CN108659132B - 一种组合蛋白制备抗人胱抑素c多克隆抗体的方法和应用 - Google Patents
一种组合蛋白制备抗人胱抑素c多克隆抗体的方法和应用 Download PDFInfo
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- CN108659132B CN108659132B CN201710196427.7A CN201710196427A CN108659132B CN 108659132 B CN108659132 B CN 108659132B CN 201710196427 A CN201710196427 A CN 201710196427A CN 108659132 B CN108659132 B CN 108659132B
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- human cystatin
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Abstract
本发明公开的是一种组合蛋白制备抗人胱抑素C多克隆抗体的方法和应用,主要解决了现有单一抗原制备的抗人胱抑素C多克隆抗体特异性差、有效浓度低,不能满足诊断试剂制备要求的问题。本发明包括在动物免疫、抗血清效价检测、多克隆抗体的抗原‑抗体亲和纯化三个步骤中均采用了重组人胱抑素C蛋白;动物免疫中采用一种重组人胱抑素C蛋白用于动物免疫;抗血清效价检测中采用一种重组人胱抑素C蛋白用于酶联免疫检测中酶标板的包被;多克隆抗体的抗原‑抗体亲和纯化中采用一种重组人胱抑素C蛋白用于偶联填料并纯化抗体。本发明制备的抗人胱抑素C多克隆抗体可作为重要生物原料应用于胱抑素C检测试剂的制备,其试剂性能满足诊断要求。
Description
技术领域
本发明属于生物工程技术领域,具体涉及的是一种利用不同重组人胱抑素C蛋白的组合制备抗人胱抑素C多克隆抗体的方法及其应用。
背景技术
血清胱抑素C浓度的检测在临床上具有非常重要的诊断意义,目前国内已逐步开展胱抑素C诊断试剂的自主研发,血清胱抑素C浓度对肾功能受损、肾小管功能失常、急性心肌梗塞等各种疾病的诊断及病情观察具有重要的临床意义。因此开发出一种制备特异性强、灵敏度高、可以满足诊断试剂性能要求的抗胱抑素C多克隆抗体的方法是非常重要的。
现有胱抑素C抗体的制备主要有两种方法,一种是单克隆抗体的制备,另一种是多克隆抗体的制备。单克隆抗体制备的开发时间较长,产量较低、成本高。多克隆抗体的制备方法相对简单、产量高,但抗人胱抑素C多克隆抗体的活性与诊断试剂的性能息息相关,而现有胱抑素C多克隆抗体的制备方法往往采用单一抗原免疫后即用硫酸铵沉淀法或Protein A柱层析法进行抗体的纯化,纯化后的抗体中夹杂着很多非特异性抗体,抗人胱抑素C多克隆抗体的有效含量低,这往往是导致不能制备出满足临床诊断要求的胱抑素C测定试剂的主要原因。
发明内容
本发明的目的在于解决现有单一抗原制备的抗人胱抑素C多克隆抗体特异性差和灵敏度低,不能满足诊断试剂制备要求的问题;提供解决上述问题的一种利用不同重组人胱抑素C蛋白的组合制备抗人胱抑素C多克隆抗体的方法及其应用。本发明的设计思路是采用不同重组胱抑素蛋白的组合,在多克隆抗体制备工艺过程中避免产生或纯化到非特异性抗体,因此获得的抗人胱抑素C多克隆抗体具有有效含量高、特异性强、灵敏度高的特点,可满足诊断试剂的制备要求。
为解决上述缺点,本发明的技术方案如下:
不同的重组人胱抑素C蛋白,包括Cys C-His蛋白、Cys C-GST蛋白和Cys C-Trx蛋白中的任意一种。所述Cys C-His基因的核苷酸序列如SEQ ID NO:3所示,Cys C-His蛋白的氨基酸序列如SEQ ID NO:4所示;Cys C-GST基因的核苷酸序列如SEQ ID NO:5所示,Cys C-GST蛋白的氨基酸序列如SEQ ID NO:6所示;Cys C-Trx基因的核苷酸序列如SEQ ID NO:7所示,Cys C-Trx蛋白的氨基酸序列如SEQ ID NO:8所示。
