC CGGGGCTGCA GGTTAA 696
<210>2
<211>231
<212>PRT
<213>刚地弓形虫(Toxoplasma gondii)
<220>
<221>CDS
<222>(1)......(231)
<220>
<221>MUTAGEN
<222>(1)......(2)，(5)，(231)
<400>2
Met Gly Phe Thr Leu Lys Cys Pro Lys Thr Ala Leu Thr Glu Pro
1 5 10 15
Pro Thr Leu Ala Tyr Ser Pro Asn Arg Gln Ile Cys Pro Ala Gly
20 25 30
Thr Thr Ser Ser Cys Thr Ser Lys Ala Val Thr Leu Ser Ser Leu
35 40 45
Ile Pro Glu Ala Glu Asp Ser Trp Trp Thr Gly Asp Ser Ala Ser
50 55 60
Leu Asp Thr Ala Gly lle Lys Leu Thr Val Pro Ile Glu Lys Phe
65 70 75
Pro Val Thr Thr Gln Thr Phe Val Val Gly Cys Ile Lys Gly Asp
80 85 90
Asp Ala Gln Ser Cys Met Val Thr Val Thr Val Gln Ala Arg Ala
95 100 105
Ser Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr Gly Ala Asp
110 115 120
Ser Thr Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro Thr Thr
125 130 135
Met Thr Leu Val Cys Gly Lys Asp Gly Val Lys Val Pro Gln Asp
140 145 150
Asn Asn Gln Tyr Cys Ser Gly Thr Thr Leu Thr Gly Cys Asn Glu
155 160 165
Lys Ser Phe Lys Asp Ile Leu Pro Lys Leu Thr Glu Asn Pro Trp
170 175 180
Gln Gly Asn Ala Ser Ser Asp Lys Gly Ala Thr Leu Thr Ile Lys
185 190 195
Lys Glu Ala Phe Pro Ala Glu Ser Lys Ser Val Ile Ile Gly Cys
200 205 210
Thr Gly Gly Ser Pro Glu Lys His His Cys Thr Val Lys Leu Glu
215 220 225
Phe Ala Gly Ala Ala Gly
230 231
Claims (4)
1.一种重组弓形虫主要表面抗原蛋白，所述的重组弓形虫主要表面抗原蛋白的氨基酸序列如下：
Met Gly Phe Thr Leu Lys Cys Pro Lys Thr Ala Leu Thr Glu Pro 15
Pro Thr Leu Ala Tyr Ser Pro Asn Arg Gln Ile Cys Pro Ala Gly 30
Thr Thr Ser Ser Cys Thr Ser Lys Ala Val Thr Leu Ser Ser Leu 45
Ile Pro Glu Ala Glu Asp Ser Trp Trp Thr Gly Asp Ser Ala Ser 60
Leu Asp Thr Ala Gly Ile Lys Leu Thr Val Pro Ile Glu Lys Phe 75
Pro Val Thr Thr Gln Thr Phe Val Val Gly Cys Ile Lys Gly Asp 90
Asp Ala Gln Ser Cys Met Val Thr Val Thr Val Gln Ala Arg Ala 105
Ser Ser Val Val Asn Asn Val Ala Arg Cys Ser Tyr Gly Ala Asp 120
Ser Thr Leu Gly Pro Val Lys Leu Ser Ala Glu Gly Pro Thr Thr 135
Met Thr Leu Val Cys Gly Lys Asp Gly Val Lys Val Pro Gln Asp 150
Asn Asn Gln Tyr Cys Ser Gly Thr Thr Leu Thr Gly Cys Asn Glu 165
Lys Ser Phe Lys Asp Ile Leu Pro Lys Leu Thr Glu Asn Pro Trp 180
Gln Gly Asn Ala Ser Ser Asp Lys Gly Ala Thr Leu Thr Ile Lys 195
Lys Glu Ala Phe Pro Ala Glu Ser Lys Ser Val Ile Ile Gly Cys 210
Thr Gly Gly Ser Pro Glu Lys His His Cys Thr Val Lys Leu Glu 225
Phe Ala Gly Ala Ala Gly 231。
2.如权利要求1所限定之蛋白编码基因的表达载体，即表达载体pET32a(+)-tSAG1。
3.一种如权利要求2所限定之表达载体PET32a(+)-tSAG1的转化菌株，即工程菌pET32a(+)-tSAG1/BL21。
4.一种用于检测和/或诊断弓形虫的试剂盒，该试剂盒包含如权利要求1所限定的蛋白，其试剂盒是ELISA试剂盒和/或胶体金试剂盒。
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| CN102323420A (zh) * | 2011-09-19 | 2012-01-18 | 厦门大学附属中山医院 | 弓形虫总抗体免疫印迹试剂盒及其制备方法 |
| CN102827292B (zh) * | 2012-09-19 | 2013-09-11 | 南京医科大学第二附属医院 | 一种融合蛋白TgMEP及其制备方法和应用 |
| CN104965086A (zh) * | 2015-05-21 | 2015-10-07 | 华南农业大学 | 一种犬弓形虫抗体间接elisa检测试剂盒 |
| CN107356762A (zh) * | 2016-05-10 | 2017-11-17 | 吉林大学 | 检测猪弓形虫病循环抗原免疫胶体金试纸条及制备方法 |
| CN108265070B (zh) * | 2016-12-30 | 2022-07-26 | 中国农业科学院上海兽医研究所 | 一种特异性的检测弓形虫的方法 |
| CN110655575B (zh) * | 2019-10-14 | 2021-01-08 | 安徽医科大学 | 一种弓形虫Actin蛋白的表位多克隆抗体及其应用 |
| CN113603760B (zh) * | 2021-09-12 | 2023-12-22 | 南京珀尔泰生物技术有限公司 | 一种人源弓形虫重组抗原蛋白的制备方法 |
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| CN1450087A (zh) * | 2003-05-09 | 2003-10-22 | 李越希 | 重组弓形虫融合蛋白抗原及其制备方法、应用 |
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| CN1450087A (zh) * | 2003-05-09 | 2003-10-22 | 李越希 | 重组弓形虫融合蛋白抗原及其制备方法、应用 |
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