CN113176406B - 一种PCV4 cap单克隆抗体及其双单抗夹心ELISA检测试剂 - Google Patents
一种PCV4 cap单克隆抗体及其双单抗夹心ELISA检测试剂 Download PDFInfo
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- CN113176406B CN113176406B CN202110458511.8A CN202110458511A CN113176406B CN 113176406 B CN113176406 B CN 113176406B CN 202110458511 A CN202110458511 A CN 202110458511A CN 113176406 B CN113176406 B CN 113176406B
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Abstract
本发明涉及PCV4cap单克隆抗体及其双单抗夹心ELISA检测试剂，具体公开了抗PCV4cap蛋白的单克隆抗体PCV4‑cap‑6D1，其重链可变区的氨基酸序列和其轻链可变区的氨基酸序列如SEQ ID NO.1‑2所示。还公开了抗PCV4cap蛋白的单克隆抗体PCV4‑cap‑4B5，其重链可变区的氨基酸序列和其轻链可变区的氨基酸序列如SEQ ID NO.3‑4所示。基于公开的单克隆抗体还进一步提供了双抗夹心ELISA检测方法试剂盒。本发明的双抗夹心ELISA检测方法具有快速、稳定、特异性强、灵敏度高的特点，无交叉反应，能够实现猪场中PCV4的流行病学检测。
Description
技术领域
本发明涉及抗体及生物技术检测技术领域，具体涉及单克隆抗体及其双单抗夹心ELISA检测试剂。
背景技术
猪圆环病毒(PCV)是一种基因组为环状、单链的DNA病毒，根据基因组特征，目前已发现PCV1、PCV2、PCV3和PCV4四种基因型。其中，PCV1可在猪群中检出，但不致病；PCV2是引起猪断奶后衰竭综合症的主要病原；PCV3是一种与母猪皮炎综合征和繁殖障碍相关的病毒；最新发现的猪圆环病毒4型(PCV4)可能引起猪呼吸道、消化道等多种疾病，且可能与猪皮炎与肾病综合征密切相关。给养猪业带来严重的威胁。然而目前尚没有针对PCV4的有效疫苗以及基于cap蛋白的快速检测PCV4特异性商品化的抗原及抗体检测试剂盒。
发明内容
本发明的目的在于提供一种PCV4 cap蛋白及其制备方法，以及一种基于制备得到的PCV4 cap蛋白的单克隆抗体的研制及其双抗夹心ELISA方法的建立。
为解决上述技术问题，本发明采用如下技术方案：
本发明一个方面提供了一种抗PCV4 cap蛋白的单克隆抗体PCV4-cap-6D1，其重链可变区的氨基酸序列如SEQID NO.1所示，其轻链可变区的氨基酸序列如SEQ ID NO.2所示。
本发明另一个方面提供了一种抗PCV4 cap蛋白的单克隆抗体PCV4-cap-4B5，其重链可变区的氨基酸序列如SEQ ID NO.3所示，其轻链可变区的氨基酸序列如SEQ ID NO.4所示。
本发明另一个方面提供了一种核苷酸序列，其编码前述的单克隆抗体抗体。
本发明另一个方面提供了一种载体，包含如前所述的核苷酸序列。
本发明另一个方面提供了一种宿主细胞，包含如前所述的载体。
本发明再一个方面提供了一种试剂盒，所述试剂盒包含如前所述的单克隆抗体抗体。
在本发明的一些技术方案中，所述的试剂盒为ELISA试剂盒。
在本发明的一些技术方案中，所述的试剂盒中包含如前所述的单克隆抗体PCV4-cap-6D1和单克隆抗体PCV4-cap-4B5。
在本发明的一些技术方案中，所述试剂盒还包括：阴性对照、阳性对照、样品稀释液、封闭液、洗涤液、显色液和反应终止液。
在本发明的一些技术方案中，单克隆抗体PCV4-cap-6D1包被在酶标板上，单克隆抗体PCV4-cap-4B5被化学发光标记物标记，例如辣根过氧化酶、碱性磷酸酶、荧光素酶及其衍生物。
