CN112538484A - 一种肝片吸虫特异抗原基因FhSAP-2及医用用途 - Google Patents
一种肝片吸虫特异抗原基因FhSAP-2及医用用途 Download PDFInfo
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Abstract
本发明涉及一种肝片吸虫特异抗原基因FhSAP‑2及医用用途,该基因是通过肝片吸虫成虫cDNA表达文库的筛选获得。制备的检测羊等动物肝片吸虫感染胶体金试纸条具有简单、快速、无需特殊仪器设备等特点;检测结果便于判读,适于临床中快速诊断;利用间接胶体金检测方法,其中纯化的肝片吸虫FhSAP‑2抗原保证了本发明的特异性;使用金黄色葡萄球菌(SPA)进行标记,成本较低。
Description
技术领域
本发明涉及一种肝片吸虫特异抗原基因FhSAP-2及医用用途,以及基于该基因抗原制备的检测动物肝片吸虫感染胶体金免疫层析试纸,可用于肝片吸虫感染的血清检测,属于免疫学技术领域。
背景技术
肝片吸虫病是由肝片吸虫(Fasciola hepatica)引起的一种重要的人畜共患寄生虫病,该虫宿主广泛,可寄生于牛、羊、鹿、骆驼等反刍动物,猪、马属动物及一些野生动物也可寄生,但较为少见,人也有感染的报道。该虫能引起肝炎和胆管炎,并伴有全身性中毒现象和营养障碍,危害相当严重,尤其对幼畜和绵羊,可引起大批死亡。在其慢性病程中,可使动物瘦弱,发育障碍,耕牛使役能力下降,乳牛产奶量减少,毛、肉产量减少和质量下降,病肝成为废弃物,给畜牧业经济带来巨大损失。早发现,早治疗是防控本病的关键。目前常用的检测方法有病原学和免疫学方法等。但病原学检查存在费时、费力、检出率低等缺点。免疫学检测方法如:酶联免疫吸附试验(ELISA),需要有专门的试验人员,相关的仪器设备,检测时间较长等弊端,所以不合适用于基层现场检测使用。临床上急需建立一种检测快速、适合基层推广、不需要使用仪器设备和专业操作人员的试验方法。其中免疫胶体金技术是近些年发展较快的一种快速现场检测技术,但目前我国尚无商品化羊肝片吸虫病胶体金快速检测试剂盒。此外,有关用于诊断的肝片吸虫特异基因报道较少,亟待挖掘。
发明内容
本发明公开一种肝片吸虫特异抗原基因FhSAP-2及医用用途,以及基于该基因抗原制备的检测动物肝片吸虫感染胶体金免疫层析试纸,可用于肝片吸虫感染的血清检测。
本发明所述的一种肝片吸虫特异抗原基因FhSAP-2(Fasciola hepatica SAP-2,简称:FhSAP-2)(GenBank:AYA57790.1),其基因序列如:SEQ No.1所示。
本发明所述的肝片吸虫特异抗原基因FhSAP-2的获取方法,包括以下步骤:
利用感染肝片吸虫绵羊血清对本实验室构建的肝片吸虫cDNA表达文库进行筛选;挑取阳性噬菌斑,PCR扩增后测序,对测序结果进行生物信息学分析;获得肝片吸虫特异抗原基因FhSAP-2。
利用本发明所述的利用肝片吸虫特异抗原基因FhSAP-2制备的一种检测羊肝片吸虫感染的胶体金免疫层析试纸条:通过表达纯化肝片吸虫特异基因抗原FhSAP-2蛋白,利用柠檬酸三钠还原法烧制胶体金溶液,之后利用胶体金溶液对SPA进行标记,制备金标垫,组装试纸条;包括玻璃纤维、硝酸纤维素膜(NC)、PVC底板、吸水板;金标垫上的金标抗体为金黄色葡萄球菌A蛋白(SPA),硝酸纤维素膜上的检测线(T)包被肝片吸虫FhSAP-2、质控线(C)包被兔抗SPA抗体,建立了一种间接胶体金试纸条检测方法。
本发明的积极效果在于:
