CN107253991B - 一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测试剂盒 - Google Patents
一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测试剂盒 Download PDFInfo
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Abstract
本发明公开了一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测试剂盒。所述试剂盒包括包被了斜带石斑鱼生长激素特异性单链单克隆抗体的微孔反应板,重组斜带石斑鱼生长激素蛋白标准品,铕标记的斜带石斑鱼生长激素特异性单链单克隆抗体,所述斜带石斑鱼生长激素特异性单链单克隆抗体的核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。本发明提供的试剂盒可以检测斜带石斑鱼血清或垂体裂解液等样品中的生长激素浓度,检测灵敏度可达到0.131ng/mL,为石斑鱼生长激素的研究提供了有效的检测手段。
Description
技术领域
本发明涉及生物分析化学、纳米生物技术领域,具体地,涉及一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测试剂盒。
背景技术
石斑鱼是海洋珊瑚礁鱼类,属鮨科(Serranidae),石斑鱼属(Epinephelus),有30多种。斜带石斑鱼是我国南方及东南亚一带重要的经济养殖鱼类,肉质鲜美,市场价值高。石斑鱼的生长主要通过生长激素(Growth hormon,GH)调节。鱼类GH为不含糖基的蛋白质,由垂体远端的嗜酸性细胞合成分泌,为一级结构中含有两个二硫键的单链。同一目鱼类中,GH氨基酸组成同源性约为80%。斜带石斑鱼生长激素的cDNA全长为615 bp,编码204个氨基酸,成熟肽含有187个氨基酸。鱼类GH参与营养物质的能量代谢,在鱼类生长、繁殖及渗透压调节等方面起到重要作用。
检测斜带石斑鱼GH大多采用放射免疫分析、酶免疫分析、Western blot等方法,但放射免疫分析会造成放射性污染,且试剂盒保存时间短。酶联免疫分析为定性或半定量方法。Western blot操作繁琐,耗时长,通量小。
时间分辨荧光免疫分析技术(TRFIA)是继放射免疫后标记物发展起来的一种技术,已成为生物医学研究和临床生化检验中一项重要的分析手段。TRFIA利用镧系荧光配合物作为荧光标记物标记配体或受体,发生免疫反应后,根据时间分辨荧光仪测到的最后产物中的荧光强度和相对荧光强度的比值,推测反应体系中分析物的浓度,达到定量分析的目的。利用镧系稀土离子荧光寿命长的特点,在每个激发光脉冲过后,通过时间延缓期,让短寿命的荧光衰变掉后,再记录稀土螯合物的荧光强度,几乎可以完全消除背景荧光的干扰,提高灵敏度。TRFIA具有操作简便、储存时间长、无放射性污染、试剂盒稳定性好、检测不受样品本底荧光干扰、标准曲线范围宽、应用范围广等优点。非常适合定量要求高,重复性好、需批量处理的检测样品。
目前TRFIA技术已广泛应用于多种临床检测项目中,但运用该技术检测斜带石斑鱼GH尚无文献报道。
发明内容
本发明的目的是为了克服现有技术的上述不足,提供一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测试剂盒。
为了实现上述目的,本发明是通过以下技术方案予以实现的:
一种斜带石斑鱼生长激素特异性单链单克隆抗体,所述抗体的核苷酸序列如SEQID NO:1所示,氨基酸序列如SEQ ID NO:2所示。
斜带石斑鱼生长激素特异性单链单克隆抗体的制备方法为:将重组斜带石斑鱼GH蛋白免疫新西兰大耳兔,提取兔血淋巴细胞RNA,反转录构建噬菌体单链单克隆抗体库,使用重组斜带石斑鱼GH蛋白筛选特异性单链抗体,并转入pET32a+质粒中进行表达纯化。
