CN111187754B - 一种杂交瘤细胞株及其产生的抗旋毛虫肠道期丝氨酸蛋白酶单克隆抗体与应用 - Google Patents
一种杂交瘤细胞株及其产生的抗旋毛虫肠道期丝氨酸蛋白酶单克隆抗体与应用 Download PDFInfo
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Abstract
一种杂交瘤细胞株及其分泌的单克隆抗体与应用,属于旋毛虫(T.spiralis)防治的技术领域。针对如何对旋毛虫病进行特异性诊断的技术问题,本发明提供了一种杂交瘤细胞株,其保藏编号为CGMCC No.18317,经试验证明由该杂交瘤细胞株所分泌的单克隆抗体Ts‑ZH68‑2A4‑Ab可以与旋毛虫感染阳性猪血清竞争结合Ts‑ZH68抗原,识别肽是222GVDRSATCQGDSGGP236。本发明的单克隆抗体以及该单克隆抗体所识别的Ts‑ZH68蛋白B细胞表位多肽可用于制备成诊断或预防旋毛虫感染的试剂或疫苗,为旋毛虫血清学诊断方法的建立奠定了基础。
Description
技术领域
本发明属于旋毛虫(T.spiralis)防治的技术领域,具体涉及一种杂交瘤细胞株及其产生的抗旋毛虫肠道期丝氨酸蛋白酶单克隆抗体与应用。
背景技术
旋毛虫病主要是由于宿主生食或半生食含有旋毛虫的肉类而引发。人旋毛虫病潜伏期平均为2周,急性期患者的主要症状为发烧、肌肉剧烈疼痛、严重的腹泻、面部水肿以及嗜酸性粒细胞增多等,症状可以持续数周并导致机体衰竭,尤其重度感染者可出现心肌及大脑严重损伤,甚至死亡。
鉴于旋毛虫对公共卫生安全和人类健康造成的极大威胁与危害,世界动物卫生组织(OIE)将旋毛虫病列为屠宰动物强制性检验及首检必检病种,更是除烈性人兽共患病之外可引起突发性公共卫生事件的典型病种之一。目前旋毛虫检测仍然采用相对费时费力,而且灵敏性低的镜检法(3条幼虫/g肉)和集样消化法(1条幼虫/g肉)进行旋毛虫检测。
国内外学者对旋毛虫检测的方法进行了大量的研究,如间接荧光免疫试验,免疫酶染色试验,免疫印迹试验,免疫吸附试验(ELISA)等,酶联免疫吸附试验(ELISA)是进行旋毛虫感染检测的最常用的免疫学方法,其具有高的敏感性,能检测底限可低至每100g肌肉组织中1条幼虫。目前,旋毛虫肌幼虫排泄分泌物ES抗原是OIE及国际旋毛虫委员会规定的唯一的用于血清学抗体检测的标准抗原。然而ES抗原成分复杂,制备繁琐、生产周期长、批次质量不均,而且存在严重的诊断盲区(感染后19d前无法检出)和交叉反应等问题,因而阻碍了其实际应用。
发明内容
针对如何对旋毛虫病进行特异性诊断的技术问题,本发明提供了一种杂交瘤细胞株,其保藏编号为CGMCC No.18317。
本发明还提供了一种抗旋毛虫肠道期丝氨酸蛋白酶的单克隆抗体,是由上述杂交瘤细胞株CGMCC No.18317所分泌。
进一步地限定,所述单克隆抗体所识别的抗原表位多肽氨基酸序列如SEQ IDNO.1所示。
本发明所述的杂交瘤细胞株可用于制备诊断旋毛虫T.spiralis感染的试剂,本发明所述的SEQ ID NO.1所示的抗原表位多肽可用于制备诊断或预防旋毛虫T.spiralis感染的疫苗。
有益效果
单克隆抗体具有与抗原结合的特异性强、纯度好、重复性强、便于质量控制、亲和力好且可大量生产的优势,因而被广泛的应用于免疫学检测方法的建立。所以,筛选出可以分泌抗Ts-ZH68蛋白特异性抗体的杂交瘤细胞株,并鉴定出其所分泌的单克隆抗体所识别的Ts-ZH68蛋白特异性B细胞表位对旋毛虫病的诊断方法的改进,及亚单位疫苗的研制具有重要的意义。
本发明所制备的单克隆抗体与旋毛虫肌幼虫粗提物抗原发生特异性反应。Ts-ZH68竞争ELISA检测结果表明,由杂交瘤细胞株ZH68-2A4所分泌的单克隆抗体Ts-ZH68-2A4-Ab可以与旋毛虫感染阳性猪血清竞争结合Ts-ZH68抗原。
