CN114717240B - 一种华支睾吸虫蛋白Cs144的制备和应用 - Google Patents
一种华支睾吸虫蛋白Cs144的制备和应用 Download PDFInfo
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Abstract
本发明公开了一种华支睾吸虫蛋白Cs144的制备和应用,涉及生物检测技术领域,华支睾吸虫基因Cs144的编码区核苷酸序列如SEQ ID NO:1所示,华支睾吸虫蛋白Cs144的氨基酸序列如SEQ ID NO:2所示;包含华支睾吸虫Cs144基因的编码区核苷酸序列的重组质粒,以及该重组质粒的制备方法;华支睾吸虫蛋白Cs144在制备华支睾吸虫感染诊断抗原的应用,在制备华支睾吸虫感染ELISA检测试剂盒中的应用。本发明提供一种重组表达的华支睾吸虫蛋白Cs144,作为抗原应用于诊断肝吸虫病的ELISA检测中,解决ELISA检测中天然抗原成本高,难以工业化生产的问题。
Description
技术领域
本发明涉及生物检测技术领域,尤其涉及一种华支睾吸虫蛋白Cs144的制备和应用。
背景技术
华支睾吸虫(Clonorchis sinensis)又称肝吸虫(liver fluke),成虫感染引起华支睾吸虫病(Clonorchiasis),又称肝吸虫病。肝吸虫成虫寄生于人等哺乳动物的肝胆管内,刺激胆道上皮来引发宿主胆道炎、胆道周围纤维化、黄疸、消化不良及肝功能低下等,宿主还会并发胆结石、肝硬化及胆囊炎等。华支睾吸虫感染引发的多种并发症非常严重,造成很大的疾病负担,极大的降低了患者的生活质量。同时,华支睾吸虫感染还是诱发胆管癌的危险因素之一。2009年世界卫生组织国际癌症研究署(IARC)将华支睾吸虫列为I类致癌物。因此在华支睾吸虫感染发展成为重症疾病之前,在早期诊断其感染,并提前治疗是非常重要的。
免疫学诊断在肝吸虫病与流行病学调查中非常重要。肝吸虫成虫浸出物及代谢产物中含有抗原成分,感染人体后会诱发多种免疫病理反应,宿主感染华支睾吸虫后可诱发较强的体液免疫应答,参与体液免疫的主要免疫球蛋白为IgG,抗华支睾吸虫抗原的IgG抗体水平在一定程度上可反映病人感染情况与免疫反应强弱。酶联免疫吸附测定(ELISA)可以利用抗原抗体特异性结合进行免疫反应的定性和定量检测方法。使华支睾吸虫抗原结合到固相载体表面,血清标本内的IgG抗体可与固相载体表面的抗原反应,再加入酶标二抗与之反应,最后结合在固相载体上的酶量与血清标本中受检物质的量成一定比例,从而可以定量分析受检物。由于酶的催化效率高,可放大反应效果,测定方法敏感度好。但肝吸虫成虫浸出物及代谢产物获取困难,来源少,成本高,难以工业化生产的问题,如果能找到一种可体外重组制备的华支睾吸虫特异性抗原蛋白靶点,应用ELISA技术,则可以简便快捷的诊断肝吸虫病。
传统上,用患者粪便中华支睾吸虫虫卵的病原学方法检测华支睾吸虫病,但是华支睾吸虫虫卵较小,漏检率高,对于轻度感染者存在漏检的可能,而且不能对华支睾吸虫病作出早期诊断。近年来,免疫检查的方法逐渐被用于华支睾吸虫病的临床诊断研究,可使用天然虫卵抗原提取物制备试剂盒,但天然抗原存在获取困难,制备困难,成本造价高,与其他寄生虫抗原有交叉性、批间差异大等缺陷,易造成试剂盒价格高、特异性较差、灵敏度较低、批间差异大等质量问题。针对肝吸虫病的诊断,目前尚无明确的生物标志物。因此,在上海市第五轮公共卫生体系建设三年行动计划 (GWV-10.1-XK13)、上海市自然科学基金项目(19ZR1428500)、国家自然科学基金面上项目(81971486)基金支持下,本领域的技术人员致力于寻找肝吸虫病新的诊断靶点,为疾病的预防和治疗提供重要的技术依据。肝吸虫感染诊断的关键点就是寻找重要的疾病靶点。
因此,本领域的技术人员致力于开发一种可体外重组制备的华支睾吸虫特异性抗原蛋白靶点,应用于诊断肝吸虫病的ELISA检测中,克服现有技术的不足。
发明内容
