CN110221065B - 一种禽滑液囊支原体间接elisa检测试剂盒 - Google Patents
一种禽滑液囊支原体间接elisa检测试剂盒 Download PDFInfo
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- CN110221065B CN110221065B CN201910449366.XA CN201910449366A CN110221065B CN 110221065 B CN110221065 B CN 110221065B CN 201910449366 A CN201910449366 A CN 201910449366A CN 110221065 B CN110221065 B CN 110221065B
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Abstract
本发明提供了一种禽滑液囊支原体间接ELISA检测试剂盒，所述试剂盒以一株禽滑液囊支原体宁夏分离株经重叠PCR扩增出的主要膜抗原MSPB重组蛋白为包被抗原。本发明的试剂盒能够快速、特异、有效地检测禽滑液囊支原体，以监测鸡群中滑液囊支原体病的流行情况。
Description
技术领域
本发明属于动物疫病病原快速诊断领域，尤其是一种禽滑液囊支原体快速血清学诊断方法。
背景技术
禽滑液囊支原体（（Mycoplasma synoviae，MS）是感染禽类和鸟类的一种重要的病原微生物，1954 年Olson 和Wills在美国于鸡体内首次发现。禽滑液囊支原体是一种能够导致禽类和鸟类发生多种疾病，且感染率极高的重要病原微生物，可以导致禽、鸟类发生关节炎、滑液囊炎和腱鞘炎、呼吸道和气囊感染等多种疾病，临床上多见禽滑液囊支原体与各种病毒、细菌、寄生虫的混合感染。1980年9月李跃庭在我国广西梧州的一批 70-85日龄表现跛行、关节肿大症状的雏鸡中首次分离得到禽滑液囊支原体，初次证实了该病在我国的存在。随着养殖业的扩大和贸易往来日益频繁，禽滑液囊支原体在我国的传播速度越来越快，流行范围也越来越广。宿主一旦感染禽滑液囊支原体便终生带毒，不能根除，所以控制此病最好的措施就是做好预防。
作为疫病综合防控的重要环节，快速、特异的诊断技术是必不可少的。要做好禽滑液囊支原体病的防控，需要用高效的检测方法对其流行病学进行调查。ELISA作为进行禽滑液囊支原体流行病学调查的重要检测方法，以其快速、便捷的优点被广泛用于禽滑液囊支原体的检测与诊断，但是已存在的ELISA试剂盒在检测禽滑液囊支原体时会出现一定的假阳性，并且我国尚无针对禽滑液囊支原体的商品化ELISA试剂盒。因此，为了尽早控制该病在我国的流行，本领域亟需快速、高效、准确的滑液囊支原体血清学诊断方法，进而开展滑液囊支原体感染的诊断、治疗与防控研究。
禽滑液囊支原体的感染宿主范围较广，在各种禽类和鸟类中普遍存在，自年1954年以来相继在已经在鸡、鹅、鸽、鸭、火鸡及其它鸟类等多种动物中分离到禽滑液囊支原体，而从不同宿主中分离到的禽滑液囊支原体在致病性、基因序列和一些主要蛋白的编码都存在差异。
发明内容
本发明在国内首次建立间接ELISA方法对禽滑液囊支原体进行抗体检测。能够对禽滑液囊支原体抗体进行快速有效地检测，是一种快速、简便的间接ELILSA检测方法。
为实现上述目的，本发明采用如下技术方案：
本发明的第一方面： MSPB蛋白的制备方法，以禽滑液囊支原体宁夏分离株NX-7的基因组作为模板，利用设计合成的四对特异性引物进行重叠PCR扩增，回收扩增的PCR产物；
所述引物对包括：
MSPB-F1 5' GCCGGATCCATGAAAAATAA 3'
MSPB-R1 5' GATTCATCTGTCCATTGGAATG 3'