上述三种的重组人胱抑素C蛋白均包含人胱抑素蛋白的序列,包括如SEQ ID NO:1所示的核苷酸序列和如SEQ ID NO:2所示的氨基酸序列。
本发明中优选的人胱抑素C蛋白具有SEQ ID NO:2所示氨基酸序列。本领域技术人员应该知晓,在本发明中所述人胱抑素C蛋白不仅局限于SEQ ID NO:2所示氨基酸序列,在其他报道中公开的、与本发明中SEQ ID NO:2所示序列类似的人胱抑素C蛋白序列也同样适用于本发明。同时,仅仅通过增加、删除或改变本发明SEQ ID NO:2所示蛋白序列中的个别氨基酸序列,该蛋白在生物学功能和制备技术上无创造性和新颖性,此类重组蛋白也同样适用于本发明。
本发明中重组人胱抑素C蛋白的表达载体分别为pET28系列、pGEX系列和pET32系列。同时本领域技术人员应该知晓,在本发明中所述重组人胱抑素C蛋白的表达载体分别为pET28系列、pGEX系列和pET32系列,仅仅通过改变酶切位点的方式将本发明中SEQ ID NO:1所示序列或类似序列与相应表达载体重组而得到的重组人胱抑素C蛋白在蛋白生物学功能和制备技术上无创造性和新颖性,此类重组蛋白也同样适用于本发明。
一种抗人胱抑素C多克隆抗体的制备方法,包括动物免疫、抗血清效价检测、多克隆抗体的抗原-抗体亲和纯化三个步骤中采用了不同重组人胱抑素C蛋白的组合:动物免疫中采用一种重组人胱抑素C蛋白用于动物免疫;抗血清效价检测中采用一种重组人胱抑素C蛋白用于酶联免疫检测中酶标板的包被;多克隆抗体的抗原-抗体亲和纯化中采用一种重组人胱抑素C蛋白用于偶联填料并纯化抗体;重组人胱抑素C蛋白为权利要求2所述的重组人胱抑素C蛋白。
其中,(1)动物免疫:选择免疫动物的种类、年龄体重、免疫剂量、免疫周期和采血方式;选择一种用于动物免疫的重组人胱抑素C蛋白。(2)抗血清效价检测:在免疫过程中抽取少量抗血清,采用酶联免疫(ELISA)方法的间接法检测抗血清效价;选择一种重组人胱抑素C蛋白用于包被酶标板。(3)多克隆抗体的抗原-抗体亲和纯化:完成抗血清的收集,然后选择一种重组人胱抑素C蛋白用于纯化填料的偶联,采用抗原-抗体亲和层析的纯化方式进行抗人胱抑素C多克隆抗体的纯化。
更进一步,重组人胱抑素C蛋白为权利要求2中Cys C-His蛋白、Cys C-GST蛋白和Cys C-Trx蛋白中的任意一种;优选地,不同重组人胱抑素C蛋白在动物免疫、抗血清效价检测、多克隆抗体的抗原-抗体亲和纯化步骤中的组合方式为组合一~组合十三中的任意一种;
组合一:动物免疫用Cys C-His蛋白,抗血清效价检测用Cys C-His蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-His蛋白;
组合二:动物免疫用Cys C-His蛋白,抗血清效价检测用Cys C-Trx蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-GST蛋白;
组合三:动物免疫用Cys C-His蛋白,抗血清效价检测用Cys C-Trx蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-Trx蛋白;
组合四:动物免疫用Cys C-His蛋白,抗血清效价检测用Cys C-GST,多克隆抗体的抗原-抗体亲和纯化用Cys C-GST蛋白;
组合五:动物免疫用Cys C-His蛋白,抗血清效价检测用Cys C-GST蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-Trx蛋白;
组合六:动物免疫用Cys C-GST蛋白,抗血清效价检测用Cys C-Trx蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-His蛋白;
组合七:动物免疫用Cys C-GST蛋白,抗血清效价检测用Cys C-Trx蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-Trx蛋白;
组合八:动物免疫用Cys C-GST蛋白,抗血清效价检测用Cys C-His蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-His蛋白;