本发明利用肽扫描技术对Ts-ZH68-2A4-Ab所识别的Ts-ZH68抗原B细胞表位进行了鉴定,确定Ts-ZH68-2A4-Ab识别肽是222GVDRSATCQGDSGGP236。
附图说明
图1 SDS-PAGE分析Ts-Zh68表达,M:蛋白分子质量标准;1:空菌2:上清3:包涵体4:纯化后ZH68蛋白;
图2单克隆抗体SDS-PAGE分析,M:蛋白相对分子质量;1:单抗2A4;
图3旋毛虫阳性血清与单抗上清的竞争结果,横坐标为血液稀释倍数,纵坐标为P/N(阳性与阴性血清的比值);
图4抗Ts-ZH68单克隆抗体对虫体可溶性抗原的Western blot分析,M:蛋白分子质量标准;1:2A4单抗;
图5 Ts-ZH68蛋白分段表达的SDS-PAGE分析,1:BL21-pET28a空菌;2:BL21-pET28a-ZH68,3:BL21-pET28a-ZH68-1,4:BL21-pET28a-ZH68-2,5:BL21-pET28a-ZH68-3;
图6 Ts-ZH68蛋白分段表达的Western blot分析,M:蛋白相对分子质量;1::Ts-Zh68-2A4与pET28a空载体诱导后菌体的反应;2:Ts-Zh68-2A4与pET28a-Ts-Zh68-1诱导后菌体的反应3:Ts-Zh68-2A4与pET28a-Ts-Zh68-2诱导后菌体的反应;4:Ts-Zh68-2A4与pET28a-Ts-Zh68-3诱导后菌体的反应;
图7 ELISA分析结果,横坐标为不同的肽段,纵坐标为OD值。
具体实施方式
旋毛虫3日龄成虫期的丝氨酸蛋白酶Ts-ZH68抗原:通过对感染后3天的旋毛虫cDNA表达文库进行免疫学筛选,获得一个高丰度强反应原性的抗原蛋白,命名为ZH68,生物信息学序列分析ZH68蛋白属于丝氨酸蛋白酶。ZH68基因注册号:EΜ263332,蛋白注册号:ABY60762.1。本发明通过进一步免疫印记表明原核表达的重组ZH68抗原可以被猪感染旋毛虫感染15天、30天和60天血清所识别,表明ZH68是旋毛虫血清学检测的理想候选抗原,可以用来改进血清学检测方法。
本发明采用TRIZOL提取旋毛虫(T.spiralis)总RNA,进而利用反转录技术克隆Ts-ZH68,将该cDNA插入原核表达载体pET28a,利用pET28a原核表达载体对Ts-ZH68基因进行原核表达,将所表达出的Ts-ZH68切胶纯化后免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,制备杂交瘤细胞。并应用亲和纯化的重组Ts-ZH68和肌幼虫期ES抗原作为检测用抗原,建立间接ELISA检测方法对阳性杂交瘤细胞进行筛选,最终获得了一株稳定分泌抗Ts-ZH68蛋白单克隆抗体的杂交瘤细胞株,ZH68-2A4,该细胞株于2019年8月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,菌种保藏编号为CGMCC No.18317。本发明所用的主要实验材料和来源如下:
1.主要试剂和药品:Ni纯化柱HisTrapHP购自美国GE公司;胎牛血清、1640培养基购自Biological Indμstries公司;HAT培养基(50×)、HT培养基(50×)和抗体亚类鉴定试剂盒购自sigma公司;Solμble TMB sμbstrate Solμtion购自TIANGEN公司;辣根过氧化物酶(HRP)标记山羊抗鼠IgG抗体购自北京博奥森公司;预染蛋白Marker购自Fermentas公司;限制性内切酶EcoRI和XhoI、反转录酶、Ex Taq DNA聚合酶、T4DNA连接酶购自宝生物(大连)有限公司;ECL发光底物购自北京索莱宝公司。