一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测试剂盒,所述试剂盒包括包被了斜带石斑鱼生长激素特异性单链单克隆抗体的微孔反应板,重组斜带石斑鱼生长激素蛋白标准品,铕标记的斜带石斑鱼生长激素特异性单链单克隆抗体,所述斜带石斑鱼生长激素特异性单链单克隆抗体的核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。
所述铕标记的斜带石斑鱼生长激素特异性单链单克隆抗体的制备方法为,将斜带石斑鱼生长激素特异性单链单克隆抗体进行Eu3+标记,抗体与Eu3+标记物的质量比例为6:1。
重组斜带石斑鱼生长激素蛋白标准品为:用含50mM Tris-HCl、0.9% NaCl、0.02%BSA、0.05% NaN3、0.05% Tween-20,pH 7.8的缓冲液,将重组斜带石斑鱼GH蛋白配制成0ng/mL、0.244 ng/mL、0.977 ng/mL、3.906 ng/mL、15.625 ng/mL、62.6 ng/mL、250ng/mL系列浓度的校准溶液,按每份500 uL配制,现配现用。
本发明提供的基于时间分辨荧光免疫分析技术和异源性抗体的罗非鱼生长激素定量检测试剂盒还包括分析缓冲液、洗涤液、增强液。
优选地,所述分析缓冲液为50mM Tris-HCl、0.9% NaCl、0.02% BSA、0.05% NaN3、0.05% Tween-20,pH 7.8的缓冲液。
优选地,所述洗涤液(25×)由1.25M Tris-HCl、22.5% NaCl、5% Tween-20、0.625%NaN3,pH 7.8的分析缓冲液组成。
优选地,斜带石斑鱼生长激素特异性单链单克隆抗体包被96孔微孔板的方法为:用包被缓冲液稀释斜带石斑鱼生长激素特异性单链单克隆抗体至适当浓度,4℃过夜包被至96孔微孔板,用封闭缓冲液封闭过夜,弃去封闭液,拍干保存;其中包被缓冲液为:pH 8.0的50mM Tris-HCl缓冲液;封闭缓冲液为含有0.05% NaN3、1% BSA,15% 蔗糖的pH 7.8的50mM Tris-HCl缓冲液。
本发明的测定方法为:往包被了斜带石斑鱼生长激素特异性单链单克隆抗体的96孔微孔反应板中加入100μL系列浓度梯度稀释的GH标准品或待检样品,25 ℃反应1 h,用洗涤液洗板6次,拍干,再加入100μL 1:200倍稀释的铕标记的斜带石斑鱼生长激素特异性单链单克隆抗体,25 ℃震荡孵育反应1 h,用洗涤液洗板6次,拍干,最后加入200uL每孔的增强液,室温振摇5min后,在时间分辨荧光检测仪上按照所编程序测定。
本发明的试剂盒的特别适合的检测对象为鱼血清、组织细胞裂解液或细胞孵育液,定量检测溶液中的斜带石斑鱼生长激素含量。
与现有技术相比,本发明具有如下有益效果:
本发明建立了夹心法测定样品中的斜带石斑鱼生长激素水平,测试灵敏度可以达到0.131 ng/mL,为石斑鱼生长激素的研究提供了有效的检测手段。
附图说明
图1是PCR扩增产物的电泳鉴定图;从图1可见PCR扩增了一段长约582bp的序列。
图2为质粒构建酶切分析图,酶切后相应大小处有条带。
图3为重组斜带石斑鱼GH蛋白表达纯化及Western blot验证;重组GH纯化,M:marker、1:菌体裂解、2:破碎上清、3:包涵体、4-6:200mM 咪唑洗脱。纯化得到约23.24 kD的目的蛋白。His抗体验证,在相应大小处有条带。
图4为多克隆抗体效价验证; A 图为M:marker、重组GH蛋白上样量从1-3分别为100 ng、50 ng、10 ng。 B 图为斜带石斑鱼垂体裂解蛋白10μL(1/10个斜带石斑鱼垂体,鱼体重约为800 g)。
图5是3F7号克隆的测序结果及预测氨基酸序列;下划线部分为Linker序列。
图6是斜带石斑鱼GH特异性单链单克隆抗体表达和纯化;M:marker、N:未诱导对照、1:菌体裂解、2:破碎上清、3:包涵体、4:200 mM 咪唑洗脱。纯化得到约52 kD的目的蛋白。
