CN111349637A - 山羊支原体山羊肺炎亚种特异性蛋白及间接elisa试剂盒 - Google Patents

山羊支原体山羊肺炎亚种特异性蛋白及间接elisa试剂盒 Download PDF

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CN111349637A
CN111349637A CN202010178955.1A CN202010178955A CN111349637A CN 111349637 A CN111349637 A CN 111349637A CN 202010178955 A CN202010178955 A CN 202010178955A CN 111349637 A CN111349637 A CN 111349637A
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储岳峰
吴娅琴
颜新敏
郝华芳
陈胜利
马丽娜
刘永生
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Abstract

本发明公开了一种山羊支原体山羊肺炎亚种特异性蛋白87.1及间接ELISA试剂盒，所述蛋白87.1具有如SEQ ID NO.1所示的核酸序列，所述山羊支原体山羊肺炎亚种特异性蛋白87.1制备的间接ELISA抗体检测试剂盒包括抗原蛋白87.1包被的酶标板、样品稀释液、阳性血清、阴性血清、洗涤液、封闭液、兔抗羊二抗、底物显色液、终止液。抗原蛋白87.1的包被量为625ng/孔，阳性血清和阴性血清的稀释度均为1/100。本发明提供的Mccp特异性蛋白87.1制备的间接ELISA试剂盒具有良好特异性，与Mccp阳性血清反应，且不与Mmc、Mcc阳性血清反应。