2.实验动物:6周龄BALB/c小鼠由长春亿斯实验动物技术有限公司提供。
下面具体描述本发明所述的技术方案,下述实施例涉及的反转录、PCR、酶切等分子生物学实验方法,如无特殊说明,均为按照相应产品试剂盒说明书或本领域常规技术方法进行。
实施例1.Ts-ZH68蛋白的原核表达及纯化。
1.引物设计:根据Genbank中已登录的ZH68基因序列(登录号:EΜ263332),设计PCR扩增引物,序列如下:
Ts-ZH68基因扩增所用模板为AD3期成虫cDNA,扩增引物如下:
TsZH68-F:5’-TAACGAATTC GAAAATTCTCCTGAAG-3’;
TsZH68-R:5’-GACGCTCGAG TTACTTAGAAAAGTG-3’。
下划线部分为引入的EcoRI、XhoI酶切位点。
2.旋毛虫T1(T.spiralis)AD3期成虫RNA的提取及反转录
取感染3天旋毛虫肌幼虫的大鼠解剖,剖开小肠,灭菌生理盐水洗涤,置于分离筛滤布上,生理盐水预热至37℃,浸没小肠,37℃孵育4h,含2%双抗的灭菌生理盐水洗涤,弃去上清,收获纯净AD3期成虫。用提取的总RNA进行反转录合成cDNA,体系如下:
RNAase-free水至25μl,
混匀后于42℃反应1h。-20℃保存。
3.表达载体的构建:以反转录获得的3日龄成虫cDNA为模板扩增Ts-ZH68。
PCR反应体系(50μL)如下:
反应条件为95℃预变性5min,95℃45s,55℃45s,72℃45s,循环30个,72℃终延伸10min。对PCR产物胶回收。将胶回收得到的目的基因Ts-ZH68以及原核表达载体pET28a分别进行双酶切,酶切体系如下:
同时,对原核表达载体pET28a进行双酶切,酶切体系如下:
将酶切反应体系置37℃水浴静置2h,之后进行胶回收。将双酶切后的目的基因与pET28a载体进行连接,体系为:10×T4DNA Ligase Bμffer 1μL,酶切后目的基因4μL,酶切后pET28a 1.5μL,T4DNA连接酶1μL,ddH2O 2.5μL。16℃连接过夜。将连接产物全部转化EcoliDH5a感受态细胞,挑取单菌落进行PCR鉴定和测序。阳性重组质粒转化BL21(DE3)感受态细胞。
4.BL21(DE3)-pET28a-Ts-ZH68诱导表达与切胶纯化。
取1ml重组菌菌液加入到100ml的LB培养基中37℃震荡培养至OD600nm约为0.5-0.8时,加IPTG至终浓度为1mmol/L,37℃诱导6-8h。将表达产物进行SDS-PAGE电泳,通过切胶方法进行纯化。将切下的胶块加入适量的PBS将其研磨成碎颗粒,可用于免疫小鼠。
5.BL21(DE3)-pET28a-Ts-ZH68亲和纯化。
诱导后菌液离心后用30mL重悬缓冲液(20mM Tris-HCL PH8.0)重悬。冰浴10min,冰上超声,超声3s间歇3s共处理30min。8000rpm离心10min收沉淀,沉淀用30ml预冷的包涵体洗涤液(2M尿素、20mM Tris-HCL、0.3M NaCL PH8.0)重悬,冰浴10min,冰上超声,超声3s间歇3s共处理10min。此步骤重复3次。8000rpm离心10min收沉淀,沉淀用30ml预冷的PBS洗涤液(含4M尿素的0.01M PBS)重悬,冰浴10min,冰上超声,超声3s间歇3s共处理5min。此步骤重复2次。8000rpm离心10min收沉淀,沉淀用5ml Binding bμffer(8M尿素、20mMTris-HCL、0.3M NaCL、5mM咪唑PH8.0)重悬,4℃溶解过夜。8000rpm离心30min收上清,上清过滤后准备上样。将带有His标签的Ts-ZH68采用AKTA pμrifier100(美国GE Healthcare公司)纯化系统纯化。分别用含有30mmol/L、50mmol/L、100mmol/L、300mmol/L咪唑的Elμtion Bμffer洗脱,纯化后的重组ZH68蛋白进行SDS-PAGE分析。可看到,纯化后的蛋白上清液只有一条清晰明显的条带,且大小与理论值相符(图1),说明本研究获得了较纯的rTs-ZH68蛋白。使用BCA蛋白定量试剂盒,对上述纯化的蛋白进行浓度测定,得出rTs-ZH68蛋白的浓度为0.85mg/mL。