提供了一种新的肝片吸虫特异抗原基因FhSAP-2(AYA57790.1)。该基因是通过肝片吸虫成虫cDNA表达文库的筛选获得。制备的检测羊等动物肝片吸虫感染胶体金试纸条具有简单、快速、无需特殊仪器设备等特点。检测结果便于判读,适于临床中快速诊断。利用间接胶体金检测方法,其中纯化的肝片吸虫FhSAP-2抗原保证了本发明的特异性。使用金黄色葡萄球菌(SPA)进行标记,成本较低。
附图说明
图1为发明肝片吸虫抗原基因免疫印迹;
图2为发明阳性噬菌体PCR扩增;
图3为发明DNA Star-Protean分析图;
图4为发明 SWISS预测蛋白三维立体图;
图5为发明 SDS-PAGE;
图6为发明 胶体金溶液图;
图7 为发明胶体金透射电镜图;
图8为发明透射电镜观察金标探针;
图9为发明特异性试验结果图;
图10为发明敏感性试验结果图;
图11为发明重复性试验结果图。
具体实施方式
通过以下实施例进一步举例描述本发明,并不以任何方式限制本发明,在不背离本发明的技术解决方案的前提下,对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
一、肝片吸虫成虫cDNA表达文库的筛选
利用感染肝片吸虫的绵羊血清对本实验室构建的肝片吸虫成虫cDNA表达文库进行筛选。挑取阳性噬菌斑,PCR扩增后测序,对测序结果进行生物信息学分析。
二、肝片吸虫特异抗原FhSAP-2蛋白的表达及纯化
1、肝片吸虫特异抗原FhSAP-2蛋白的诱导表达
(1)将FhSAP-2基因与原核表达载体pET-32a连接,并将连接产物转入到表达感受态BL21中,挑取阳性菌落并置于事先分装并高压灭菌后的LB培养基(5mL/管)中,同时加入5μl氨苄青霉素。转入37℃恒温振荡培养箱中,150r/min,振荡过夜。
(2)取振荡过夜的菌液转移至高压灭菌后的300mL LB培养基中同时加入300μl的氨苄青霉素,封口后转入37℃恒温振荡培养箱中进行恒温振荡培养1.5h~2h。
(3)加入300μl的IPTG进行诱导,4~5h后4℃离心机12000 r/min 10min,收集菌液。
(4)将菌液重悬后制样,80μl菌液加入20μl蛋白buffer,沸水中煮10min,离心后进行SDS-PAGE。
(5)取PBS对菌液进行重悬,超声破碎,离心取上清。
2、肝片吸虫特异抗原FhSAP-2蛋白的纯化
(1)收集超离后的上清, 将其转入装有Ni2+-NTA树脂的层析柱中,上样完毕后静置2 h, 以便6个组氨酸标签与填料中Ni2+充分结合;
(2)分别用10倍柱体积的结合缓冲液和6倍柱体积的漂洗缓冲液(1mol/L咪唑,3mol/LNaCl ,10mmol/L Tris-HCl ,pH8.0)过柱以洗去未结合的杂蛋白;
(3)用洗脱缓冲液 (1mol/L咪唑,3mol/L NaCl ,10mmol/LTris.HCl, pH8.0) 将FhSAP-2蛋白洗脱下来。纯化的蛋白用0.22μm的滤膜除菌后, 分别用1.5mL离心管分装,-20℃保存备用;
(7)SDS-PAGE电泳分析。
三、胶体金试纸条的制备
取21mm的NC膜贴于PVC底板上,保证NC膜表面的清洁。裁剪好的金标垫(8mm×3.2mm),金标垫压在NC膜上2mm,样品垫(22mm×32mm),压在金标垫上2mm,吸水纸(32mm×32mm),压在NC膜上2mm,粘在底板上,按压保证各组状部分粘好。每个试纸4mm,密封干燥保存。取试纸条进行抗体的包被划线,检测线(T)划纯化的FhSAP-2蛋白、质控线(C)划兔抗SPA抗体。
实施例2
肝片吸虫特异抗原基因FhSAP-2(AYA57790.1)的获取