Description

山羊支原体山羊肺炎亚种特异性蛋白及间接ELISA试剂盒
技术领域
本发明属于生物技术领域，尤其涉及一种山羊支原体山羊肺炎亚种特异性蛋白及间接ELISA试剂盒。
背景技术
山羊传染性胸膜肺炎(contagious caprine pleuropneumonia,CCPP)是由山羊支原体山羊肺炎亚种(Mycoplasma capricolumsubsp.capripneumoniae,Mccp) 引起山羊的一类严重的呼吸道疾病，被世界动物组织(OIE)列为法定报告动物传染病之一。Mccp属于丝状支原体簇，该簇成员具有高度的血清学和基因组学相似性，除Mccp外，还包括：丝状支原体丝状亚种(M.mycoides subsp. mycoides,Mmm)，丝状支原体山羊亚种(M.mycoidessubsp.capri,Mmc)，山羊支原体山羊亚种(M.capricolumsubsp.Capricolum,Mcc)，李奇氏支原体(M.Leachii, Ml)，其中Mmm和Ml是牛源支原体，Mmc、Mcc以及Mccp为羊源支原体，均能引起羊肺炎。与Mccp亲缘关系最近的是Mcc和Ml，易与Mccp在血清学检测中产生交叉反应。决定血清学诊断方法特异性和敏感性的关键是抗原，主要通过两个方向挖掘Mccp特异性抗原，一方面，随着基因组测序技术的快速发展，10株Mccp菌株的完整基因组序列已被解析。有报道指出26个潜在毒力因子，并表明其能被宿主免疫系统识别，可作为潜在的血清学诊断靶标。另一方面，从蛋白水平上进行，通过IgG免疫印迹试验，发现实验感染血清和25份田间血清均在44、40和23Kd处出现条带，大多数血清中观察到108、70 和62Kd条带；又通过2D电泳质谱分析以及western blot比较蛋白组学方法得到丝状支原体山羊亚种6个候选诊断蛋白，这些蛋白包括丙酮酸脱氢酶复合物、磷酸甘氨酸激酶、吡啶-核苷磷酸酶、30S核糖体蛋白S6、二磷酸三乙酯和酰胺乙酰转移酶。这些都可作为Mccp特异性抗原候选靶标，但是目前还没有报道指出以这些候选抗原是否能够进一步加以应用。
发明内容
针对上述背景技术中指出的不足，本发明提供了一种山羊支原体山羊肺炎亚种特异性蛋白及间接ELISA试剂盒，能与Mccp阳性血清反应且不与Mmc、 Mcc阳性血清反应，具有良好的特异性。
为实现上述目的，本发明采用的技术方案是：
一种山羊支原体山羊肺炎亚种特异性蛋白，其表达序列为蛋白87.1序列，所述蛋白87.1具有如SEQ ID NO.1所示的核酸序列。
本发明进一步提供了一种山羊支原体山羊肺炎亚种特异性蛋白在制备山羊支原体山羊肺炎亚种间接ELISA抗体检测试剂盒中的应用。
本发明进一步提供了上述山羊支原体山羊肺炎亚种特异性蛋白制备的间接ELISA抗体检测试剂盒，该试剂盒包括抗原蛋白87.1包被的酶标板、样品稀释液、阳性血清、阴性血清、洗涤液、封闭液、兔抗羊二抗、底物显色液、终止液。
优选地，所述抗原蛋白87.1的包被量为625ng/孔，阳性血清和阴性血清的稀释度均为1/100。
相比于现有技术的缺点和不足，本发明具有以下有益效果：
本发明提供了Mccp特异性蛋白87.1，通过生物信息学分析，得到Mccp 特异性序列87.1，再通过原核表达技术获得重组蛋白87.1，并建立间接ELISA 试剂盒对87.1的特异性以及反应性进行了初步评估，发现87.1与Mccp阳性血清反应，能够反映抗体水平的变化，且不与Mmc、Mcc阳性血清反应。
附图说明
图1是本发明实施例提供的山羊支原体山羊肺炎亚种特异性蛋白87.1的表达形式；图中，M：Protein Marker，1-3为87.1菌液，其中1：菌液超声后上清，2：超声后沉淀，3：未超声菌液；4：pET-30a空载对照。
图2是本发明实施例提供的山羊支原体山羊肺炎亚种特异性蛋白87.1包涵体纯化效果图。图中，C：8M尿素裂解液PH6.3，第1/2/3次洗杂液；D：8M 尿素裂解液PH5.9，第1/2/3次洗杂液；E：8M尿素裂解液PH4.5，第1/2/3/4 次洗脱液。
图3是本发明实施例提供的山羊支原体山羊肺炎亚种特异性蛋白反应原性评估结果。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
1、实验材料
主要试剂：质粒pET-30a，感受态BL21(DE3)，LB，LA，IPTG，QIGEN 树脂，脱脂奶粉，二抗(兔抗羊，sigma)，Mccp抗体检测cELISA试剂盒(IDEXX 公司)，BCA试剂盒，尿素。
主要仪器：摇床，温箱，超声破碎仪，离心机，蛋白电泳仪，酶标仪。血清：采自疫苗/感染实验中不同时间点。
2、序列获得
将Mccp 1601基因组序列(NZ_CP017125.1)与丝状支原体簇其他成员Mmc、 Mcc、Mmm、Ml基因组序列BLAST，得到部分Mccp特有序列，在NCBI进行线上比对，发现序列全部存在于已完成测序10株Mccp基因组序列中。
3、序列分析筛选
通过分子量大小进行初步筛选，再通过线上预测软件对这些序列的结构功能进行预测，包括抗原指数、表面可及性、亲水指数、细胞定位、跨膜区以及信号肽。选择抗原指数高、亲水性好、表面可及性高、无信号肽、跨膜区少的序列更容易通过原核表达技术获得重组蛋白的序列，最终确定87.1 (NZ_CP017125.1，complement(740269..740802))序列作为表达序列，87.1具有如SEQ ID NO.1所示的核酸序列。
4、pET-30a-87.1质粒构建
在支原体中，密码子TGA不作终止密码子使用，而是翻译为色氨酸，所以先将序列中TGA密码子用TGG替代，然后经原核表达密码子优化系统进行优化，N、C端分别插入BamHI、XhoI酶切位点，N端再插入His标签，送公司合成序列，选择pET-30a作为表达载体，构建表达载体pET-30a-87.1，并进行酶切以及测序验证。
5、87.1蛋白原核表达
将构建好的pET-30a-87.1质粒转入感受态BL21(DE3)进行表达，转化步骤同说明书，挑取卡纳抗性平板上单菌落接入LB，进行诱导表达，经过不同诱导温度(16/26/37℃)、诱导时间(4/8/12/16h)以及IPTG浓度(0.1/0.5/1mM) 的条件摸索，结果表明所有的条件下，87.1表达形式均以包涵体为主，大小约为26Kd，如图1所示，虽有少量的上清表达但纯化效果不佳，根据表达量，最终选择37℃，4h，1mM IPTG的作为诱导条件，以不同PH洗脱液进行洗脱，并进行蛋白电泳验证，BCA定量。最终成功纯化获得87.1包涵体蛋白，结果如图2所示。
6、间接ELISA体系建立
通过对抗原抗体稀释度以及封闭液条件进行优化，iELISA反应条件为：抗原625ng/孔，每孔100μl，以0.05M PH9.6的碳酸盐包被缓冲液进行稀释，37℃温箱孵育1h，再4℃过夜；0.01M PBST洗板3次，封闭液37℃温箱封闭2h； 0.01M PBST洗板3次，阳性血清也用样品稀释液进行100倍稀释，每孔100μl， 37℃孵育1h；0.01M PBST洗板4次，二抗用样品稀释液5000倍稀释，100μl 每孔，37℃孵育1h；0.01M PBST洗板4次,每孔加入100μl TMB显色，37℃孵育10min；100μl 2M H2SO4每孔终止反应；酶标仪上读取OD450 nm值。
7、特异性以及反应原性评估
取疫苗实验期间不同时间点的血清进行Mccp抗体检测，8只羊5个不同时间点的血清，包括免疫0天，免疫3周，攻毒0天/免疫1月，攻毒2周，攻毒 3周，共计40份。又因为丝状支原体簇中，羊源支原体Mcc和Mmc与Mccp 在血清学检测方法中易出现交叉反应，所以检测了Mmc、Mcc阳性血清各2份，评估87.1特异性。
反应原性评估结果如图3所示，经初步验证，87.1可检测出Mccp抗体，并能够反应出不同时间点的抗体水平变化，具有反应原性；且不与Mmc、Mcc 阳性血清反应，如表1所示，再次证明87.1蛋白具有特异性。
表1 87.1特异性评估
Figure RE-GDA0002485498020000051
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (4)