本发明再一个方面提供了如前所述的单克隆抗体在制备检测猪圆环病毒4型试剂的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测，优选为双抗夹心酶联免疫吸附检测。
在本发明的一些技术方案中，所述用途为检测猪圆环病毒4型。
在本发明的一些技术方案中，如前所述的单克隆抗体抗体为单克隆抗体PCV4-cap-6D1和单克隆抗体PCV4-cap-4B5联合使用；其中单克隆抗体PCV4-cap-6D1作为包被抗体，单克隆抗体PCV4-cap-4B5作为酶标抗体。
本发明再一个方面提供了一种猪圆环病毒4型的检测方法，所述检测方法包括以下步骤：
1)以单克隆抗体PCV4-cap-6D1包被酶标板，将单克隆抗体PCV4-cap-4B5以化学发光标记物标记；
2)以双夹心酶联免疫吸附检测法检测待测物；
3)显色后读取数据。
在本发明的一些技术方案中，所述的检测方法为非诊断或治疗目的的。
与现有技术相比，本发明的有益技术效果在于：
1、本发明的单克隆抗体是由单个细胞株产生，具有高度特异性、均一性等特性，单抗纯度高、特异性强、能持续无限的供应，在免疫检测中具有更广泛的研究应用价值和商业使用价值。
2、本发明的单克隆抗体具有较强的特异性，与PCV1、PCV2、PCV3均无交叉反应，可用于PCV4的病原检测。
3、本发明的单克隆抗体具有较高的灵敏度，只与PCV4反应，IFA效价不小于1:6400。
4、本发明的一种检测猪圆环病毒4型的试剂盒中，所述阴性对照包括PCV2病毒，所述阳性对照包括PCV4 cap蛋白。
5、本发明的一种检测猪圆环病毒4型检的试剂盒中，当所述捕获抗体PCV4-cap-6D1的包被浓度为1.8μg/ml时，酶标抗体稀释度为1:1000时，若OD450>0.09，判定结果为阳性。
本发明提供了原核表达的PCV4 cap蛋白以及基于cap蛋白的PCV4病毒检测用单克隆抗体，能够实现PCV4病毒的双抗夹心高效检测。本发明所述应用检测特异性好，不与其它猪圆环病毒包括PCV1、PCV2和PCV3存在交叉反应，可用于临床样品中PCV4病毒的检测，能够快速、特异、高通量地用于PCV4病毒检测，具有良好的临床应用价值；而且本发明应用相关的试剂盒可用于大规模临床样品中PCV4病毒的检测，具有特异性好、灵敏度高、检测时间短、通量高等技术优势，具有重要的临床实用价值和广阔的市场应用前景。
附图说明
图1为PCR扩增PCV4 cap片段。泳道M，DNAMarker；泳道1，PCV4 cap片段PCR产物。
图2为SDS-PAGE分析PCV4病毒cap蛋白的表达。泳道M，蛋白Marker；泳道1，空载对照；泳道2，cap诱导后的总蛋白；泳道3，cap超声裂解沉淀；泳道4，cap超声裂解上清；泳道5，纯化后的cap超声裂解上清。
图3为SDS-PAGE分析PCV4-cap-6D1和PCV4-cap-4B5腹水纯化效果图。泳道M：蛋白Marker；泳道1：纯化后的PCV4-cap-6D1；泳道2：纯化后的PCV4-cap-4B5；泳道3：未纯化的PCV4-cap-6D1腹水；泳道4：未纯化的PCV4-cap-4B5腹水
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
为了阐明本发明的技术方案及技术目的，下面结合附图及具体实施方式对本发明做进一步的介绍。
实施例1：制备猪圆环病毒4型cap蛋白
1)设计扩增PCV4 cap基因的引物：
表1：扩增PCV4 cap基因的引物设计：
2)PCV4 cap基因片段PCR扩增以及pCold-TF载体的酶切：
以PCV4阳性组织DNA为模板，表1所述引物为引物进行PCR扩增。PCR扩增反应体系为：25μL PrimeSTAR Max Premix(2×)，1μL 10μmol上游引物，1μL 10μmol下游引物，200ngPCV4阳性组织DNA，加ddw至50μL。PCR扩增循环反应参数为：95℃变性5分钟，随后进行35个循环(98℃变性10秒，55℃退火15秒，72℃延伸10秒)，最后72℃延伸10分钟。PCR结束后，PCR产物在1％的琼脂糖凝胶中进行电泳。电泳结果如图1所示。同时，对pCold-TF载体进行酶切。酶切体系为：5μL Cut Smart buffer，2μL XhoⅠ内切酶，2μL BamHⅠ内切酶，1μg pCold-TF载体质粒，加ddw至50μL，37℃反应30分钟。