鉴于现有技术的上述缺陷,本发明所要解决的技术问题是开发一种重组表达的华支睾吸虫蛋白,应用于诊断肝吸虫病的ELISA检测中,解决ELISA检测中天然抗原成本高,难以工业化生产的问题。华支睾吸虫感染者体内存在针对这种抗原的特异性抗体,针对抗体进行检测可以用于评测华支睾吸虫感染与否,具有高特异性和高灵敏度的检测特点。我们通过吸虫家族基因表达谱的比较,寻找到一种华支睾吸虫成虫钙结合蛋白Cs144,首次在体外对其进行了重组表达,及功能测定。我们证实该蛋白可被肝吸虫感染者血清识别,以其作为诊断抗原,应用于诊断肝吸虫病的ELISA检测中,可区分肝吸虫感染者与未感染者,由此可用于肝吸虫病的诊断。
为实现上述目的,本发明提供了一种华支睾吸虫基因Cs144,其编码区核苷酸序列如SEQ ID NO:1所示。
进一步地,基因Cs144编码的氨基酸序列如SEQ ID NO:2所示。
本发明还提供一种重组质粒,包含基因Cs144编码区核苷酸序列。
本发明还提供一种含华支睾吸虫基因Cs144的重组质粒的制备方法,包括以下步骤:
步骤一、提取华支睾吸虫成虫的RNA,反转录RNA获得华支睾吸虫cDNA;
步骤二、设计Cs144基因的正向引物和反向引物,以步骤一获得的cDNA为模板扩增,得获得Cs144基因序列;将扩增产物纯化回收,并酶切连接至pET302/NT-His 载体上,筛选阳性克隆并进行双向序列测定,得到重组质粒。
进一步地,步骤二中正向引物和反向引物分别为Cs144-F和Cs144-R,其中 Cs144-F的DNA序列SEQ ID NO:3所示,Cs144-R的DNA序列如SEQ ID NO:4 所示。
进一步地,步骤二中还包括在正向引物和反向引物上分别引入XhoI和AvrII酶切位点。
本发明还提供华支睾吸虫Cs144基因编码的华支睾吸虫蛋白Cs144在制备华支睾吸虫感染诊断抗原的应用。
本发明还提供华支睾吸虫Cs144基因编码的华支睾吸虫蛋白Cs144在华支睾吸虫感染ELISA检测试剂盒中的应用。
进一步地,华支睾吸虫感染ELISA检测试剂盒,其主要试剂包括抗原,所述抗原为华支睾吸虫蛋白Cs144,溶于磷酸盐缓冲液中。
进一步地,磷酸盐缓冲液组分为0.03M Na2CO3,0.07M NaHCO3。
在本发明的较佳实施方式1中,详细说明制备Cs144基因的重组质粒和华支睾吸虫蛋白Cs144的过程;
在本发明的另一较佳实施方式2中,试验验证华支睾吸虫蛋白Cs144刺激宿主产生特异性抗体。
本发明的技术效果如下:
(1)本发明提供了一个华支睾吸虫感染检测的靶点-华支睾吸虫蛋白Cs144,华支睾吸虫患者体内存在针对这种抗原的抗体,应用ELISA技术对该抗体的检测具有高敏感性和高特异性的特点,可成为肝吸虫检测的有效指标;
(2)本发明的华支睾吸虫蛋白Cs144可大量重组表达,为其大规模生产与应用提供了可能性;
(3)本发明的华支睾吸虫蛋白Cs144为制备华支睾吸虫感染检测试剂盒提供了基础,为华支睾吸虫的防治提供了重要的技术依据
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明的一个较佳实施例1的华支睾吸虫基因Cs144编码区核苷酸序列的扩增结果示意图;
图2是本发明的一个较佳实施例1的构建的重组质粒pET302/NT-His-Cs144测序结果与参考序列对比示意图;
图3是本发明的一个较佳实施例1的制备的重组质粒pET302/NT-His-Cs144 的结构示意图谱;
图4是本发明的一个较佳实施例2的利用SDS-PAGE检测华支睾吸虫蛋白 Cs144表达的结果示意图;
图5是本发明的一个较佳实施例2的利用免疫印迹法检测华支睾吸虫蛋白 Cs144表达的结果示意图;
图6是本发明的一个较佳实施例2的利用ELISA检测华支睾吸虫感染与未感染者血清抗华支睾吸虫蛋白Cs144特异性IgG抗体水平的结果示意图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例,使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现,本发明的保护范围并非仅限于文中提到的实施例。
实施例1制备包含华支睾吸虫基因Cs144的重组质粒
(1)华支睾吸虫成虫总RNA的提取