组合九:动物免疫用Cys C-GST蛋白,抗血清效价检测用Cys C-His蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-Trx蛋白;
组合十:动物免疫用Cys C-Trx蛋白,抗血清效价检测用Cys C-GST,多克隆抗体的抗原-抗体亲和纯化用Cys C-His蛋白;
组合十一:动物免疫用Cys C-Trx蛋白,抗血清效价检测用Cys C-GST,多克隆抗体的抗原-抗体亲和纯化用Cys C-GST蛋白;
组合十二:动物免疫用Cys C-Trx蛋白,抗血清效价检测用Cys C-His蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-GST蛋白;
组合十三:动物免疫用Cys C-Trx蛋白,抗血清效价检测用Cys C-His蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-His蛋白。
本发明利用多个重组人胱抑素C蛋白的组合制备抗人胱抑素C多克隆抗体,通过该方法能提高纯化后的人胱抑素C多克隆抗体的有效含量以及抗人胱抑素C多克隆抗体的特异性,进而可制备出灵敏度、特异性均满足临床检测性能要求的胱抑素C检测试剂。
所述免疫的动物可为优选为山羊或绵羊。优选的动物年龄约6~10个月,体重25Kg左右。
本发明中免疫的动物可为优选为山羊或绵羊。但本领域技术人员应该知晓,本发明方法也可在其它动物中得到相同的效果,因此仅仅通过改变免疫动物的方法而获得的抗人胱抑素C多克隆抗体也同样适用于本发明。
所述免疫方案为初次免疫,采用重组胱抑素C蛋白与等体积的弗氏完全佐剂混合,免疫计量为2~10mg/只羊,充分乳化后在羊背上皮下多点免疫;之后每隔2周进行加强免疫,采用重组胱抑素C蛋白与等体积的弗氏不完全佐剂混合,免疫计量为1~5mg/只。从第3免疫之后,每次免疫后的第七天采血1ml用于检测抗血清的效价。第6次免疫之后,羊可获得高效价的免疫效果,一般效价可达到1:16×104~1:1024×104。在此效价区间内的抗血清经过本方法纯化后获得的多克隆抗体活性可以满足自主试剂盒性能的要求。免疫效价达到该范围之后即可采集抗血清用于抗体的纯化。一般一只羊可以通过直接颈部静脉放血的方式获得1000ml左右的抗血清,如果希望每只羊能获得更多的抗血清,可以在抗血清效价在前述区间范围内时,每隔四周静脉采血200ml来获得血清100ml,同时每隔两周加强免疫一次并采血进行抗血清效价检测。如果抗血清效价不明显降低,则可继续每隔四周采血200ml一次;如果出现抗血清效价明显下降,在效价低出区间范围之前,直接颈部静脉放血方式获得剩余的抗血清。在此效价范围内的抗血清均可用于抗人胱抑素C多克隆抗体的纯化,纯化后的抗人胱抑素C多克隆抗体具有特异性强、灵敏度高的特点。
所述胱抑素C抗血清效价的检测方法为采用ELISA间接法检测抗血清效价,具体为将重组人胱抑素C蛋白用碳酸包被液稀释到1~10ug/ml的浓度,以0.1~1ug/孔的蛋白量包被于96孔酶标板上,取少量抗血清以10倍的稀释方式逐一稀释4次,再以2倍的稀释方式逐一稀释10次;同时以稀释1000倍的免疫前的羊血清作为阴性对照。样品37℃孵育40~60分钟后,用ELISA洗涤液清洗酶标板,清洗后加入1:500~1:2000稀释的兔抗羊-酶标记物,37℃孵育30分钟。ELISA洗涤液清洗后加入显色液,37℃孵育10~15分钟。最后加入2M H2SO4终止反应,用酶标仪450nm比色读数值。样本OD值≥2×阴性对照OD值时,结果判为阳性。检测值最低阳性孔所对应的稀释比例即为对应抗血清效价。免疫后血清效价达1:16×104~1:1024×104范围内可收获血清并用于抗人胱抑素C多克隆抗体的纯化。
所述抗人胱抑素C多克隆抗体的纯化方式为以5~10mg重组人胱抑素C蛋白对应1ml活化的CNBr activated SepharoseTM4 FastFlow填料的方式,将重组人胱抑素C蛋白偶联到CNBr activated SepharoseTM4 FastFlow填料上,用于抗原-抗体亲和纯化。抗人胱抑素C多克隆抗体在一定缓冲液的条件下与填料上偶联的重组人胱抑素C蛋白牢固结合。完成抗原-抗体的充分结合后,再通过改变缓冲液pH使得抗体和抗原分离,即可获得高纯度的抗人胱抑素C多克隆抗体。