3)PCV4 cap基因片段快速克隆进pCold-TF线性化载体：
将以上纯化的pCold-TF酶切产物以及PCV4 cap基因片段PCR产物在商品化重组酶ExnaseⅡ的作用下进行重组。具体重组反应体系如下：纯化的PCV4 cap基因片段PCR产物50-100ng，pCold-TF酶切产物50ng，2μL商品化的ExnaseⅡ酶，4μL 5×CE Buffer，加ddw至20μL，37℃反应30分钟后，置冰上5分钟。随后将连接产物转化到常规感受态细菌，涂氨苄抗性的LB板，次日挑取细菌克隆进行质粒制备，并进行测序鉴定。
4)PCV4 cap蛋白的诱导表达及纯化：
将获得的鉴定正确的PCV4 cap基因阳性质粒(命名为pCold-TF-PCV4-Cap)转化Transetta大肠杆菌后，以25mg/L IPTG在15℃条件下诱导5h后进行超声波裂解，裂解的上清和沉淀经SDS-PAGE验证发现cap蛋白可在上清中以可溶性形式存在。如图2所示，泳道M，蛋白Marker；泳道1，空载对照；泳道2，cap诱导后的总蛋白；泳道3，cap超声裂解沉淀；泳道4，cap超声裂解上清；泳道5，纯化后的cap超声裂解上清。
实施例2：抗PCV4 cap蛋白的单克隆抗体的制备
1.将纯化的cap蛋白腹腔免疫7周龄BALB/c小鼠，免疫3次，每次间隔14天，剂量均为50μg/只/次。首免时蛋白与等体积的弗氏完全佐剂混合至油包水的状态，二免三免均与弗氏不完全佐剂混合。三免后采集小鼠血清，以IFA的方法测定血清效价；进行细胞融合前3天，用50μg不含佐剂的cap蛋白进行冲击免疫。
2.IFA测定免疫小鼠血清抗体效价：
构建pEGFP-C1-cap真核表达质粒，将其转染293T细胞30小时后，固定作为检测原，经通透、封闭后，每孔加入用PBS以2倍倍比稀释的小鼠血清作为一抗，37℃孵育1小时后，PBS洗3遍，加入1000倍稀释的Alexa Fluor 555红色荧光染料作为二抗，37℃孵育1小时后，PBS洗3遍，在荧光显微镜下判定结果。
IFA结果显示小鼠血清效价为1:6400，可用于细胞融合制备单克隆抗体。
3.细胞融合
1)提前一天制备饲养细胞
a.取1只ICR小鼠引颈致死后浸泡在75％酒精消毒液中体表消毒；
b.将75％酒精浸泡过的小鼠固定，在超净台中用无菌手术剪剪开腹部皮肤，暴露腹膜；
c.用无菌镊子提起腹膜，用无菌注射器将5mLHAT选择培养基缓慢注入小鼠腹腔，轻轻按压腹腔再将注入的培养基重新吸出；
d.将饲养细胞每孔100μL铺至96孔细胞培养板中，37℃，5％CO2培养箱中培养。
2)先准备好8瓶正处于对数生长期的骨髓瘤细胞，使用不含双抗血清的DMEM吹下备用。
3)取冲击免疫的小鼠，小鼠脱颈椎致死后将其放入75％酒精浸泡10min，在无菌条件下，解剖小鼠，小心将脾脏剪下，放入提前准备的含有双抗的DMEM里，去除脾脏周围的结缔组织，将其放入另一个盛有DMEM的平皿中，用弯头针压住脾脏，用针头戳破脾脏，完全释放脾细胞。将吹好的骨髓瘤细胞与取到的脾淋巴细胞混合于50m1融合管里，1000rpm离心10min，倒掉上清。将融合管放于手心中，轻轻拍打底部，使两种细胞充分混匀，将预热的PEG在60s内匀速滴加入融合管里，一边加一边摇匀并拍打融合管。加完后，立即滴加预热的DMEM，终止融合。放入37℃里静置10min后，1000rpm离心10min，弃掉上清，加入5mLHAT培养基，轻轻吹打，悬浮沉淀细胞。分装于铺有饲养细胞的96孔板，放入37℃，5％C02培养箱培养。5天后观察细胞，半换液HAT培养基。融合后10天左右，在大多数细胞液变黄或者克隆分布孔底面积1/3时，可以进行抗体检测，通过间接免疫荧光方法，用转染pEGFP-C1-cap的293T细胞作为抗原检测、筛选分泌抗cap蛋白抗体的阳性细胞孔。通过筛选，获得了两株抗PCV4 cap的单克隆抗体，将其命名为PCV4-cap-6D1，PCV4-cap-4B5。