1、方法
将XL-Blue菌11200g离心10min,收集菌体,用100μl 10mol/L MgSO4重悬;取200μl菌液与100μl SM缓冲液(含104噬菌体肝片吸虫cDNA表达文库)混匀,37℃孵育30 min;加入4 ml顶层琼脂,充分混匀后迅速倒入37℃预热的NZY琼脂平板上,冷却后封板,37℃温箱中倒置培养6h-8h。取10 mol/L IPTG处理的NC膜覆盖于琼脂平板表面,继续培养6h。从平板上剥离NC膜,加入DAB显色。阳性噬菌斑为周围深、中央浅的环形斑。用记号笔标识出阳性噬菌斑的位置。收集阳性噬菌斑收集于1.5 mL离心管中,重新测定噬菌体的滴度,再用上述方法进行一次筛选,直至得到一致的阳性重组噬菌体。
用SM Buffer洗脱阳性噬菌斑,以噬菌体测序通用引物扩增,插入片段:
上游引物:5'-CTCGGGAAGCGCGCCATTGTGTTGGT-3;
下游引物:5'-ATACGACTCACTATAGGGCGAATTGGCG3';
PCR反应体系:模板1μl,上、下游引物各1μl, 2×Master Mix 10μl, ddH2O 8μl。PCR条件:94℃5min;94℃60s, 61℃60s,72℃1min,共35个循环;72℃延伸10min, PCR产物4℃保存并进行1%琼脂糖电泳鉴定PCR产物;
阳性克隆的序列分析将回收纯化的PCR产物送生物工程北京公司测序,并登陆NCBI进行Blast比对。
、 结果
(1)免疫筛选结果
经过扩增培养,琼脂培养基上出现了明显均一的噬菌体斑。在培养基表面覆盖一层经浸有IPTG的NC膜,继续培养一段时间后,噬菌体中插入的肝片吸虫的cDNA片段在NC膜上进行了翻译表达,并通过感染肝片吸虫绵羊血清和HRP标记的兔抗羊IgG与这些表达的蛋白进行免疫印迹反应,结果NC膜上出现了明显均一的阳性噬菌体斑点印迹,这些印迹与固体琼脂培养基上的噬菌体斑点位置、大小相符(参见图1);
(2)阳性噬菌体斑的PCR扩增
用SM Buffer溶解的阳性噬菌斑溶液为模板,加入通用引物,设定PCR程序,扩增复筛获得的阳性噬菌体插入片段。1~12都是阳性是菌斑PCR的结果(参见图2);
(3)生物信息学分析
NCBI Blast分析显示肝片吸虫特异抗原FhSAP-2,其基因全长为306bp,编码101个氨基酸。利用DNAStar软件分析预测基因编码蛋白的抗原特性分子量大小值之为11495.43ku,理论等电点为4.66。亲水性总平均值为0.236 (>0表示疏水性,<O表示亲水性),表明该蛋白为疏水性蛋白,无跨膜区域;DNAStar结果显示:亲水区域为18-26, 39-40,45-53,55-56,63-67,69-70,76-77,80-81,84-93;潜在的抗原决定簇区域为17-27,29-32,34-37,39-42,44-46,48-52,64-71,75-79,85-91,92-93(图3)。经TMHMM分析,FhSAP-2蛋白无跨膜区。经ProtScale分析,亲水性总平均值为0.236 (>0表示疏水性,<0表示亲水性),表明该蛋白为疏水性蛋白。经Swiss-Model分析,该蛋白寡聚状态为同源二聚体,并预测了该蛋白的三维空间立体结构(参见图4)。
实施例3
羊肝片吸虫感染免疫胶体金试纸条的制备
1、方法
(1)胶体金溶液的制备
选择柠檬酸三钠还原法来进行制备,取99mL四馏水中加入1mL的氯金酸溶液(1%),加入1mL柠檬酸三钠溶液(1%),制备40nm胶体金颗粒;
(2)胶体金颗粒的鉴定