MSPB-F2 5' CATTCCAATGGACAGATGAATC 3'
MSPB-R2 5' ATTTTTCTCTAGCTTTGGTCCAAG 3'
MSPB-F3 5' CTTGGACCAAAGCTAGAGAAAAAT 3'
MSPB-R3 5' ATAAACCCGTCCCAGTATAGTGT 3'
MSPB-F4 5' ACACTATACTGGGACGGGTTTAT 3'
MSPB-R4 5' CTTCTCGAGTTAATCTTCGTGAGT 3'
将扩增的PCR产物、pET-30a表达载体先用BamHI与XhoI酶切后再连接，构建重组表达质粒；将重组表达质粒转化BL 21(DE3)感受态细胞，并采用异丙基硫代半乳糖苷诱导表达，将获得的表达产物进行纯化，即得所述MSPB蛋白。
作为优选，所述禽滑液囊支原体MSPB蛋白重组抗原的包被量为1μg/ml/孔。
进一步的，所述间接ELISA检测试剂盒还包含酶标抗体、样品稀释液、洗涤液、阴性对照血清、阳性对照血清、底物显色液和终止液。
作为优选，所述试剂盒还包括封闭液，所述封闭液为1%脱脂奶粉。
作为优选，所述试剂盒还包括包被缓冲液，所述包被缓冲液为0.01M pH 9.6的碳酸盐缓冲液。
作为优选，所述试剂盒还包括血清稀释液，所述血清稀释液为1%脱脂奶粉。
作为优选，所述试剂盒酶标抗体为辣根过氧化物酶标羊抗鸡抗体。
上述间接ELISA检测试剂盒在禽滑液囊支原体的流行病学调查中的应用也是本发明的保护范围；
所述禽滑液囊支原体为宁夏分离株NX-7。
本发明的第二方面，提供一种禽滑液囊支原体抗体的间接ELISA检测方法，包括以下步骤：（1）包被以宁夏本地株NX-7 MSPB重组蛋白作为包被抗原，将抗原稀释后加入到96孔板中，37℃孵育1 h后4℃过夜孵育，采用PBST进行洗涤。
（2）封闭：采用PBST稀释的1%脱脂奶粉100μL/孔进行封闭，37℃条件下孵育60 min后甩干，用PBST洗涤；
（3）血清作用条件：每孔加入待检血清稀释液，37℃条件下孵育60 min后甩干，用PBST洗涤；
（4）二抗孵育条件：将酶标羊抗鸡IgG用按1:3000稀释，每孔加100 μL/孔，37℃条件下作用60 min后甩干，用PBST洗涤；
（5）底物显色：底物显色液100 μL/孔，37℃条件下避光作用20 min；
（6）终止反应：每孔加终止液50 μL终止显色反应采用酶标仪吸光度450 nm下读取数据；
（7）阴阳性临界值的确定：根据公式阴阳性临界值=阴性样本OD450平均值标准偏差3SD，得到阴阳性临界值；当在OD450在0.324以上，判定为阳性。
步骤（1）中，所述禽滑液囊支原体MSPB重组蛋白由如下方法制备而成：
以禽滑液囊支原体宁夏分离株NX-7的全基因组作为模板，利用设计合成的特异性引物对MSPB基因进行重叠PCR扩增，回收扩增的PCR产物，将扩增的PCR产物、pET-30a表达载体先用BamHI与XhoI酶切后再连接，构建重组表达质粒；将重组表达质粒转化BL 21(DE3)感受态细胞，并采用异丙基硫代半乳糖苷诱导表达，将获得的表达产物进行纯化，即得所述MSPB蛋白,
所述引物对包括：
MSPB-F1 5' GCCGGATCCATGAAAAATAA 3'
MSPB-R1 5' GATTCATCTGTCCATTGGAATG 3'
MSPB-F2 5' CATTCCAATGGACAGATGAATC 3'
MSPB-R2 5' ATTTTTCTCTAGCTTTGGTCCAAG 3'
MSPB-F3 5' CTTGGACCAAAGCTAGAGAAAAAT 3'
MSPB-R3 5' ATAAACCCGTCCCAGTATAGTGT 3'
MSPB-F4 5' ACACTATACTGGGACGGGTTTAT 3'
MSPB-R4 5' CTTCTCGAGTTAATCTTCGTGAGT 3'.