上述抗人胱抑素C多克隆抗体可以制备成胶体金颗粒增强免疫比浊法检测试剂盒,并应用于胱抑素C的检测。该抗人胱抑素C多克隆抗体所制备的检测试剂盒的具体参数如下:
该试剂盒由配比为4:1的R1试剂和R2试剂组成;
R1试剂的缓冲液组成为:pH 7.5~8.5的Tris-HCl,0.05~0.1mol/L;PEG20000,0.5~2%(W/V);Tween20,0.01~0.05%(V/V);Proclin 300,0.02~0.05%(V/V);
R2试剂的缓冲液组成为:pH 7.5~8.5的Tris-HCl,0.01-0.05mol/L;BSA,0.5~2%(W/V);PEG20000,0.1~0.5%(W/V);Proclin 300,0.02~0.05%(V/V);标记抗人胱抑素C多克隆抗体的均相胶体金颗粒。所述胶体金粒径大小为30~60nm。
本发明与现有技术相比,具有以下优点及有益效果:
1、由单一重组抗原制备的人胱抑素C抗血清通常采用硫酸铵沉淀或Protein A纯化,所得的多克隆抗体中有很大一部分为非特异性的抗体,抗人胱抑素C多克隆抗体的有效含量很低。本发明利用不同重组人胱抑素C蛋白的组合制备出的抗人胱抑素C多克隆抗体的方式,设计思路就是采用不同重组胱抑素蛋白的组合,在多克隆抗体制备工艺过程中避免产生或纯化到非特异性抗体,因此获得的抗人胱抑素C多克隆抗体具有有效含量高、特异性强、灵敏度高的特点;
2、本发明制备出的抗人胱抑素C多克隆抗体所制备的检测试剂与由单一抗原制备出的抗体所制备的检测试剂相比较具有准确性好、灵敏度高的特点,本发明制备出的抗人胱抑素C多克隆抗体可作为重要生物原料应用于胱抑素C检测试剂的制备,试剂性能符合产品要求;
3、本发明制备的胱抑素检测试剂与重庆中原生物技术有限公司的胶体金免疫比浊试剂盒平行检测20个临床样本进行比较,结果显示出良好的一致性,效果显著。
附图说明
图1为本发明检测试剂与对照试剂检测结果的对比示意图。
图2为本发明检测试剂与单一抗原制备试剂检测结果的对比示意图。
图3为本发明胱抑素C检测试剂盒的制备流程示意图。
具体实施方式
下面结合实施例及其附图,对本发明作进一步地详细说明,但本发明的实施方式不限于此。
实施例1
本实施例公开的是一种重组人胱抑素C蛋白的制备方法,该重组人胱抑素C蛋白是Cys C-His蛋白,其具体制备方法如下:
(1)人工合成人胱抑素C基因序列并构建表达载体
按照人胱抑素C基因序列信息进行人工基因合成,其序列为SEQ ID NO:1。该基因完成合成后与pMD-18载体链接,人胱抑素C基因序列的5’端设计有BamH I和EcoR I酶切位点,3’端设计有终止子和XhoI酶切位点。利用BamH I和XhoI酶切位点组合或者EcoR I和XhoI酶切位点组合将人胱抑素C基因序列从pMD-18载体上双酶切下来,同时用BamH I和XhoI酶切位点组合或者EcoR I和XhoI酶切位点组合对pET28a(+)表达载体进行双酶切。胶回收这两个基因片段。用连接酶将双酶切后的人胱抑素C基因序列与pET28a(+)表达载体链接,转化大肠杆菌DH5α。挑取单克隆菌落进行培养,提取重组质粒并进行双酶切鉴定,确认阳性重组质粒。将重组质粒送生工测序。经过DNA测序验证,确定重组质粒的序列和阅读框是正确的。将获得的重组质粒转化宿主细胞Rosetta(DE3)。通过重组质粒转化宿主细胞Rosetta(DE3)即可有效表达出具有SEQ ID NO:3所示核苷酸序列,SEQ ID NO:4所示氨基酸序列的Cys C-His蛋白。
(2)筛选高效的表达重组胱抑素C蛋白的菌株