通过透射电镜技术对胶体金进行鉴定,取胶体金溶液滴于镍网上,用滤纸吸干后,电镜进行观察。优质的胶体金颗粒大小均一,成分散状分布无聚集;
(2)金标抗体最佳pH确定
利用0.2mol/L的K2CO3溶液来对胶体金溶液pH进行调试,通过观察颜色的变化来进行判断;
(3)金标抗体的制备
利用摸索好的最佳K2CO3加入量,对胶体金进行标记,标记后的金标抗体溶于复溶液中4℃保存待用;
(4)检测线T包被浓度的选择
对T线包被的蛋白进行1mg/mL,0.5 mg/mL,0.25 mg/mL,0.125 mg/mL进行稀释。按照常规条件进行操作,观察结果T线的显色情况,确定T线包被浓度;
(5)质控线C包被浓度的选择
在确定T线包被浓度的情况下对C线进行摸索,分别按2mg/mL,1mg/mL,0.5 mg/mL,0.25 mg/mL进行稀释。按照常规条件进行操作,观察结果C线的显色情况,确定C线抗体的最佳包被浓度。
、结果
(1)SDS-PAGE
纯化后的FhSAP-2蛋白,经SDS-PAGE电泳分析后,FhSAP-2目的条带单一,大小为28.5KDa(图5);
(2)胶体金溶液的制备
胶体金溶液颜色呈酒红色,溶液透明无杂质(参见图6);
(3)胶体金颗粒的鉴定
透射电镜观察颗粒大小基本一致,约40nm左右,分散均匀,没有聚集(参见图7);
(4)金标抗体最佳pH确定
确定胶体金标记抗体的最佳pH值为加入4μl的0.2mol/L K2CO3;
(5)金标抗体的制备
通过颜色变化和透射电镜观察,在加入6μg的SPA时胶体金颜色不再变化。电镜观察金颗粒周围有一圈蛋白晕,无聚集现象出现,表明标记成功(参见图8);
(6)检测线T包被浓度的选择
将FhSAP-2蛋白稀释到0.5mg/ml时依然可以看到较为清晰的条带,故选择0.5mg/ml为T线的最佳包被浓度;
(7)质控线C包被浓度的选择
结果显示当抗体浓度在1mg/ml时C线颜色较深,所以最终确定C线的最佳包被浓度为1mg/ml。
实施例4
胶体金免疫层析试纸条性能测试
1、方法
测试试纸条的性能,需要进行一系列的试验,主要包括:特异性试验、敏感性试验、重复性试验、保存期试验;
(1)特异性试验
本试验分别选取了羊肝片吸虫阳性参照血清、羊阴性参照血清、双腔吸虫阳性血清、华支睾吸虫阳性血清,进行交叉反应试验;
(2)敏感性试验
选取羊肝片吸虫阳性参照血清按照1:10、1:20、1:40、1:80、1:160、1:320、1:640比例进行倍比稀释。取健康羊血清做阴性参照。等待5-10min观察结果;
(3)重复性试验
选择三个批次的试纸条进行重复性试验,分别滴加羊肝片吸虫阳性参照血清和阴性参照血清进行重复性试验。根据显色情况来评判试纸条的重复性;
(4)稳定性试验
按摸索的最佳条件进行试纸条的组装,放于室温、4℃、37℃,进行封闭干燥储存。分别在1d、7d、30d、60d、90d、120d后进行试验。观察试纸条显色深浅的变化来确定最佳保存期限;
(7)试纸条的初步应用
应用本发明建立好的胶体金检测方法来进行初步临床样品检测。共检测了66份羊血清。计算样品的阳性率。
、结果
(1)特异性试验
本发明分别选取了羊肝片吸虫阳性参照血清、羊阴性参照血清、双腔吸虫阳性血清和华支睾吸虫阳性血清,进行试纸条特异性检测。羊肝片吸虫阳性参照血清T、C线均显色;羊阴性参照血清、双腔吸虫阳性血清和华支睾吸虫阳性血清检测线(T)均不显色,质控线(C)均发生颜色变化,说明试纸条特异性较好(参见图9);
(2)敏感性试验
当稀释至1:160时可看到T线有轻微的条带,在1:640时T线已经没有颜色变化。所以本试验的敏感性为1:160(参见图10);