4.单克隆抗体的制备及特性鉴定
包被在酶标板的捕获抗体(抗PCV4 cap蛋白单克隆抗体)为杂交瘤细胞株PCV4-cap-6D1分泌获得，检测抗体为杂交瘤细胞株PCV4-cap-4B5分泌获得。将杂交瘤细胞株注入小鼠腹腔制备PCV4-cap-6D1和PCV4-cap-4B5的单克隆抗体腹水。利用GE公司的Protein G预装柱进行单克隆抗体IgG纯化。纯化后利用SDS-PAGE鉴定，结果显示获得了正确的且纯度高的单克隆抗体，纯化后PCV4-cap-6D1和PCV4-cap-4B5单抗的SDS-PAGE鉴定如图3所示，其中，泳道M：Marker；泳道1：纯化后的PCV4-cap-6D1；泳道2：纯化后的PCV4-cap-4B5；泳道3：未纯化的PCV4-cap-6D1腹水；泳道4：未纯化的PCV4-cap-4B5腹水。由图3可知，获得了正确的且纯度高的单克隆抗体PCV4-cap-6D1和PCV4-cap-4B5。
(2)酶标单克隆抗体的制备
使用加强型活化过氧化物酶标记试剂盒说明书进行杂交瘤细胞PCV4-cap-4B5分泌的单克隆抗体进行HRP酶标记。PCV4-cap-4B5这株单克隆抗体经过酶标记后，记为PCV4-cap-4B5-HRP。
(3)捕获抗体包被浓度及酶标抗体工作浓度的确定
采用方阵滴定法来测定捕获抗体及检测抗体最佳工作浓度，最终确定捕获抗体包被浓度为1.8μg/mL，每孔100μL；酶标抗体稀释度为1:1000，每孔100μL。
(4)双单抗夹心ELISA抗原检测试剂盒的构建和临床样品检测结果
1.包被酶标板：单抗PCV4-cap-6D1纯化后，得到捕获抗体，所述捕获抗体稀释为1.8μg/mL，100μL/孔对酶标板进行包被，4℃放置过夜；
2.封闭：取出包被酶标板，用PBST洗涤1次；用5％脱脂乳封闭，360μL/孔，37℃放置2h，再用PBST洗涤；
3.加样：取样品加入上述封闭好的酶标板，100μL/孔，其中，阴阳性对照分别以1:50的体积比进行稀释，37℃反应60min，用PBST洗涤5次；
4.加酶标单抗：将酶标抗体用PBST 1:1000稀释，100μL/孔，37℃
反应1h，PBST洗涤5次；
5.显色：弃去二抗，用洗涤液洗涤3次，加入底物显色液，每孔100μL，37℃避光作用10分钟，加入50μL终止液终止反应。
6.读板：酶标仪上测定各孔OD450nm，进行结果判定，OD450nm大于0.09判为阳性，小于0.09判为阴性。
ELISA包被液：
NaCO3 1.59g
NaHCO3 2.93g
溶于1L蒸馏水中，调节pH＝9.6
ELISA洗涤液：
用去离子水定容至1L，调节pH值为7.4。
封闭液：
BSA 0.5g
酪蛋白 0.025g
定溶于10mL样品洗涤液中。
底物显色液：
A液：柠檬酸21g溶于1L去离子水中。
B液：磷酸氢二钠71.6g溶于1L去离子水中。
使用前取4.86mLA液、5.14mLB液，加入邻苯二胺10mg混匀后加入37.5μL 30％H2O2。
终止液：
在177.8mL的蒸馏水中加入22.2mL浓硫酸混匀。
(5)双单抗夹心ELISA特异性验证
取猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、日本乙型脑炎病毒(JEV)、猪伪狂犬病毒(PRV)、猪细小病毒(PPV)、PCV1、PCV2、PCV3阳性病毒，使用建立的双抗夹心ELISA方法进行检测，结果均为阴性，OD450见表2。结果表明该ELISA方法均有良好的特异性。
表2双抗夹心ELISA抗原检测试剂盒特异性验证结果
实施例3
抗PCV4 Cap蛋白单克隆抗体可变区基因的扩增
根据鼠源单克隆抗体的序列特征，设计重链可变区引物序列：
上游引物：TTCTCTTGACGT TGTACTGG SEQ ID NO.7
下游引物：TTTTGAGGAGACGGTGA SEQ ID NO.8
设计轻链可变区引物序列：
上游引物：TTATGGGTAGTTCATGGAGACA SEQ ID NO.9