较之现有技术而言，本发明的优点在于：
目前国内尚未有针对禽滑液囊支原体间接ELISA检测方法的研究报道。本发明利用原核表达的禽滑液囊支原体MSPB蛋白作为包被抗原，建立了检测禽滑液囊支原体病血清抗体的间接ELISA方法。
1、本发明以MSPB蛋白作为包被抗原建立间接ELISA检测方法，可以快速检测禽滑液囊支原体抗体，具有良好的特异性。对临床样品进行检测，结果表明本方法可以作为禽滑液囊支原体抗体检测的一种方法而运用到生产实际中。
2、本发明的检测方法还具有检测快速、便捷的优点。
附图说明
图1是MSPB蛋白纯化的SDS-PAGE分析。
图2是MSPB蛋白的Western blotting分析。
具体实施方式
下面实施例对本发明内容进行详细说明：
MSPB蛋白的制备方法，以禽滑液囊支原体宁夏分离株NX-7的核酸作为模板，利用设计合成的特异性引物进行重叠PCR扩增，回收扩增的PCR产物。
所述产物序列如SEQ ID NO.1所示
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。
在本发明的一个实施方案中，所给出的禽滑液囊支原体间接ELILSA检测方法，包括以下步骤：
（1）包被抗原的制备
以禽滑液囊支原体宁夏分离株NX-7的全基因组作为模板，根据该株禽滑液囊支原体序列中的MSPB蛋白编码区域，设计四对引物：
MSPB-F1 5' GCCGGATCCATGAAAAATAA 3'
MSPB-R1 5' GATTCATCTGTCCATTGGAATG 3'
MSPB-F2 5' CATTCCAATGGACAGATGAATC 3'
MSPB-R2 5' ATTTTTCTCTAGCTTTGGTCCAAG 3'
MSPB-F3 5' CTTGGACCAAAGCTAGAGAAAAAT 3'
MSPB-R3 5' ATAAACCCGTCCCAGTATAGTGT 3'
MSPB-F4 5' ACACTATACTGGGACGGGTTTAT 3'
MSPB-R4 5' CTTCTCGAGTTAATCTTCGTGAGT 3'
用上述引物对MSPB进行重叠 PCR进行扩增，并通过1%凝胶电泳和胶回收的方式纯化，将纯化后MSPB基因的克隆到原核表达载体pET-30a中，构建重组原核表达载体pET30a-MSPB。将重组原核表达载体pET30a-MSPB。转化至感受态细胞中，经0.5 mM IPTG诱导成功表达了重组蛋白。该蛋白主要以包涵体的形式存在，包涵体经过变性和复性，将纯化的禽滑液囊支原体MSPB蛋白作为包被抗原。
因为MSPB蛋白的有一段富含脯氨酸的序列，蛋白的大小较预期大。
（2）以纯化后的禽滑液囊支原体MSPB重组蛋白做包被抗原建立间接ELISA检测方法：
①包被抗原按所需浓度用CBS（pH=9.6）稀释后包被到96孔酶标板中，保鲜膜封口包好，37℃孵育1 h后4℃过夜孵育，采用PBST洗涤三次，每遍3 min。
②封闭：采用PBST稀释的1%脱脂奶粉100 μL/孔进行封闭，37℃条件下孵育60 min后甩干，用PBST洗涤三次，每遍3 min；
③血清作用条件：每孔加入待检血清稀释混合液，37℃条件下孵育60 min后甩干，用PBST洗涤三次，每遍3 min；
④二抗孵育条件：将酶标羊抗鸡IgG用按1:3000稀释，每孔加100μL/孔，37℃条件下作用60 min后甩干，用PBST洗涤三次，每遍3 min；
⑤底物显色：底物显色液100 μL/孔，37℃条件下避光作用20 min；
⑥终止反应：每孔加终止液50 μL终止显色反应采用酶标仪吸光度450nm下读取数据；
⑦阴阳性临界值的确定：根据公式阴阳性临界值=阴性样本OD450平均值标准偏差3SD，得到阴阳性临界值；当在OD450在0.324以上，判定为阳性。
对于该禽滑液囊支原体间接ELISA检测而言，包被抗原的选择非常关键，直接决定检测方法的特异性。为优选包被抗原，本发明在试验过程中选择禽滑液囊支原体基因序列中具有主要膜抗原特征的MSPB蛋白编码区，分别设计引物对，扩增得到基因片段，构建重组表达载体并转化至感受态细胞中，加入IPTG进行诱导表达，将表达产物经过变性和复性后，获得纯化重组蛋白，将获得的重组蛋白作为包被抗原，建立间接ELISA检测试剂盒。应用临床上已确诊的血清样本对不同包被抗原制备的间接ELISA检测试剂盒的特异性进行考察。结果显示，以本发明纯化的禽滑液囊支原体MSPB蛋白作为包被抗原制备的间接ELISA检测试剂盒对已确诊血清样本检测的准确率达100%，抗原的敏感性和特异性较高。
为了使得本领域技术人员能够更加清楚地了解本申请的技术方案，以下将结合具体的实施例详细说明本申请的技术方案。