图7是斜带石斑鱼GH标准曲线;A图:系列浓度梯度稀释的GH标准品和血清稀释(1:2/4/8)的曲线基本平行,B图:使用梯度稀释的重组斜带石斑鱼Somatolactin蛋白(Gp-SL)及Prolactin蛋白(Gp-PRL)、重组罗非鱼TSH β蛋白(Ti-TSHβ)进行测定,结果表明重组斜带石斑鱼Somatolactin蛋白、Prolactin蛋白及重组罗非鱼TSHβ蛋白稀释曲线不与标准曲线平行且测定值较低,说明无交叉反应。
具体实施方式
下面结合具体实施例对本发明作出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1
重组斜带石斑鱼生长激素蛋白的表达纯化
一、斜带石斑鱼重组GH蛋白表达菌株的构建
(1)斜带石斑鱼GH成熟肽序列的扩增
根据已发表的斜带石斑鱼GH基因的序列(GenBank no. AY155226.1)及限制性内切酶Bam HI和Hind III的识别序列,设计合成一对特异性引物(invitrogen公司),上游引物为 5’ CGGGGTACCCAGCCAATCACAGACGGCCA 3’共29bp, 是在GH基因的成熟肽序列前添加了BamHI酶切识别位点,下游引物为5’ CCCAAGCTTCTACAGGGTACAGTTGGCCT 3’共29bp,是在GH基因成熟肽序列后添加了Hind III酶切识别位点。取斜带石斑鱼垂体,提取总RNA,反转录为cDNA,再作为模版进行PCR反应,使用KOD Plas Neo高保真PCR酶(TOYOBO)进行PCR,反应条件为:95℃预变性3分钟;接着为35个循环,95℃变性15秒,65℃退火15秒,68℃延伸30秒;最后68℃延伸5分钟。
PCR扩增产物的电泳鉴定图见图1。从图1可见PCR扩增了一段长约582 bp的序列。
(2)含斜带石斑鱼GH基因的大肠杆菌表达载体PQE30-GH的构建
PCR产物用限制性内切酶BamHI和Hind III(NEB)双酶切后,酶切产物用E.Z.N.A.® Gel Extraction Kit回收,进行琼脂糖凝胶电泳,分离纯化约582 bp的斜带石斑鱼GH基因片段;载体质粒PQE30经限制性内切酶BamHI和Hind III双酶切后分离纯化3.6 kb的大片段,与582 bp的斜带石斑鱼GH基因片段按1:3混合,用NEB T4连接酶16℃连接16小时后用标准氯化钙转化法转入大肠杆菌Trans Jm109中,用LB平板筛选具Ampicillin抗性的转化子,用E.Z.N.A.® Plasmid Mini Kit提取质粒,筛选大小约4.2 kb的重组质粒,用限制性内切酶BamHI和Hind III(NEB)双酶切重组质粒,得到582 bp和3.6 kb的两个片段,大小分别与斜带石斑鱼GH基因和表达载体PQE30大小相同,测序结果与预期相同,证明斜带石斑鱼GH基因已克隆入大肠杆菌表达载体PQE30中,重组质粒命名为PQE30-GH。质粒构建酶切验证图见图2,测序由invitrogen公司完成。
(3)斜带石斑鱼生长激素基因大肠杆菌表达菌株M15-PQE30-GH的构建
将PQE30-GH质粒用标准氯化钙转化法转入大肠杆菌M15中,用LB平板筛选具Ampicillin和Kanamycin抗性的转化子,得到M15-PQE30-GH菌株。
二、斜带石斑鱼重组GH蛋白的制备
(1)挑取M15-PQE30-GH单菌落接种在10mL带有Ampicillin和Kanamycin 的LB液体培养基中,37℃,200 rpm/min培养16 h。
(2)将菌液按照1:100的比例接种到1L带有Ampicillin和Kanamycin抗性的LB液体培养基中,37℃,200 rpm/min培养至OD为0.5-0.6,加入IPTG至终浓度为1 mM,取10 mL菌液作为未诱导的对照。诱导4h后,6000×g离心15min,收集菌体。
(3)按培养基比binding buffer(不含尿素)为100:1的比例向菌体中加入bindingbuffer,重悬菌体,置于冰上,用超声波细胞粉碎仪进行超声破碎30 min。用Beckman冷冻超速离心机12000×g离心15 min,上清为细胞内可溶蛋白,沉淀为包涵体。