实施例2.杂交瘤细胞株的制备方法。
1.小鼠免疫
以实施例1切胶纯化的重组Ts-ZH68蛋白免疫5只6周龄雌性BALB/c小鼠,共免疫4次,每次免疫间隔两周,免疫剂量为50μg/只加入等体积的佐剂,第一次免疫为弗氏完全佐剂,其他三次为弗氏不完全佐剂,免疫途径为腹腔免疫。
分别在第3次免疫和第4次免疫后1周对小鼠进行断尾采血,分离血清(4℃,3000rpm离心30min),用亲和纯化后的Ts-ZH68-间接ELISA方法检测抗体水平。Ts-ZH68间接ELISA方法操作如下:重组的Ts-ZH68蛋白用包被液(0.1M的碳酸盐缓冲液,PH9.6,Na2CO363.6g,NaHCO333.6 g定容至1000mL)稀释,包被量为0.1μg/孔,每孔100μl。于37℃包被1h后4℃过夜。之后PBST(含0.05%吐温20)洗板3次。用封闭液(5%脱脂乳)37℃封闭2h。之后PBST洗板3次。待检血清倍比稀释(或者杂交瘤上清1:2倍稀释)后加入微孔板,每孔100μl。37℃孵育1h。之后PBST洗板3次。羊抗鼠二抗1:1000倍稀释,与37℃孵育30min。之后PBST洗板4次。37℃显色10min。2M H2SO4终止反应,读取OD450nm处光吸收值。
在细胞融合前3天,对免疫效果好的BALB/c小鼠进行加强免疫,每只小鼠尾静脉注射50μg免疫原。
2.细胞融合
融合前1天准备小鼠饲养层细胞,按照常规方法取BALB/c小鼠腹腔巨噬细胞铺于96孔细胞培养板中待用。断颈处死待取脾的小鼠,无菌取脾并分离脾细胞,按照脾细胞1×108与SP2/0骨髓瘤细胞2.5×107(4:1)的比例混合,用1ml PEG1000进行细胞融合,1min内滴加结束。然后在1min内将预热至37℃的1ml 1640培养基滴加进细胞悬液中,边滴加边搅拌。在3min内将预热至37℃的1ml 1640培养基滴加进细胞悬液中,边滴加边搅拌。最后将10ml37℃的1640培养基缓慢的加入细胞悬液中,整个过程在37℃水浴中操作。1000r/min离心10min,弃上清,用HAT培养基重悬细胞,将细胞均匀的铺至预先铺有饲养细胞的96孔细胞培养板中,放置于37℃,5%CO2培养箱中培养。
3.阳性杂交瘤细胞株的筛选及克隆
利用Ts-ZH68间接ELISA方法筛选阳性杂交瘤细胞株,对反应阳性的杂交瘤细胞进行扩大培养,同时用有限稀释法进行阳性杂交瘤细胞的第1次亚克隆。参考OIETerrestrial Manμal2017 Chapter2.1.20-Trichinellosis所述ES间接ELISA方法再次筛选阳性杂交瘤细胞株,对反应阳性的杂交瘤细胞进行扩大培养,同时用有限稀释法进行阳性杂交瘤细胞的亚克隆,至少亚克隆3次,将亚克隆好的阳性杂交瘤细胞及时冻存。最终获得可以稳定分泌抗Ts-ZH68蛋白单克隆抗体的杂交瘤细胞株,于2019年8月15日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,菌种保藏编号为CGMCC No.18317,命名为ZH68-2A4,并将其分泌的单克隆抗体命名为ZH68-2A4-Ab以下简称为2A4。
实施例3.抗Ts-ZH68蛋白单克隆抗体腹水的制备。