1.一种山羊支原体山羊肺炎亚种特异性蛋白87.1，其特征在于，所述蛋白87.1具有如SEQ ID NO.1所示的核酸序列。
2.一种山羊支原体山羊肺炎亚种特异性蛋白87.1在制备山羊支原体山羊肺炎亚种间接ELISA抗体检测试剂盒中的应用。
3.一种如权利要求1所述的山羊支原体山羊肺炎亚种特异性蛋白87.1制备的间接ELISA抗体检测试剂盒，其特征在于，所述试剂盒包括抗原蛋白87.1包被的酶标板、样品稀释液、阳性血清、阴性血清、洗涤液、封闭液、兔抗羊二抗、底物显色液、终止液。
4.如权利要求3所述的间接ELISA抗体检测试剂盒，其特征在于，所述抗原蛋白87.1的包被量为625ng/孔，阳性血清和阴性血清的稀释度均为1/100。
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CN116536284B (zh) * 2023-04-26 2024-07-05 中国农业科学院兰州兽医研究所 Mccp pdhC单克隆抗体、杂交瘤细胞株及试剂盒

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CHU Y. 等: ""Mycoplasma capricolum subsp. capripneumoniae M1601 chromosome, complete genome"" *
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吴娅琴: "虹鳟源鲁氏耶尔森菌主要生物学特性及减毒活疫苗研究" *
王慧等: "绵羊肺炎支原体DnaK B细胞抗原表位预测及其蛋白结构分析" *

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Publication number Priority date Publication date Assignee Title
CN116536284A (zh) * 2023-04-26 2023-08-04 中国农业科学院兰州兽医研究所 Mccp pdhC单克隆抗体、杂交瘤细胞株及试剂盒
CN116536284B (zh) * 2023-04-26 2024-07-05 中国农业科学院兰州兽医研究所 Mccp pdhC单克隆抗体、杂交瘤细胞株及试剂盒

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