取适量转化后的宿主细胞涂布到带抗性的LB固体培养基中,37℃培养过夜,第二天挑选10株以上的单克隆菌落,接种于5ml带抗性的LB液体培养基中,37℃,200rpm培养过夜,同时转化pET28a(+)空载体的宿主菌作为对照。第三天,上述各样品各取5μl培养后的菌液接种于新的5ml带抗性的液体LB培养基中,以37℃,200rpm培养5小时后,每个样品加入终浓度为1mM的IPTG,再以37℃,200rpm培养5小时。取1ml培养产物,离心收集菌体,用50μl20mM的PBS缓冲液重悬菌体,加入12.5μl蛋白电泳上样缓冲液(60mM Tris-HCl,25%甘油(W/V),2%SDS(W/V),5%2-巯基乙醇(V/V),0.1%溴酚蓝(W/V),pH 6.8),沸水煮10分钟,以12000rpm离心10分钟后,取20μl上清液进行SDS-PAGE电泳。考马斯亮蓝染色,在预测分子量大小的地方有一条明显的蛋白条带,而转化pET28a(+)空载体的菌没有这条带。进一步将诱导后的菌体超声破碎后离心取上清液和沉淀,SDS-PAGE电泳显示,目标蛋白主要以可溶形式存在于上清中。
比较不同的重组表达细胞的表达量差异,选择表达量较高的菌株作为后续大规模表达的菌株。
(3)表达菌株的大规模发酵及蛋白纯化
三角瓶的发酵:将筛选的高表达菌接种于200ml带抗性的LB液体培养基中,37℃,200rpm培养过夜。第二天,按照1:50~1:100的比例接种于1~8个内有1000ml带抗性的LB培养基的三角瓶中,以37℃,220rpm培养4~6小时至OD值0.6~0.8,加入IPTG至终浓度为1mM,37℃,220rpm诱导4~6小时,或者25℃,220rpm诱导16~20小时。
生物反应器的发酵:将筛选的高表达菌株接种于500ml带抗性的LB液体培养基中,以37℃,500rpm培养过夜。第二天,在总体积为7L的生物反应器中加入3~4L的LB液体培养基,煮沸灭菌后降温至37℃。将前一天的培养菌液按照1:50~1:100的比例接种于生物反应器的培养基中,同时加入相应抗生素,以37℃,200~300rpm培养4~6小时至OD值0.6~0.8,加入IPTG至终浓度为1mM,以37℃,200~300rpm诱导4~6小时,或者以23~25℃,200~300rpm诱导16~20小时。
诱导后的菌液用大型高速离心机以6000rpm转速离心10分钟,收集菌体,然后以1g菌体对应10ml缓冲液的比例加入His Loading buffer(Na2HPO4 3.58g/L,NaH2PO4 1.56g/L,NaCl 29.22g/L,咪唑2g/L,pH 7.4)重悬菌体,同时加入一定量的PMSF防止蛋白降解,用超声破碎或高压匀浆的方式裂解菌体。裂解完成后,用大型高速离心机以4℃,12000rpm转速离心20分钟,收集上清液。上清液用His Loading Buffer稀释5-10倍后用0.22μm滤器过滤,待纯化。
采用AKTA UPC10纯化仪进行蛋白纯化。将AKTA纯化仪的A泵管,B泵管放入已经用0.22um滤膜过滤的纯水中,进样阀4,5出口管和收集阀的废液出口接到废液瓶中,在Systemcontrol窗口下,点击Manual打开pump菜单,选择PumpWashBasic命令,指定冲洗液A泵管,B泵管,执行命令。运行前,在Alarm&Mon中设定报警压力为0.3MPa,设定流速为2ml/min,用水冲洗仪器管路约5min。
根据纯化量与亲和填料最大载量的关系,选择已装好的Ni SepharoseTM6FastFlow亲和柱。将亲和柱的上端和下端管路与AKTA UPC10纯化仪器链接,用约5倍柱体积的纯水冲洗,分别将A泵管放入His Loading Buffer中,B泵管放入His elution Buffer(Na2HPO43.58g/L,NaH2PO4 1.56g/L,NaCl 29.22g/L,咪唑20.4g/L,pH7.4)中。先用100%B泵冲洗约20ml,再切换到100%A泵冲洗柱子约10倍柱体积,同时冲洗干净所有收集管道。平衡好后,在Alarm&Mon菜单中选autozeroUV将紫外调零,并在Frc中设置峰收集150mAu,50ml/管,点击pause暂停仪器,将A泵管从His Loading Buffer中移入到重组抗原的上清液中,点击
continue开始以2ml/min的流速上样。待上样完成后,将A泵管放入His LoadingBuffer中冲洗亲和柱,洗去未结合的杂蛋白,直至紫外基线平衡。当紫外基线平衡后,切换到100%B泵用His elution Buffer洗脱目的抗原,收集洗脱液。洗脱液装入3000分子量的透析袋,用20mM Tris缓冲液(Tris 2.42g/L,pH 8.0)在2-8℃透析2天,更换透析液3次以上。