本发明实施例中所用的未进行具体说明试验材料均为本领域常规的试验材料，均可通过商业渠道购买得到。本发明中所用到的部分试剂及组分如下：
包被缓冲液CBS：称取1.59 g Na2CO3、2.93 g NaHCO3，倒入锥形瓶中，向锥形瓶中加入400mL蒸馏水使其彻底溶解混匀，调节pH至9.6，定容至500mL，置于４℃储存。
PBS缓冲液：8.5 g NaCl、0.2 g KCl、0.27 g KH2PO4、1.42 g Na2HPO4，于800 mL的蒸馏水中溶解，调pH至7.4，定容至1000 mL，置于４℃储存。
PBST洗液：1000 mL PBS，加入0.05 mL的Tween20，充分混匀，置4℃保存。
封闭液：将1g脱脂奶粉溶解于100mL PBST稀释液中，短期保存于4℃，长期保存-20℃。
终止液：将浓硫酸以体积比1:5的比例加入超纯水中，混匀冷却至室温即为终止液(浓度为2 M H2SO4)。
本发明实施例中未注明具体实验条件和方法的，通常按照常规条件。
实施例1：包被抗原的制备：
1.1 特异性引物设计和合成：根据该禽滑液囊支原体NX-7全基因序列中的MSPB区域设计四对引物，引物两端分别设有BamHI和XhoI酶切位点，预计可以扩增出1398bp大小的基因片段。
MSPB-F1 5' GCCGGATCCATGAAAAATAA 3'
MSPB-R1 5' GATTCATCTGTCCATTGGAATG 3'
MSPB-F2 5' CATTCCAATGGACAGATGAATC 3'
MSPB-R2 5' ATTTTTCTCTAGCTTTGGTCCAAG 3'
MSPB-F3 5' CTTGGACCAAAGCTAGAGAAAAAT 3'
MSPB-R3 5' ATAAACCCGTCCCAGTATAGTGT 3'
MSPB-F4 5' ACACTATACTGGGACGGGTTTAT 3'
MSPB-R4 5' CTTCTCGAGTTAATCTTCGTGAGT 3'
1.2 pET30a-MSPB原核表达载体的构建：
采用特异性上下游引物通过Overlap扩增出MSPB基因片段，将纯化后的片段与pET-30a载体，用BamHI和XhoI酶限制性内切酶（购自NEB公司）分别对重组质粒进行双酶切，用T4连接酶（购自NEB公司）连接两者酶切产物。将连接产物转化到BL 21（DE3）感受态细胞中，挑取阳性菌落摇菌提取重组质粒，双酶切鉴定阳性克隆，阳性克隆送至生工生物工程（上海）股份有限公司测序。
挑取单个阳性克隆菌落鉴定结果表明能扩增到1500左右的目的条带，与预计的MSPB基因片段大小一致。挑取单个阳性克隆菌落进行增菌培养后提质粒，用限制性内切酶和进行酶切鉴定，酶切条带正确。阳性克隆测序结果显示。基因插入位置、插入方向和阅读框正确，结果表明重组载体pET30a-MSPB构建成功。
1.3 重组蛋白pET30a-MSPB诱导表达与鉴定：
取上述含有重组质粒的BL 21(DE3)菌，于含卡那霉素的 LB 固体平板中进行划线，37℃倒置培养 12 h；挑取单个菌落，转接于2 mL含抗生素的 LB 液体培养基中，37℃，220 r/min 振荡培养12 h；取过夜培养的菌液，按照1:100的比例转接至含抗生素的 LB 液体培养基中，37℃， 220 min振摇 2 h左右，测定菌液 OD 600 达到0.7左右时，加入0.5 mM的IPTG诱导表达5 h；4℃，12000 r/min 离心1 min，弃掉上清收集菌体沉淀；将沉淀用PBS缓冲液悬浮，-20℃保存备用。注意：以 BL 21（DE3）pET-30a 空质粒诱导表达作为对照组。取10 µL 上述PBS悬浮的菌液于EP管中，随后加入10 µL的2×蛋白上样缓冲液，混匀，煮沸5min，进行SDS-PAGE电泳，鉴定重组蛋白是否表达成功。取5 mL诱导表达产物，10000 r/min离心，PBS 洗涤，反复三次，最终以250 µL悬浮，后超声破碎，条件为：5 s，5 s，20 min，冰浴；12000 r/min离心1 min，分别收集沉淀和裂解上清，沉淀以一定量的PBS悬浮，取10 µL上述PBS悬浮的菌液于EP管中，随后加入10 µL的2×蛋白上样缓冲液，混匀，煮沸5 min，SDS-PAGE电泳鉴定重组蛋白表达形式。
1.4 蛋白纯化：
按照1.3中蛋白诱导表达条件进行大量诱导培养。取包涵体参照试剂盒说明书进行蛋白纯化，去回收蛋白质样品处理后，进行SDS-PAGE鉴定蛋白纯度。
SDS-PAGE分析显示MSPB重组蛋白在BL 21（DE3）感受态细胞中成功表达，主要以包涵体的形式存在，蛋白分子量大小为70 KD，大小较预期的重组MSPB大（参见图1）。