(4)弃去上清,包涵体用含有8M尿素的binding buffer溶解。
(5)各取1mL未诱导及诱导后的菌体,12000×g离心5min,弃去上清,加入50uL PBS作为样品,上清和包涵体溶解液分别取20ul,加入20ul 2×SDS蛋白上样缓冲液,沸水浴5min,进行SDS-PAGE,考马斯亮蓝染色10min,脱色液脱色至背景清晰。M15-PQE30-GH在23.24 kD处有条带,而未诱导对照不出现此带。包涵体蛋白用GE公司的镍柱进行纯化,用3kD超滤管(PALL)脱盐。使用碧云天的BCA蛋白浓度测定试剂盒,按说明书方法操作,进行蛋白浓度的测定。
将重组斜带石斑鱼生长激素蛋白采用免疫印迹(Western blot)方法进行免疫活性鉴定。一抗为His单克隆抗体(Novagen公司)和抗GH兔多克隆抗体(实验室制备),二抗为羊抗兔IgG-HRP(博士德生物工程有限公司)。显示抗His单克隆抗体和抗GH兔多克隆抗体均能识别我们用大肠杆菌表达的重组GH蛋白,证明得到的蛋白是重组斜带石斑鱼GH,蛋白表达纯化及Western blot验证见图3。
实施例2
斜带石斑鱼GH特异性单链单克隆抗体的制备
一、重组斜带石斑鱼生长激素兔多克隆抗体的制备及纯化
(1)从广州中医药大学实验动物中心购买新西兰大耳兔3只(雄性,3-4月龄,体重2.5-3kg),将家兔编号为A /B /C,A/B注射斜带石斑鱼重组GH蛋白,C为对照注射等量的PBS与佐剂的混合物。每次注射取一定量纯化的斜带石斑鱼重组GH蛋白用PBS稀释,与等体积弗氏佐剂(Sigam)混匀乳化,在家兔背部皮下多点注射,共免疫四次,除第一次用弗氏完全佐剂外,再次免疫均用弗氏不完全佐剂。
(2)免疫方案:首次注射前,分别取2ml血液作为空白对照。四次免疫分别在第1天、第11天、第18天、第25天。其中第1天和第25天蛋白用量为500 ug,第11天和第18天蛋白用量为200 ug。
(3)每次注射前取2mL血清采用免疫印迹(Western blot)方法进行抗体效价验证,效价达到1:10000以上时,第34天禁食一天,在第35天通过颈动脉放血,离心取得血清,即兔抗GH多克隆抗体。用免疫印迹(Western blot)方法进行抗体效价验证,一抗为制备的兔抗GH多克隆抗体,二抗为羊抗兔IgG-HRP(博士德生物工程有限公司),抗体效价验证结果如图4,制备的GH多克隆抗体可做为之后筛选单链单克隆抗体的阳性对照。
二、斜带石斑鱼GH单链单克隆初级抗体库的构建
(1)采用Ficoll密度梯度离心法分离兔外周血淋巴细胞,取得实施例2(一)中家兔抗凝血后,加入与血液等体积的D-Hank’s溶液,混匀,小心将10mL血液稀释液加到预先加入5mL淋巴细胞分离液(天津灏洋)的15mL离心管液面上,使用水平转子500×g离心40min。离心后吸取单核细胞层的细胞,使用D-Hank’s洗涤2次,提取RNA,使用Olligo dT引物(Life公司)进行反转录备用。
(2)根据NCBI上兔IgG序列,比对保守区,设计兔IgG重链和轻链可变区基因片段(VH、VL)克隆引物,重链可变区上游引物:VHF1 TCCGGGGGTCGCCT、VHF2 TCCGGGGGAGRC、VHF3TCCGGGGGT、VHF4 GAGTCCGGGGG;重链可变区下游引物:VHR1 TGARGAGAYGGTGACSAGGG、VHR2GACTGATGGAGCCTTAGGTT;轻链可变区上游引物:VLF1 GWKATGACCCAGACTCCA;轻链可变区下游引物:VLR1 AGGTGCAACTGGATCACC,引物由invitrogen公司合成。
(3)克隆VH和VL基因片段,通过Linker进行连接,组成单链抗体(scFv片段),Linker编码的一段富含Gly的连接肽的序列为:SGGGSGGGGGGGSGGGGS,可将VH和VL肽段连接并且不会对两个肽段的空间结构有太大影响。
(4)将scFv片段连接到pCANTAB5e载体上,电转化到TG1宿主菌中,即为斜带石斑鱼GH单链单克隆初级抗体库,噬菌体抗体库构建试剂盒购自北京宝科维食安公司。