采用经典的Trizol法提取样品总RNA。取华支睾吸虫成虫用玻璃匀浆器在冰上迅速匀浆,匀浆充分后转入离心管,取100μL组织匀浆加入1mL Trizol,混匀,室温放置5min。
加入200μL氯仿,剧烈振荡15s,混匀后室温放置5min,4℃,12000×g,离心 15min。
取上层水相至另一新的离心管,加入500μL异丙醇,颠倒混匀,室温放置10min, 4℃,12000×g,离心10min。
弃上清,加入冰上预冷的75%的乙醇1mL洗涤沉淀,4℃,7500×g,离心5min。
弃上清,室温晾干,用无RNA酶的水溶解RNA沉淀,立即进行反转录。
(2)华支睾吸虫成虫cDNA的合成;
采用Invitrogen公司的反转录试剂盒,按照说明书操作步骤进行cDNA合成,具体步骤如下:
在PCR反应管中依次加入1000μg上述提取的RNA、1μL随机六聚体引物、补充无RNA酶的水至总体积12μL;于65℃反应5min,立即置于冰上,然后加入4μL 5×ReactionBuffer、1μL RiboLock RNase Inhibitor、2μL 10mM dNTP Mix和1μL RevertAid M-MuLVRT,42℃反应60min,25℃反应5min,之后70℃反应5min获得 cDNA。
(3)华支睾吸虫Cs144基因的扩增
首先利用Primer Express 5.0和BioEdit 7.0软件,根据GenBank中华支睾吸虫Cs144 基因序列保守区设计引物,并分别在正向引物和反向引物上引入Xho I和Avr II酶切位点,Cs144-F的DNA序列如SEQ ID NO:3所示,Cs144-R的DNA序列如SEQ ID NO:4所示,以华支睾吸虫cDNA为模板进行PCR扩增,引物DNA序列具体为:
Cs144-F:CCGCTCGAGCGGATGTGCCACGGCATTTCT;
Cs144-R:CCGCCTAGGCGGTTACTGCGCAATTTGTT。
PCR反应体系如下:ddH2O 37.5μL、10×Ex Taq Buffer(Mg2+plus)(20mM)5μL、dNTP Mixture(各2.5mM)4μL、上下游引物(10μmol/L)各1μL、TaKaRa Ex Taq(5U/μL)0.5μL、cDNA模板1μL,总体积为50μL。
PCR反应条件为:98℃预变性1min;98℃变性10s,60℃退火30s,72℃延伸 45s,共35个循环;72℃延伸5min;4℃保温。
PCR扩增产物经1%琼脂糖凝胶电泳,切胶后纯化回收。如图1所示,华支睾吸虫基因Cs144编码区核苷酸序列的扩增结果示意图,其中,M和1通道分别为DNA Marker 2000和华支睾吸虫基因Cs144扩增产物,华支睾吸虫基因Cs144扩增产物的大小为435bp。
(4)重组质粒pET302/NT-His-Cs144的构建
分别将回收的片段和pET302/NT-His载体(购于Invitrogen公司)用Xho I和AvrII限制性内切酶37℃酶切过夜,酶切片段纯化回收,在T4 DNA连接酶作用下于16℃连接过夜。连接产物热击转化至大肠杆菌感受态DH5α中,转化后利用菌液PCR筛选阳性克隆,并送擎科生物科技有限公司进行双向序列测定,所构建的重组质粒 pET302/NT-His-Cs144测序结果与参考序列对比示意图如图2所示;重组质粒命名为pET302/NT-His-Cs144,图谱如图3所示。
(5)重组质粒pET302/NT-His-Cs144和空载体pET302/NT-His的制备
将30μL测序正确的单克隆菌液接种到3mL含50μg/mL氨苄青霉素的LB液体培养基中,37℃、220转/分钟,摇床培养过夜,用质粒提取试剂盒(购于天根生化科技有限公司)提取重组质粒pET302/NT-His-Cs144和空载体质粒pET302/NT-His,具体步骤如下:
将培养过夜的菌液置于1.5mL离心管中,室温、13,400×g离心1min,弃尽上清,重复以上操作直至所有菌液离心完毕;
在细菌沉淀中加入500μL溶液P1悬浮沉淀;