1.5 Western Bloting分析：将纯化后的重组蛋白按照Western Bloting方法进行操作，最后用底物显色剂进行显色。结果显示：与在纯化蛋白大小一致的位置出现了一条免疫条带，表明重组蛋白MSPB能与阳性血清中抗体发生特异性抗原抗体反应(参见图2)。
实施例2：间接ELISA检测禽滑液囊支原体抗体
取实施例1纯化重组蛋白MSPB做包被抗原建立间接ELISA方法，采用方阵法摸索ELISA最佳蛋白包被浓度和血清稀释浓度，以及ELISA最佳反应条件。最终确定条件如下：
2.1包被：采用碳酸盐缓冲液（pH=9.6）将抗原稀释，取100μL稀释液包被到96孔酶标板，保鲜膜，封口包好，37℃孵育1 h后4℃过夜，PBST洗涤三次，每次3 min。
2.2 封闭：采用PBST稀释的1%脱脂奶粉37℃孵育1 h后4℃过夜后，100 μL /孔进行封闭，37℃条件下孵育60 min后甩干，PBST洗涤三次，每次3 min。
2.3 血清作用条件：每孔加入待检血清稀释混合液，37℃条件下孵育60 min后甩干，用PBST洗涤三次，每次3 min；
2.4 二抗孵育条件：将酶标羊抗鸡IgG用按1:3000稀释，每孔加100 μL/孔，37℃条件下作用60 min后甩干，PBST洗涤三次，每次3 min；
2.5 底物显色：底物显色液100 μL/孔，37℃条件下避光作用20 min；
2.6 终止反应：每孔加终止液50 μL终止显色反应；
2.7采用酶标仪吸光度450 nm下读取数据；
3、结果判定标准：
阴阳性临界值=阴性样本OD450平均值+标准偏差3SD，得到阴阳性临界值；当在OD450在0.324以上，判定为阳性。
4、特异性：经交叉反应试验，本发明建立的方法只对禽滑液囊支原体阳性血清反应呈阳性，说明该方法具有很好的特异性。
以上所述仅为本申请的优选实施例而已，并不用于限制本申请，对于本领域的技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本申请的保护范围之内。
序列表
<110> 宁夏大学
宁夏晓鸣农牧股份有限公司
<120> 一种禽滑液囊支原体间接ELISA检测试剂盒
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1398
<212> DNA
<213> Mycoplasma synoviae
<400> 1
atgaaaaata aaaaaattaa attactatta gcagctagtg cagtggccat tgctcctgct 60
gttatagcaa tttcatgtgg tgatcaaact ccagcacctg ctccaacacc tggaaaccca 120
aatactaata atcctcaaaa cccaaatcca ggaaacccag aaaacccagg tactgataat 180
actcaaaact caaatccagg aaacccaggg ggtggtacag ttgaccctgt agaggctgct 240
aaaacagaag ctaaaactgc tattgatgct gcagcagaat tatcagattc agttaaagaa 300
gcattaaaaa gacaagttga agcaactaca acagaatctg cagccagaga tttaaaagct 360
aaagcagaag ctcttgtttc agctgtaaaa gcattaagcg gatcggttac tgctgctaaa 420
gcagttaaag atgatgctga atactcaaaa gtaacagata ctcttagaac tgatttagaa 480
gcaaaattaa cagcagcagc atcattacta gaaggtgaaa caaaacttgc aaatctagat 540
gcttcaagta atttagacac aacaaaagct acattagaat ctgcaaaaac agctcttgat 600
gcagcagttg cagcagttaa accaaatctt gaattccaaa aaacaaaaac tagcgctgca 660
gctaaagtta cagaacttga atctcttgtt aactcagctt taaaagctga actagaaaga 720
caagttaatg aattaacaaa agaacaagct gctcaagcta caacaatgtt agttaaccta 780
acatctttaa aagaatcact agaatctctt caaactttag tatcagatgg cctaaaaatg 840
caagtggatt atccacaaaa atactacgat gctgacaata aagaagcatt cgatgcagcg 900