(3)重复性试验
选取三个不同批次的试纸条来检测羊肝片吸虫阳性参照血清和阴性参照血清,分别进行批间和批内重复,结果显示较一致,说明试纸条的重复性较好(参见图11);
(4)保存期试验
按优化好的条件进行试纸条的组装后,分别封闭干燥存放于室温、37℃、4℃环境当中。分别在1d、7d、30d、60d,进行试验。试纸条在4℃保存的效果较好,可达到60d。室温保存的试纸条相对较好,保存期可达到30d(依然可见轻微条带)。37℃保存的试纸条效果相对差些,保存期在60d时已经失效;
(5)试纸条的初步应用
应用组装好的免疫胶体金试纸条检测方法来对临床66份样品进行初步检测,其中34份显示为阳性,阳性率为51.5%。
序列表
<110> 吉林大学
<120> 一种肝片吸虫特异抗原基因FhSAP-2及医用用途
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 306
<212> DNA
<213> 肝片吸虫FhSAP-2(Fasciola hepatica FhSAP-2,AYA57790.1)
<400> 1
atgaacccac tgttcgtgtt aatgctggcg gctgtgactt tcgcaagctt tgatgcgcca 60
tcaaagcaac caactattga cattgaccta tgtgatatat gcacgaacac gatggatgta 120
ataaagaaaa tgctcgcaga tcagactgtc gaggagcaca ttggatactt ggtgaaatac 180
ttgtgcaaaa tagctcgctc tcaggatgca tgcattgaat ttgttcaaca ggaagtcgat 240
tatattatcg atcacgtaga gcaacacaac gcaacggaaa tctgccgttt aatcaagctg 300
tgctag 306
<210> 2
<211> 26
<212> DNA
<213> 肝片吸虫(肝片吸虫)
<400> 2
ctcgggaagc gcgccattgt gttggt 26
<210> 3
<211> 28
<212> DNA
<213> 肝片吸虫(肝片吸虫)
<400> 3
atacgactca ctatagggcg aattggcg 28
Claims (3)
1.一种肝片吸虫特异抗原基因FhSAP-2,其基因序列如:SEQ No.1所示。
2.如权利要求1所述的肝片吸虫特异抗原基因FhSAP-2的获取方法:包括以下步骤:
利用感染肝片吸虫绵羊血清对本实验室构建的肝片吸虫cDNA表达文库进行筛选;挑取阳性噬菌斑,PCR扩增后测序,对测序结果进行生物信息学分析;获得肝片吸虫特异抗原基因FhSAP-2。
3.利用权利要求1所述利用肝片吸虫特异抗原基因FhSAP-2制备的一种检测羊肝片吸虫感染的胶体金免疫层析试纸条,其特征在于:
通过表达纯化肝片吸虫特异基因抗原FhSAP-2蛋白,利用柠檬酸三钠还原法烧制胶体金溶液,之后利用胶体金溶液对SPA进行标记,制备金标垫,组装试纸条;包括玻璃纤维、硝酸纤维素膜(NC)、PVC底板、吸水板;金标垫上的金标抗体为金黄色葡萄球菌A蛋白(SPA),硝酸纤维素膜上的检测线(T)包被肝片吸虫FhSAP-2、质控线(C)包被兔抗SPA抗体,建立了一种间接胶体金试纸条检测方法。
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