三、斜带石斑鱼GH单链单克隆抗体的筛选和表达纯化
(1)将纯化后的重组斜带石斑鱼GH蛋白包被在5mL免疫管(NUNC)中,对斜带石斑鱼GH单链单克隆初级抗体库进行3轮筛选,得到斜带石斑鱼GH单链单克隆三级抗体库。
(2)将3级抗体库的单克隆进行菌液PCR,阳性的菌落用Phage ELISA及scFv ELISA筛选使用二抗分别为HRP-anti M13 antibody (GE公司)和HRP-anti E Tag antibody(abCam公司),得到阳性最高的一个菌,将其编号为3F7,3F7号克隆的测序结果如图5。
(3)将3F7片段克隆到pET32a+载体上,命名为pET32-GHab-3F7,将其转到BL21Trxb菌株中进行表达,纯化得到52 kD的斜带石斑鱼GH特异性单链单克隆抗体,如图6。将抗体透析并超滤浓缩备用。
实施例3
斜带石斑鱼GH时间分辨荧光免疫检测试剂盒的制备
(1)斜带石斑鱼GH特异性单链单克隆抗体包被96孔微孔反应板: 用包被缓冲液稀释斜带石斑鱼GH特异性单链单克隆抗体至适当浓度,4℃过夜包被至96孔微孔板,用封闭缓冲液封闭过夜,弃去封闭液,拍干保存。其中包被缓冲液为:pH 8.0的50mM Tris-HCl缓冲液;封闭缓冲液为含有0.05% NaN3、1% BSA,15% 蔗糖的pH 7.8的50mM Tris-HCl缓冲液。
(2)纯化后的斜带石斑鱼GH特异性单链单克隆抗体进行Eu3+标记,抗体与Eu3+标记物的比例为6:1(质量比)为最佳比例。铕标记斜带石斑鱼GH特异性单链单克隆抗体的制备步骤为:
斜带石斑鱼GH特异性单链单克隆抗体的纯化和浓缩:将1.2mg重组斜带石斑鱼GH蛋白加入0.5 mL标记缓冲液(50mM Na2CO3,pH 9.0),混匀后,用Millipore公司的离心柱进行浓缩,8500 rpm离心5min,重复6次,弃去滤出液,将滤膜反转离心收集重组蛋白,体积控制在200 uL左右。
斜带石斑鱼GH特异性单链单克隆抗体的标记:将纯化的重组蛋白加入0.2 mg Eu3+标记试剂中(广州达瑞公司),充分混匀,25℃震荡20 h。
标记蛋白的纯化:用Sephadex G-50层析柱(1×45cm,GE公司)分离纯化,洗脱缓冲液(50mM Tris-HCl,0.9% NaCl,0.05% NaN3,pH 7.8)洗脱,收集流出液1mL/管,用BCA法(碧云天公司)逐管测定蛋白浓度,用增强液稀释1000-10000倍测定荧光强度。合并蛋白浓度最高的几管,混匀并计算标记率。
确定稀释度:合并后的铕标记物,进行稀释度摸索,选择线性较好,灵敏度较高的稀释度。
分装保存:向标记物中加入高纯度的BSA至终浓度为0.1 %,将标记物分为100 uL/管,-20℃保存。
(3)标准品制备步骤为:用含50mM Tris-HCl、0.9% NaCl、0.02% BSA、0.05% NaN3、0.05% Tween-20, pH 7.8的缓冲液,将重组斜带石斑鱼GH蛋白配制成0 ng/mL、0.244 ng/mL、0.977 ng/mL、3.906 ng/mL、15.625 ng/mL、62.6 ng/mL、250ng/mL系列浓度的校准溶液,按每份500 uL配制,现配现用。
(4)试剂盒包含的溶液:
分析缓冲液:50mM Tris-HCl、0.9% NaCl、0.02% BSA、0.05% NaN3、0.05% Tween-20,pH 7.8的缓冲液。
洗涤液母液(25×):由1.25M Tris-HCl、22.5% NaCl、5% Tween-20、0.625% NaN3,pH 7.8的分析缓冲液组成。
增强液购于(广州达瑞公司)。
实施例4
本发明的试剂盒的使用方法
(1)样品收集:取斜带石斑鱼血清或其他含GH待检样品100uL,2-8℃保存一周,长时间保存在-80℃。
(2)试剂的准备:洗涤液:将80mL洗涤液母液(25×)和1920mL去离子水混合,作为工作洗涤液。标记物工作液:取标记物,按1:200稀释,作为铕标重组蛋白标记物工作液。GH蛋白标准品:按上述标准品制备步骤制备浓度梯度的标准品。