ctacttaaag cttcatcagt atttccagca ttccaatgga cagatgaatc aattatggtt 960
cctgctccag aaggagacgc gcttccaaac cctagagctt ggaccaaagc tagagaaaaa 1020
tcagaattca aactacaaaa cttcttaatg gctccagctc aagcagctgc tccaacacca 1080
acacagcctg cagcagttga atcaacttta gctacagtta gacttgcaaa tggtgaatca 1140
gtttcagcag atggcacagc atcaagagaa gcacaaacaa ctcctgattt agcatcaact 1200
gcttcttatc taaaaacttt agaaacagag cttaaagcgc aaactgcagc actaaatggt 1260
gatactacaa ctaataagac agcatactac aaaccagtta acggtcgtac actatactgg 1320
gacgggttta tgcctaaaat agttgtagaa ggttatgaag cagatggtga aggaaatggt 1380
aaagcaactc acgaagat 1398
<210> 2
<211> 466
<212> PRT
<213> 滑液囊支原体(Mycoplasma synoviae)
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Pro Ala Pro Thr Pro Gly Asn Pro Asn Thr Asn Asn Pro Gln Asn Pro
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Asn Pro Gly Asn Pro Glu Asn Pro Gly Thr Asp Asn Thr Gln Asn Ser
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Asn Pro Gly Asn Pro Gly Gly Gly Thr Val Asp Pro Val Glu Ala Ala
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Lys Thr Glu Ala Lys Thr Ala Ile Asp Ala Ala Ala Glu Leu Ser Asp
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Ser Val Lys Glu Ala Leu Lys Arg Gln Val Glu Ala Thr Thr Thr Glu
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Ser Ala Ala Arg Asp Leu Lys Ala Lys Ala Glu Ala Leu Val Ser Ala
115 120 125
Val Lys Ala Leu Ser Gly Ser Val Thr Ala Ala Lys Ala Val Lys Asp
130 135 140
Asp Ala Glu Tyr Ser Lys Val Thr Asp Thr Leu Arg Thr Asp Leu Glu
145 150 155 160
Ala Lys Leu Thr Ala Ala Ala Ser Leu Leu Glu Gly Glu Thr Lys Leu
165 170 175
Ala Asn Leu Asp Ala Ser Ser Asn Leu Asp Thr Thr Lys Ala Thr Leu
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Glu Ser Ala Lys Thr Ala Leu Asp Ala Ala Val Ala Ala Val Lys Pro
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Asn Leu Glu Phe Gln Lys Thr Lys Thr Ser Ala Ala Ala Lys Val Thr
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Glu Leu Glu Ser Leu Val Asn Ser Ala Leu Lys Ala Glu Leu Glu Arg