加入500μL P2温和并充分地上下翻转6-8次使菌体充分裂解,直至形成透亮的溶液,此步骤不宜超过5min;
加入500μL溶液P4,立即温和地上下翻转6-8次,充分混匀,此时会出现白色絮状沉淀,然后室温放置10min左右,13,400×g离心10min,此时在离心管底部形成沉淀;
吸取上清并转移至过滤柱,室温13,400×g离心2min,滤液收集在干净的2ml离心管中;
向滤液中加入0.3倍滤液体积的异丙醇,上下颠倒混匀后转移到吸附柱中;
室温13,400×g离心1min,倒掉收集管中的废液,将吸附柱重新放回收集管中;
用600μL漂洗液PW洗两次,弃滤液;室温13,400×g离心1min,离心空柱2min;
吸附柱移入新的1.5mL离心管中,在吸附柱膜中央加100μL洗脱缓冲液TB,室温放置2min,13,400×g离心1min;质粒浓度用分光光度计进行测定。
(6)华支睾吸虫蛋白Cs144的原核诱导表达
将30μL测序正确的单克隆菌液接种到3mL含50μg/mL氨苄青霉素的LB液体培养基中,37℃、220转/分钟,摇床培养过夜;
将菌液转移至300mL含50μg/mL氨苄青霉素的LB液体培养基中,37℃、220 转/分钟,摇床培养至OD=0.8,取少量菌液作为诱导前对照;
加入1M IPTG诱导16h,20℃,取少量菌液后剩余菌液4000rpm,离心30min;
将沉淀重悬于裂解液(50mM NaH2PO4,300mM NaCl,10mM imidazole,pH 8)中,每克湿重加入5ml裂解液,加入溶菌酶至1mg/ml,冰上孵育30分钟;
将5μL 2X SDS-PAGE样品缓冲液添加到5μL裂解样品中作为超声破碎前对照;
冰上超声裂解,然后在4℃,15,000rpm离心20min;
将5μL 2X SDS-PAGE样品缓冲液添加到5μL样品中作为超声破碎后对照;
将上清液加入1ml预平衡的Ni-琼脂糖凝珠中,4℃孵育2h,将裂解液-Ni混合物装入色谱柱,取下底盖并收集色谱柱流出液,将流出液保存,将5μL 2X SDS-PAGE样品缓冲液添加到5μL样品中作为镍柱结合后上清对照;
用4ml清洗液(50mM NaH2PO4,300mM NaCl,20mM imidazole,pH 8)洗涤两次;
用0.5ml洗脱液(50mM NaH2PO4,300mM NaCl,250mM imidazole,pH 8)将蛋白质洗脱,收集洗脱液,进行BCA蛋白浓度测定,将5μL 2X SDS-PAGE样品缓冲液添加到5μL样品中作为镍柱纯化后样品通过SDS-PAGE凝胶进行分析。
实施例2验证华支睾吸虫蛋白Cs144刺激宿主产生特异性抗体
(7)聚丙烯酰胺凝胶电泳(SDS-PAGE)
取实施例1中步骤(6)中超声破碎前对照样品,超声破碎后对照样品,镍柱结合后上清对照样品和镍柱纯化后样品,分别涡旋混匀后,在95℃煮5min,进行 SDS-PAGE。
使用考马斯染剂染色30min,脱色过夜。
聚丙烯酰胺凝胶电泳结果如图4所示,其中,1-4通道分别为华支睾吸虫蛋白Cs144超声破碎前、超声破碎后、镍柱结合后上清和镍柱纯化后样品,表明华支睾吸虫蛋白 Cs144被成功诱导表达并纯化。
(8)免疫印迹实验(Western blot)
取实施例1中步骤(6)的5μL诱导前和诱导后菌液,加入20μL 5X SDS-PAGE样品缓冲液,95℃煮5min,进行SDS-PAGE。
裁剪与分离胶大小完全吻合的滤纸和NC膜,在转移缓冲液中平衡15min;
凝胶电泳结束后,拆卸凝胶夹层,分离胶于适量转移缓冲液中平衡10min;
组装转印夹层,在恒压100V,60min条件下转移蛋白;
转膜完毕,将膜取出,放入杂交盒,加10mL 5%脱脂牛奶的TBST封闭液,37℃孵育1h;
倒掉封闭液,加入针对重组质粒中与华支睾吸虫蛋白Cs144共表达的His标签的抗体(一抗,以体积比1:1000稀释),4℃孵育过夜;
TBST洗3次,每次10min,加入HRP标记的山羊抗兔IgG抗体(1:3000),室温孵育1h;
倒掉二抗,TBST洗3次,每次10min;
滴加化学发光液显影,将膜置于凝胶成像仪显影。