(3)操作步骤:往包被了斜带石斑鱼GH特异性单链单克隆抗体的96孔微孔反应板中加入100ul系列浓度梯度稀释的GH标准品或待检样品,25 ℃反应1 h,用洗涤液洗板6次,拍干,再加入100μL 1:200倍稀释的铕标记的斜带石斑鱼生长激素特异性单链单克隆抗体,25 ℃震荡孵育反应1 h,用洗涤液洗板6次,拍干,最后加入200uL每孔的增强液,25 ℃振摇5min后,在时间分辨荧光检测仪(如PerkinElmer VICTOR X5多功能酶标仪)上按照所编程序测定。
实施例5
本发明的试剂盒的方法学检定
按照本领域中常规的制造和检定规程对通过实施例4中制备成的试剂盒进行检定,结果如下:
(1)准确度:对系列浓度梯度稀释的标准品与血清稀释样品(1:2/4/8)同时进行分析测定后,用双对数数学模型拟合,两条曲线基本平行(图7-A)。GH标准曲线的相关系数R=0.977。
(2)分析灵敏度和线性范围:将零参考标准品测量9次,计算其荧光值及标准差。以该点荧光测定平均值加2倍标准差所得的荧光值代入标准曲线方程计算得出的浓度值为其灵敏度,经测定本试剂盒灵敏度为0.131 ng/mL。将抗原稀释成不同浓度进行测定,测得标准曲线线性范围为0.131-250 ng/mL。
(3)精密度(CV%):用本发明试剂盒对自制GH质控品(预期浓度为35 ng/mL)进行测定,各3个重复。结果本发明试剂盒的组内变异系数CV%≤12.8,组间变异系数CV%≤10.4。
表1 组内精密度测试结果(n=3)
测定值 | GH(ng/mL) | CV% | GH(ng/mL) | CV% | GH(ng/mL) | CV% |
质控品 | 34.958 | 12.8 | 36.875 | 7.8 | 30.002 | 3.1 |
表2 组间精密度测试结果(三批,n=3*3)
测定值 | GH(ng/mL) | CV% |
质控品 | 33.945 | 10.4 |
(4)特异性:使用斜带石斑鱼重组Somatolactin及Prolactin蛋白以及重组罗非鱼TSHβ蛋白作为样品用本试剂盒进行测定,结果表明稀释曲线不与标准曲线平行且测定值低(图7-B),说明无交叉反应,如表3。
表3 GH试剂盒特异性检测结果
检测项目 | Somatolactin | Prolactin | TSHβ |
样品浓度(ng/mL) | 1000 | 1000 | 1000 |
测定浓度(ng/mL) | 0.42 | 4.72 | 0.36 |
以上内容是结合具体的优选实施方式对本发明所做的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,可以做出若干简单推演或替换,都应当视为属于本发明的保护范围。
SEQUENCE LISTING
<110> 中山大学,中山大学深圳研究院
<120> 一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测
试剂盒
<130>
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 795
<212> DNA
<213> GHab-3F7核苷酸序列
<400> 1
atgtccgggg gtcgcctggt aacgcctgga ggatccctga cactcacctg cacagtctct 60
ggaatcgacc tcaataccta ctatatgagc tgggtccgcc aggctccagg aaaggggctg 120
gaatacatcg gattcattga tactggtggt gccacatact acgcgacctg ggcgaaaggc 180
cgattcacca tctccaaaac ctcgaccacg gtgaatctga agatcaccag tccgacaacc 240
gaggacacgg ccacctattt ctgtgccaga gggggtactg gtagtaattt gtggggccca 300