给12周龄左右健康的BALB/c小鼠腹腔注射石蜡油,0.5ml/只,1周后腹腔注射1×106个实施例2制备的杂交瘤细胞,7~10天后当小鼠腹腔极度膨胀时抽取腹水,隔2天抽取一次,收集腹水。腹水利用protein G亲和层析介质进行纯化,纯化前10000rpm离心10min去除红细胞及脂肪,吸取上清液进行纯化。首先将层析柱与注射器用接合器连接,用注射器吸取5-10ml结合缓冲液,注入层析柱,流速为1mL/min,吸取准备好的腹水注入至层析柱中,用10-15mL的洗涤缓冲液冲洗柱子,洗脱杂蛋白,最后用5mL洗脱缓冲液洗脱,收集至离心管中,SDS-PAGE分析(图2)证实单克隆抗体2A4被成功纯化,-20℃保存备用。
单克隆抗体的鉴定:
1.单克隆抗体的亚类鉴定。
按照抗体亚类鉴定试剂盒操作说明书对实施例2所得到单克隆抗体进行亚类鉴定。结果显示本发明单克隆抗体2A4的重链为IgG2a轻链为kappa链。
表1单克隆抗体亚型的鉴定
单抗名称 | lgG1 | lgG2a | lgG2b | lgG3 | lgM | lgA | κ链 | λ链 |
ZH68-2A4-Ab | 0.313 | 1.312 | 0.271 | 0.306 | 0.293 | 0.305 | 0.841 | 0.302 |
2.Ts-ZH68竞争ELISA试验
包被封闭同实施例2所述的Ts-ZH68间接ELISA方法,从1:1开始2倍倍比稀释猪旋毛虫感染阳性和阴性血清,稀释后的待检血清各取50μl分别与抗ZH68单抗上清50μl等体积混合,取混合后溶液100μl加入微孔板,37℃作用1h,之后同间接ELISA,分析单抗上清能否与猪旋毛虫感染阳性血清竞争结合Ts-ZH68抗原。
试验结果证实,本发明所制备的单克隆抗体2A4能够与猪旋毛虫感染阳性血清竞争结合Ts-ZH68蛋白,图3。
表2 2A4单克隆抗体与猪旋毛虫感染阳性血清竞争结合Ts-ZH68抗原的ELISA检测结果
3.Western blot鉴定
将旋毛虫肌幼虫用ddH2O洗涤3次,加入少量ddH2O,在冰浴中以组织研磨器研磨虫体至碎片,再于冰浴中用超声波细胞粉碎仪破碎虫体碎片。电压300V,超声5s间隔9s,工作5次,超声3个循环,光镜下观察虫体已粉碎成较小碎片时,置4℃和-20℃交替冻融5次,冻融后的虫体4℃1600g离心30min,吸取上清即为可溶性抗原。处理后进行SDS-PAGE,然后经电转印将蛋白转移到硝酸纤维素膜上,5%脱脂乳4℃封闭过夜,加入单克隆抗体室温孵育1h,用PBST洗涤3次,再与3000倍稀释的HRP标记的羊抗鼠IgG抗体室温孵育1h,PBST洗涤3次后,用ECL发光底物显色。试验结果证实,本发明所制备的单克隆抗体2A4能与旋毛虫(T.spiralis)肌幼虫可溶性抗原发生特异性反应,图4。
实施例4.B细胞表位多肽的鉴定。
1.Ts-ZH68蛋白的分段截短表达。
以重组质粒pET28a-ZH68的基因序列为模板,设计3对引物,ZH68-1-μp和ZH68-1-down用于扩增Ts-ZH68-1,ZH68-2-μp和ZH68-2-down用于扩增Ts-ZH68-2,ZH68-3-μp和ZH68-3-down用于扩增Ts-ZH68-3,经PCR扩增后将他们分别连接到pET28a上构建重组质粒,分别命名为pET28a-ZH68-1、pET28a-ZH68-2和pET28a-ZH68-3。对含有pET28a-ZH68-1、pET28a-ZH68-2和pET28a-ZH68-3的重组菌液按实施例1的方法分别进行诱导表达,并对其进行SDS-PAGE电泳分析,确定短肽表达成功(图5)。三段蛋白处理后进行SDS-PAGE,按照实施例3的方法将他们分别与单克隆抗体进行Western blot试验。试验结果表明单克隆抗体所针对的B细胞表位位于pET28a-ZH68-2分段蛋白上(图6)。SDS-PAGE分析表明3个蛋白均获得成功表达。经Western blot鉴定,该单抗可与Ts-ZH68-2产生特异性反应,且反应性较强。