透析完成后,取10μl进行SDS-PAGE电泳检测,结果显示Cys C-His蛋白纯度大于85%,蛋白浓度大于2mg/ml。
实施例2
本实施例与实施例1的区别在于,本实施例公开一种重组人胱抑素C蛋白是Cys C-GST蛋白,其具体制备方法与实施例1的区别仅仅在于:
步骤(1)中的表达载体为pGEX-4T-1;步骤(2)中对照采用转化pGEX-4T-1空载体的宿主菌;步骤(3)中加入GST Loading buffer替换His Loading buffer,GST elutionBuffer替换His elution Buffer,Glutathione Sepharose 4Fast Flow亲和柱替换NiSepharoseTM6 Fast Flow亲和柱,PBS缓冲液替换Tris缓冲液。
其中,GST Loading buffer的组成为:NaCl 8.18g/L,KCl 0.2g/L,Na2HPO4 3.58g/L,KH2PO4 0.25g/L,pH7.3。GST elution Buffer的组成为:Tris 6.06g/L,还原性谷胱甘肽3.04g/L,pH 8.0。PBS缓冲液的组成为:Na2HPO4 5.8g/L,NaH2PO4 0.6g/L,NaCl 9.0g/L,pH7.4。
本实施例中通过重组质粒转化宿主细胞Rosetta(DE3)即可有效表达出具有SEQID NO:5所示核苷酸序列、SEQ ID NO:6所示氨基酸序列的Cys C-GST蛋白。
本实施例中,透析完成后,取10μl进行SDS-PAGE电泳检测,结果显示Cys C-GST蛋白纯度大于80%,蛋白浓度大于2mg/ml。
实施例3
本实施例与实施例1的区别在于,本实施例公开一种重组人胱抑素C蛋白是Cys C-Trx蛋白,其具体制备方法与实施例1的区别仅仅在于:
步骤(1)中的表达载体为pET32a(+);步骤(2)中对照采用转化pET32a(+)空载体的宿主菌。
本实施例中通过重组质粒转化宿主细胞Rosetta(DE3)即可有效表达出具有SEQID NO:7所示核苷酸序列、SEQ ID NO:8所示氨基酸序列的Cys C-Trx蛋白。
本实施例中,透析完成后,取10μl进行SDS-PAGE电泳检测,结果显示Cys C-Trx蛋白纯度大于80%,蛋白浓度大于2mg/ml。
实施例4
本实施例公开了利用三种重组人胱抑素C蛋白的组合制备抗人胱抑素C多克隆抗体的方法,如图3所示。本实施例中优选以组合四的方案来制备抗人胱抑素C多克隆抗体,即,动物免疫用Cys C-His蛋白,抗血清效价检测用Cys C-GST蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-GST蛋白。具体过程如下:
(1)动物免疫和抗血清的采集:
免疫动物优选为年龄约6~10个月,体重20-25Kg左右的山羊。
免疫方案为初次免疫,采用1ml浓度为5mg/ml的Cys C-His蛋白与体积的弗氏完全佐剂混合,免疫计量为5mg/只羊,充分乳化后在羊背上进行皮下多点免疫;之后每隔2周进行加强免疫,采用0.5ml浓度为5mg/ml的Cys C-His蛋白与体积的弗氏不完全佐剂混合,免疫计量为2.5mg/只。从第3免疫之后,每次免疫后的第七天采血2ml用于抗血清效价的检测。第6次免疫之后,免疫动物获得高效价的免疫效果,一般效价可达到1:16×104~1:1024×104。
免疫效价达到上述区间范围之后即可采集抗血清用于纯化。采血方式有以下两种:
采血方式一:若第7次免疫效价与第6次免疫效价比较没有显著升高,则可通过直接颈部静脉放血方式获得1000ml左右的抗血清,如表1所示;
采血方式二:如果希望每只羊能获得更多的抗血清,可以在抗血清效价在1:16×104~1:1024×104区间范围内时,每四隔周静脉采血200ml来获得血清100ml,同时每两周加强免疫一次并采血进行抗血清效价检测。如果抗血清效价不明显降低,则可继续每隔四周采血200ml一次;如果出现抗血清效价明显下降,在效价低出区间范围之前,直接颈部静脉放血方式获得剩余的抗血清,如表2所示。