ggcaccctgg tcaccgtctc ctcagggcaa cctaaggctc catcagtcgg ccaaggccac 360
ggtcaccgtc tcctcagtgg aggcggttca ggcggaggtg gcggaggtgg ctctggcggt 420
ggcggatcgg acattgagct cacccagtct ccagatatga cccagactcc agcctccgtg 480
gaggcagctg tgggaggcac agtcaccatc aagtgccagg ccagtcagag cattggtagt 540
aatttagcct ggtatcagca gaaaccaggg cagcctccca agctcctgat ctattctaca 600
tccactctgg catctggggt ctcatcgcgg ttcaaaggca gtggatctgg gacacacttc 660
actctcacca tcggcggcgt gcagtgtgac gatgctgcca cttactactg tcaaggcggt 720
tattataata gtggttggta cttggctttc ggcggaggga ccgaggtggt ggtcaaaggt 780
gatccagttg cacct 795
<210> 2
<211> 265
<212> PRT
<213> GHab-3F7氨基酸序列
<400> 2
Met Ser Gly Gly Arg Leu Val Thr Pro Gly Gly Ser Leu Thr Leu Thr
1 5 10 15
Cys Thr Val Ser Gly Ile Asp Leu Asn Thr Tyr Tyr Met Ser Trp Val
20 25 30
Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile Gly Phe Ile Asp Thr
35 40 45
Gly Gly Ala Thr Tyr Tyr Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile
50 55 60
Ser Lys Thr Ser Thr Thr Val Asn Leu Lys Ile Thr Ser Pro Thr Thr
65 70 75 80
Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Gly Gly Thr Gly Ser Asn
85 90 95
Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys
100 105 110
Ala Pro Ser Val Gly Gln Gly His Gly His Arg Leu Leu Ser Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
130 135 140
Ile Glu Leu Thr Gln Ser Pro Asp Met Thr Gln Thr Pro Ala Ser Val
145 150 155 160
Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln
165 170 175
Ser Ile Gly Ser Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro
180 185 190
Pro Lys Leu Leu Ile Tyr Ser Thr Ser Thr Leu Ala Ser Gly Val Ser
195 200 205
Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile
210 215 220
Gly Gly Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly
225 230 235 240
Tyr Tyr Asn Ser Gly Trp Tyr Leu Ala Phe Gly Gly Gly Thr Glu Val
245 250 255
Val Val Lys Gly Asp Pro Val Ala Pro
260 265
<210> 3
<211> 29
<212> DNA
<213> GH上游引物
<400> 3
cggggtaccc agccaatcac agacggcca 29
<210> 4
<211> 29
<212> DNA
<213> GH下游引物
<400> 4
cccaagcttc tacagggtac agttggcct 29
<210> 5
<211> 14
<212> DNA
<213> VHF1
<400> 5
tccgggggtc gcct 14
<210> 6
<211> 12
<212> DNA
<213> VHF2
<400> 6
tccgggggag rc 12
<210> 7
<211> 9
<212> DNA
<213> VHF3
<400> 7
tccgggggt 9
<210> 8
<211> 11
<212> DNA
<213> VHF4
<400> 8
gagtccgggg g 11
<210> 9
<211> 20
<212> DNA
<213> VHR1
<400> 9
tgargagayg gtgacsaggg 20
<210> 10
<211> 20
<212> DNA
<213> VHR2
<400> 10
gactgatgga gccttaggtt 20
<210> 11
<211> 18
<212> DNA
<213> VLF1
<400> 11
gwkatgaccc agactcca 18
<210> 12
<211> 18
<212> DNA
<213> VLR1
<400> 12
aggtgcaact ggatcacc 18
<210> 13
<211> 18
<212> PRT
<213> 连接肽
<400> 13
Ser Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly
1 5 10 15
Gly Ser
Claims (4)
1.一种斜带石斑鱼生长激素特异性单链单克隆抗体,其特征在于,所述抗体的核苷酸序列如SEQ ID NO:1所示,氨基酸序列如SEQ ID NO:2所示。
2.一种基于时间分辨荧光免疫分析技术的斜带石斑鱼生长激素特异性定量检测试剂盒,其特征在于,所述试剂盒包括包被了斜带石斑鱼生长激素特异性单链单克隆抗体的微孔反应板,重组斜带石斑鱼生长激素蛋白标准品,铕标记的斜带石斑鱼生长激素特异性单链单克隆抗体,所述斜带石斑鱼生长激素特异性单链单克隆抗体的核苷酸序列如SEQ IDNO:1所示,氨基酸序列如SEQ ID NO:2所示。
3.根据权利要求2所述的试剂盒,其特征在于,所述铕标记的斜带石斑鱼生长激素特异性单链单克隆抗体的制备方法为,将斜带石斑鱼生长激素特异性单链单克隆抗体进行Eu3+标记,抗体与Eu3+标记物的质量比例为6:1。
4.根据权利要求2所述的试剂盒,其特征在于,所述重组斜带石斑鱼生长激素蛋白标准品为:用含50mM Tris-HCl、0.9%NaCl、0.02%BSA、0.05%NaN3、0.05%Tween-20,pH 7.8的缓冲液,将重组斜带石斑鱼GH蛋白配制成0ng/mL、0.244ng/mL、0.977ng/mL、3.906ng/mL、15.625ng/mL、62.6ng/mL、250ng/mL系列浓度的校准溶液。
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CN104371007A (zh) * | 2014-11-14 | 2015-02-25 | 中山大学 | 一种能促进罗非鱼生长激素表达的多肽pp1及其应用 |
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