免疫印迹结果如图5所示,其中,1和2通道分别为华支睾吸虫蛋白Cs144诱导前和诱导后样品,表明pET302/NT-His-Cs144诱导表达后,华支睾吸虫蛋白Cs144的表达水平得到了显著提高。
(9)酶联免疫吸附测定(ELISA)
本例中,共采集了17位健康对照和65位华支睾吸虫感染患者的血清样本,华支睾吸虫感染情况由粪便虫卵检查确定。样品来源于中国疾病预防控制中心寄生虫病预防控制所,本研究中所用样本,均对患者进行知情告知并经伦理委员会通过。
取实施例1步骤(6)中镍柱纯化后样品。取Cs144加入磷酸盐缓冲液(0.03M Na2CO3,0.07M NaHCO3)中至终浓度100μg/mL,100μL/孔进行铺板,4℃孵育过夜;
PBST(PBS,0.3%Tween-20),250μL/孔洗三次;
含有0.2%BSA的PBST,200μL/孔,室温孵育1h;
250μL/孔PBST洗三次;
100μL/孔PBST,每孔分别加入1μL血清样品,37℃孵育1h;
250μL/孔PBST洗三次;
含有0.2%BSA的PBST,100μL/孔,每孔分别加入IgG-HRP(1:5000)(购自 SIGMA公司),37℃孵育1h;
250μL/孔PBST洗三次;
显色底物溶液(10mL 0.5M醋酸钠+200μL 10mg/mL TMB+2μL H2O2)100μL/ 孔,室温10min;
2mol/L H2SO4 50μL/孔终止反应;
2mol/L H2SO4 50μL/孔终止反应;
使用自动酶标仪检测450nm波长的吸光度。
两个样本间的差异使用Mann Whitney test(two-tailed),实验结果用GraphPadPrism 9软件分析。*P<0.05,**P<0.01,***P<0.001,NS表示无统计学差异。当P<0.05 时,实验结果的差异有统计学意义。
结果如图6所示,华支睾吸虫感染者体内的Cs144抗体水平明显高于未感染者,健康人血清与华支睾吸虫病患者血清抗体检测结果差异均有统计学意义(P<0.05), Cs144抗体水平是华支睾吸虫感染的有效的检测指标。
实施例1通过基因克隆技术从华支睾吸虫中分离出Cs144基因,实施例2通过一系列的试验验证,表明本发明获得的Cs144蛋白具有优异的刺激宿主产生特异性抗体的特点,可通过测试宿主抗体产生情况来间接检测感染情况,具有高特异性高灵敏度,操作简便快捷的优势。本发明的Cs144蛋白及其基因为华支睾吸虫检测及防治提供了新的思路,也为其它蠕虫的感染的检测提供了借鉴意义。
以上详细描述了本发明的较佳具体实施例。应当理解,本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此,凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案,皆应在由权利要求书所确定的保护范围内。
序列表
<110> 上海交通大学医学院
<120> 一种华支睾吸虫蛋白Cs144的制备和应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 435
<212> DNA
<213> 华支睾吸虫 (Clonorchis sinensis)
<400> 1
atgtgccacg gcatttctgc tcaacagtta gaagagctct tccaaagtct ggaccatgac 60
ggtagcggtg ctgtggacag agaagaactg actgtcgctc tcaaagaagc cggaattcca 120
gaaaaaaatg cagagcgcat aatccaagag ttggacgtaa accaagatgg acatataact 180
ttaggtgaat acagactcgt catgggatta accgatgagc caatagccga atggaaacgt 240
ttgtttgtca cgctggactc tgatcgatcg gggaaagtgg acaaacgaga attgcagcaa 300