在血清效价范围1:16×104~1:1024×104内的抗血清均可用于抗人胱抑素C多克隆抗体的纯化,纯化后的抗人胱抑素C多克隆抗体具有特异性强、灵敏度高的特点。
表1
表2
备注:免疫第八次到第十七次为预设的抗血清效价还在1:16×104~1:1024×104的范围内。如果出现抗血清效价明显下降,在效价低出区间范围之前,直接颈部静脉放血方式获得剩余抗血清。
(2)抗血清效价检测:
在免疫过程中,抽取2ml血液,室温放置,待血清分离出来后,以1000rpm低速离心2分钟,取出血清用于后续抗血清效价的检测。抗血清的效价采用ELISA间接法检测。
将Cys C-GST蛋白用碳酸包被液(Na2CO3 1.59g/L,NaHCO3 2.94g/L,pH9.6)稀释成2.5ug/ml,每孔取100μl加入到96孔酶标板中进行包被,于4℃冰箱中放置过夜。第二天,抛弃孔中液体,拍干。每孔加入150μl封闭液(10mM PBS,20%新生牛血清(V/V),pH7.4),37℃恒温培养箱中封闭2小时,抛弃孔中液体,拍干,室温吹干。取少量抗血清进行检测,同时以稀释1000倍的免疫前血清作为阴性对照。酶标板第一个孔中加样10μl抗血清和90μl PBS(第一孔即稀释10倍),然后以10倍的稀释方式逐一稀释3次,再以2倍的稀释方式逐一稀释10次。置37℃恒温培养箱中孵育40分钟。ELISA洗液(10mM PBS,0.05%吐温-20,pH7.4)洗板五次,拍干。将兔抗羊-酶标记物用10mM PBS按1:500稀释后,每孔加100μl,置37℃恒温培养箱中孵育30分钟。ELISA洗液洗板五次,拍干。显色液A(EDTA-2Na 0.17g/L,柠檬酸0.875g/L,12.5%甘油(V/V),TMB 0.25g/L,12.5%丙酮(V/V))、显色液B(Na2HPO426.9g/L,柠檬酸10.2g/L,0.1%H2O2(30%)(V/V))按1:1比例配制成显色液,每孔加100μl显色液,置37℃恒温培养箱中孵育10分钟。加2M硫酸终止液50μl,酶标仪450nm比色读数值。结果判定:样本OD值≥2×阴性对照OD值时,为阳性标本;反之为阴性标本。检测值最低阳性孔所对应的稀释比例即为抗血清效价。
抗血清效价检测结果:第6次免疫之后,免疫动物获得高效价的免疫效果,效价为1:128×104。在抗血清效价范围内,以直接颈部静脉放血方式获得1000ml左右的抗血清。
(3)抗人胱抑素C多克隆抗体的纯化:
本案例采用抗原-抗体亲和层析的纯化方式进行抗人胱抑素C多克隆抗体的纯化。
CNBr activated SepharoseTM4FastFlow填料的蛋白载量为20~60mg/ml。依据需要纯化蛋白量的大小确定析空柱型号及亲和填料的用量。例如:用XK 50/20装填100ml CysC抗原亲和层析柱,理论上每批次可以纯化600mg以内的抗人胱抑素C单克隆抗体或者1600mg以内的抗人胱抑素C多克隆抗体。
100ml的Cys C-GST抗原亲和层析柱的制备:称取30g干粉剂CNBr activatedSepharoseTM4FastFlow填料溶于100ml 1mM HCl中,再加入500ml 1mM HCl冲洗填料,待填料充分溶胀。用500ml Coupling Buffer(0.1M NaHCO3,0.5M NaCl,pH8.3)冲洗溶胀后的CNBractivated SepharoseTM4FastFlow,然后抽干填料中的多余液体。先将1000mg的Cys C-GST蛋白在Coupling Buffer中进行透析换液,然后将溶胀后的CNBr activatedSepharoseTM4FastFlow填料加入Cys C-GST蛋白溶液中,在滚轴混匀仪上2~8℃混动过夜。第二天,将偶联Cys C-GST蛋白的CNBr activated SepharoseTM4FastFlow填料装入层析空柱XK 50/20中,并将纯化柱的上端和下端与AKTA纯化仪链接,用Coupling Buffer以10ml/min流速冲洗填料至无蛋白洗出,收集洗出液A。用300ml Blocking Buffer(0.1M Tris-HCl,0.5M NaCl,pH8.0)以10ml/min流速冲洗填料后,室温静置2小时。用C液(0.1M乙酸钠/乙酸,0.5M NaCl,pH 4.0)和Blocking Buffer以10ml/min流速交替冲洗填料5个循环,每次5个柱体积。用纯水以10ml/min流速冲洗填料10个柱体积。用20%乙醇以10ml/min流速冲洗填料10个柱体积。偶联验证:检测洗出液A的体积V和蛋白浓度C,偶联效率公式为=(1000-C*V)/1000×100%。偶联效率应不低于70%,否则需要重新偶联。