下游引物：ACACATGGTGCAGCATCAGCC SEQ ID NO.10
通过分子克隆技术分别获得单克隆抗体PCV4-cap-6D1的可变区序列,送上海生工生物有限公司测序。测定单克隆抗体PCV4-cap-6D1的重链可变区和轻链可变区氨基酸序列分别为SEQ ID NO.1、SEQ ID NO.2所示；单克隆抗体PCV4-cap-4B5的重链可变区和轻链可变区氨基酸序列分别为SEQ ID NO.3、SEQ ID NO.4所示
SEQ ID NO.1
SEQ ID NO.2
SEQ ID NO.3
SEQ ID NO.4
实施例4双抗夹心ELISA抗原检测试剂盒对临床血清样品的检测
实验室分别从江苏、河南、广东、广西、安徽、浙江、福建、河北等省份猪场收集93份猪组织病料用于PCV4双抗夹心ELISA试剂盒进行评价，检测结果(表3)为10份病料为阳性，阳性率为10.75％，表明我国部分猪群已感染PCV4。
表3临床组织样品的检测结果
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
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Thr Gly Tyr Asn Gln Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp
50 55 60
Ser Ser Thr Ala Tyr Met Glu Ile Arg Arg Leu Thr Ser Glu Asp Ser
65 70 75 80
Cys Ala Tyr Ala Arg Pro Asn Tyr Gly Tyr Leu Asp Tyr Trp Val Tyr
85 90 95
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Lys
100 105
<210> 4
<211> 125
<212> PRT
<213> 人工序列
<400> 4
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala
20 25 30
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser
35 40 45
Val Ser Thr Ser Gly Tyr Ser Tyr Met His Trp Asn Gln Gln Lys Pro
50 55 60
Gly Gln Pro Pro Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser
65 70 75 80
Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Thr Tyr Cys Gln His
100 105 110
Ile Arg Glu Leu Thr Arg Ser Gly Gly Pro Ser Trp Lys
115 120 125
<210> 5
<211> 35
<212> DNA
<213> 人工序列
<400> 5
atggagctcg gtaccatgcc aatcagatct aggta 35
<210> 6
<211> 35
<212> DNA
<213> 人工序列
<400> 6
cttgaattcg gatccttatc cctgtttggg gtagt 35
<210> 7
<211> 20
<212> DNA
<213> 人工序列
<400> 7
ttctcttgac gttgtactgg 20
<210> 8
<211> 17
<212> DNA
<213> 人工序列
<400> 8
ttttgaggag acggtga 17
<210> 9
<211> 22
<212> DNA
<213> 人工序列
<400> 9
ttatgggtag ttcatggaga ca 22
<210> 10
<211> 21
<212> DNA
<213> 人工序列
<400> 10