ZH68-1-μp 5’-GCTCTAGAATGAGCAGCCATCATCATC-3’
ZH68-1-down 5’-CCCAAGCTTTAAGAGGTCATAAACTAC-3’
ZH68-2-μp 5’-CCGGAATTCATGCTCTCTCTAGTGAT-3’
ZH68-2-down 5’-CCGCTCGAGTAACACAGTAGAATCAATGCT-3’
ZH68-3-μp 5’-CCGGAATTCATGACTGCCCGAAGC-3’
ZH68-3-down 5’-CCGCTCGAGTAAAGTGAATGATGGATCATT-3’
2.单克隆抗体抗原表位的鉴定
应用生物学软件DNAstar对ZH68进行亲水性、抗原性预测,同时利用表位预测程序ABCpred和Bepipred进行表位预测,利用PepScan技术合成如下短肽(表3),再进行间接ELISA试验检测,包被量为0.25μg/孔。结果显示2A4与Ts-ZH68-W5肽段发生特异性反应,结合Western blot结果推断2A4所识别的Ts-ZH68蛋白B细胞表位初步定位于222
GVDRSATCQGDSGGP236,(表4中W5)核酸序列:
GGTGTTGACCGCTCTGCAACATGTCAGGGTGATTCTGGTGGACCA。
表3合成短肽
表4 ELISA分析结果
综上所述,本发明制备并鉴定出了一种针对Ts-ZH68蛋白的单克隆抗体,本发明的单克隆抗体以及该单克隆抗体所识别的Ts-ZH68蛋白B细胞表位多肽可用于制备成诊断或预防旋毛虫(T.spiralis)感染的试剂或疫苗,为旋毛虫(T.spiralis)血清学诊断方法的建立奠定了基础。
氨基酸和核苷酸序列表
<110> 吉林大学
<120> 一种杂交瘤细胞株及其产生的抗旋毛虫肠道期丝氨酸蛋白酶单克隆抗体与应用
<130>
<160> 17
<170> PatentIn version 3.5
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ccggaattca tgactgcccg aagc 24
<210> 16
<211> 30
<212> DNA
<213> ZH68-3-down
<400> 16
ccgctcgagt aaagtgaatg atggatcatt 30
<210> 17
<211> 45
<212> DNA
<213> 识别肽核苷酸序列
<400> 17
ggtgttgacc gctctgcaac atgtcagggt gattctggtg gacca 45
Claims (3)
1.一种杂交瘤细胞株,其保藏编号为CGMCC No.18317。
2.一种抗旋毛虫(Trichinella spiralis)肠道期丝氨酸蛋白酶的单克隆抗体,是由权利要求1所述的杂交瘤细胞株CGMCC No.18317所分泌。
3.权利要求1所述的杂交瘤细胞株在制备诊断旋毛虫(Trichinella spiralis)感染的试剂中的应用。
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US17/111,608 US11459402B2 (en) | 2020-01-21 | 2020-12-04 | Hybridoma cell strain and monoclonal antibody produced therefrom against serine protease of Trichinella spiralis in intestinal stage and application thereof |
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