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Gln Val Asn Glu Leu Thr Lys Glu Gln Ala Ala Gln Ala Thr Thr Met
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Leu Val Asn Leu Thr Ser Leu Lys Glu Ser Leu Glu Ser Leu Gln Thr
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Leu Val Ser Asp Gly Leu Lys Met Gln Val Asp Tyr Pro Gln Lys Tyr
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Tyr Asp Ala Asp Asn Lys Glu Ala Phe Asp Ala Ala Leu Leu Lys Ala
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Ser Ser Val Phe Pro Ala Phe Gln Trp Thr Asp Glu Ser Ile Met Val
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Pro Ala Pro Glu Gly Asp Ala Leu Pro Asn Pro Arg Ala Trp Thr Lys
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Ala Arg Glu Lys Ser Glu Phe Lys Leu Gln Asn Phe Leu Met Ala Pro
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Ala Gln Ala Ala Ala Pro Thr Pro Thr Gln Pro Ala Ala Val Glu Ser
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Thr Leu Ala Thr Val Arg Leu Ala Asn Gly Glu Ser Val Ser Ala Asp
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Gly Thr Ala Ser Arg Glu Ala Gln Thr Thr Pro Asp Leu Ala Ser Thr
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Ala Ser Tyr Leu Lys Thr Leu Glu Thr Glu Leu Lys Ala Gln Thr Ala
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Ala Leu Asn Gly Asp Thr Thr Thr Asn Lys Thr Ala Tyr Tyr Lys Pro
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Val Asn Gly Arg Thr Leu Tyr Trp Asp Gly Phe Met Pro Lys Ile Val
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Val Glu Gly Tyr Glu Ala Asp Gly Glu Gly Asn Gly Lys Ala Thr His
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Glu Asp
465
Claims (2)
1.一种禽滑液囊支原体抗体间接ELISA检测试剂盒，其特征在于：所述试剂盒以MSPB蛋白作为ELISA酶标板的包被抗原；所述MSPB蛋白的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的禽滑液囊支原体抗体间接ELISA检测试剂盒，其特征在于包括：包被禽滑液囊支原体MSPB蛋白的酶标板、血清标准液、酶标记抗体、TMB底物显色液、样品稀释液、洗涤液和终止反应液。
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