acacatggtg cagcatcagc c 21
Claims (12)
1.一种抗PCV4 cap蛋白的单克隆抗体PCV4-cap-6D1，其特征在于，其重链可变区的氨基酸序列如SEQID NO.1所示，且其轻链可变区的氨基酸序列如SEQ ID NO.2所示。
2.一种抗PCV4 cap蛋白的单克隆抗体PCV4-cap-4B5，其特征在于，其重链可变区的氨基酸序列如SEQ ID NO.3所示，且其轻链可变区的氨基酸序列如SEQ ID NO.4所示。
3.一种核苷酸序列，其特征在于，其编码权利要求1所述的单克隆抗体PCV4-cap-6D1或权利要求2所述的单克隆抗体PCV4-cap-4B5。
4.一种载体，其特征在于，包含权利要求3所述的核苷酸序列中的至少一种。
5.一种宿主细胞，其特征在于，包含权利要求4所述的载体中的至少一种。
6.一种试剂盒，其特征在于，所述试剂盒包含权利要求1所述的单克隆抗体PCV4-cap-6D1和/或权利要求2所述的单克隆抗体PCV4-cap-4B5。
7.权利要求6所述的试剂盒，其特征在于，所述的试剂盒为ELISA试剂盒。
8.权利要求7所述的试剂盒，其特征在于，所述的试剂盒中包含如前所述的单克隆抗体PCV4-cap-6D1和单克隆抗体PCV4-cap-4B5。
9.权利要求8所述的试剂盒，其特征在于，单克隆抗体PCV4-cap-6D1包被在酶标板上，单克隆抗体PCV4-cap-4B5被化学发光标记物标记。
10.权利要求1所述的单克隆抗体PCV4-cap-6D1和/或权利要求2所述的单克隆抗体PCV4-cap-4B5在制备检测猪圆环病毒4型试剂的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测。
11.根据权利要求10所述的用途，其特征在于，所述试剂用于以下用途的试剂：双抗夹心酶联免疫吸附检测。
12.根据权利要求10所述的用途，其特征在于，单克隆抗体抗体为单克隆抗体PCV4-cap-6D1和单克隆抗体PCV4-cap-4B5联合使用；其中单克隆抗体PCV4-cap-6D1作为包被抗体，单克隆抗体PCV4-cap-4B5作为酶标抗体。
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| CN108586607A (zh) * | 2018-04-13 | 2018-09-28 | 郑州大学 | 抗hpv16 l1蛋白单克隆抗体的制备方法及其应用 |
| CN108752471A (zh) * | 2018-04-13 | 2018-11-06 | 河南中泽生物工程有限公司 | 抗pcv2单克隆抗体的制备方法及其应用 |
| CN109406788A (zh) * | 2018-11-13 | 2019-03-01 | 昆明理工大学 | 一种单克隆抗体及其应用 |
| CN111848786A (zh) * | 2020-07-29 | 2020-10-30 | 中牧实业股份有限公司 | 一种单克隆抗体、其制备方法及用途 |
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| CN108586607A (zh) * | 2018-04-13 | 2018-09-28 | 郑州大学 | 抗hpv16 l1蛋白单克隆抗体的制备方法及其应用 |
| CN108752471A (zh) * | 2018-04-13 | 2018-11-06 | 河南中泽生物工程有限公司 | 抗pcv2单克隆抗体的制备方法及其应用 |
| CN109406788A (zh) * | 2018-11-13 | 2019-03-01 | 昆明理工大学 | 一种单克隆抗体及其应用 |
| CN111848786A (zh) * | 2020-07-29 | 2020-10-30 | 中牧实业股份有限公司 | 一种单克隆抗体、其制备方法及用途 |
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