atgttcgacg aaatgggaat gcattttgcc ctaagcactc tggaagactg gatagctgat 360
cacgatgtgg atggcgacgg aaaactaacg tatcaggaat tcatgggctt tgttgctgaa 420
caaattgcgc agtaa 435
<210> 2
<211> 144
<212> PRT
<213> 华支睾吸虫 (Clonorchis sinensis)
<400> 2
Met Cys His Gly Ile Ser Ala Gln Gln Leu Glu Glu Leu Phe Gln Ser
1 5 10 15
Leu Asp His Asp Gly Ser Gly Ala Val Asp Arg Glu Glu Leu Thr Val
20 25 30
Ala Leu Lys Glu Ala Gly Ile Pro Glu Lys Asn Ala Glu Arg Ile Ile
35 40 45
Gln Glu Leu Asp Val Asn Gln Asp Gly His Ile Thr Leu Gly Glu Tyr
50 55 60
Arg Leu Val Met Gly Leu Thr Asp Glu Pro Ile Ala Glu Trp Lys Arg
65 70 75 80
Leu Phe Val Thr Leu Asp Ser Asp Arg Ser Gly Lys Val Asp Lys Arg
85 90 95
Glu Leu Gln Gln Met Phe Asp Glu Met Gly Met His Phe Ala Leu Ser
100 105 110
Thr Leu Glu Asp Trp Ile Ala Asp His Asp Val Asp Gly Asp Gly Lys
115 120 125
Leu Thr Tyr Gln Glu Phe Met Gly Phe Val Ala Glu Gln Ile Ala Gln
130 135 140
<210> 3
<211> 30
<212> DNA
<213> 人工序列 (Artifical sequence)
<220>
Claims (9)
1.一种华支睾吸虫基因Cs144,其特征在于,所述基因Cs144的编码区核苷酸序列如SEQID NO:1所示。
2.一种如权利要求1所述的基因Cs144的重组质粒,其特征在于,所述重组质粒包含基因Cs144编码区核苷酸序列。
3.一种如权利要求2所述的重组质粒的制备方法,包括以下步骤:
步骤一、提取华支睾吸虫成虫的RNA,反转录所述RNA获得华支睾吸虫cDNA;
步骤二、设计一对正向引物Cs144-F和反向引物Cs144-R,以步骤一获得的cDNA为模板扩增,将扩增产物纯化回收,并酶切连接至载体pET302/NT-His上,筛选阳性克隆并进行双向序列测定,得到所述重组质粒。
4.如权利要求3所述的方法,其特征在于,所述步骤二中所述正向引物为Cs144-F的DNA序列SEQ ID NO:3所示,所述反向引物Cs144-R的DNA序列如SEQ ID NO:4所示,所述扩增产物为包含基因Cs144的DNA序列。
5.如权利要求3所述的方法,其特征在于,所述步骤二中还包括在所述正向引物和所述反向引物上分别引入XhoI和AvrII酶切位点。
6.一种如权利要求1所述的华支睾吸虫基因Cs144编码的蛋白Cs144在制备所述华支睾吸虫感染诊断抗原的应用。
7.一种如权利要求1所述的华支睾吸虫基因Cs144编码的蛋白Cs144在制备华支睾吸虫感染ELISA检测试剂盒中的应用。
8.如权利要求7所述的应用,其特征在于,所述华支睾吸虫感染ELISA检测试剂盒的试剂包括抗原,所述抗原为所述华支睾吸虫蛋白Cs144,溶于磷酸盐缓冲液中。
9.如权利要求8所述的应用,其特征在于,所述磷酸盐缓冲液组分为0.03MNa2CO3,0.07MNaHCO3。
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