抗人胱抑素C多克隆抗体的纯化:根据100ml抗原-抗体亲和柱的载量,取适当体积的人胱抑素C抗血清,以12000rpm离心20分钟后,去油脂和沉淀,上清用Mab LoadingBuffer(Na2HPO4 5.12g/L,NaH2PO4 0.872g/L,NaCl 9g/L,pH 7.2)稀释5~10倍,再用0.22μm滤膜过滤。打开电脑及AKTA纯化仪,等待AKTA纯化仪自检完成后,点击的UNICORN软件,输入用户名和密码进入软件控制系统。将AKTA纯化仪的A泵管和B泵管放入已经用0.22um滤膜过滤的纯水中,进样阀4,5出口管和收集阀的废液出口接到废液瓶中,在System control窗口下,点击Manual打开pump菜单,选择PumpWashBasic命令,指定冲洗液A泵管,B泵管,执行命令。运行前,在Alarm&Mon中设定报警压力为0.3MPa,设定流速为2ml/min,将A泵管和B泵管放入纯水中,用水冲洗仪器管路约5分钟。将已装好的100ml亲和柱的上端和下端管路与AKTA UPC10蛋白纯化仪器链接,用约5倍柱体积的纯水冲洗,分别将A泵管放入Mab LoadingBuffer中,B泵管放入Mab Elution Buffer(柠檬酸17.22g/L,柠檬酸三钠5.3g/L,pH3.0)中。先用100%B泵冲洗亲和柱约2倍柱体积,再切换到100%A泵冲洗柱子约10倍柱体积,同时冲洗干净所有收集管道。平衡好后,在Alarm&Mon菜单中选autozeroUV将紫外调零,并在Frc中设置峰收集150mAu,50ml/管,点击pause暂停仪器,将A泵管从Mab Loading Buffer中移入到装有上述稀释好的人胱抑素C抗血清稀释液的试剂瓶中,点击continue开始以2~10ml/min的流速上样。上样完成后,将A泵管放入Mab Loading Buffer中继续冲洗亲和柱,洗去未结合的杂蛋白,直至紫外基线平衡。当纯化仪的紫外基线平衡后,切换到100%B泵用Mab elution Buffer洗脱目的抗体,收集抗体洗脱液,并以约4ml洗脱液加1ml中和液(Tris12.114g/L,pH 10.0)的方式调节pH到7.0左右。将收集的抗体洗脱液并装入3000分子量的透析袋,用5L体积的20mM的PB缓冲液(Na2HPO4 5.8g/L,NaH2PO4 0.6g/L,pH7.4)在2~8℃透析2天,换液3次以上。透析后的抗体可以用聚乙二醇进行适当的浓缩,使抗体浓度在5mg/ml以上。抗人胱抑素C多克隆抗体制备完成后,用BCA法检测抗体浓度,并用SDS-PAGE法检测抗体纯度。
实施例5
本实施例与实施例4的区别在于,本实施例是利用不同的三种重组人胱抑素C蛋白的组合制备抗人胱抑素C多克隆抗体。本实施例中优选以组合九的方式来制备抗人胱抑素C多克隆抗体,即,本实施例中动物免疫用Cys C-GST蛋白,抗血清效价检测用Cys C-His蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-Trx蛋白。
本实施例与实施例4的区别在于:
(1)抗血清效价检测结果:第6次免疫之后,免疫动物获得高效价的免疫效果,效价为1:32×104。在抗血清效价范围内,以直接颈部静脉放血方式获得1000ml左右的抗血清。
(2)经抗原-抗体亲和纯化后,得到的抗人胱抑素C多克隆抗体的总产量低于实施例4,但抗体活性基本无差异。
实施例6
本实施例与实施例4的区别在于,本实施例是利用不同的三种重组人胱抑素C蛋白的组合制备抗人胱抑素C多克隆抗体。本实施例中优选以组合十二的方式来制备抗人胱抑素C多克隆抗体,即,本实施例中动物免疫用Cys C-Trx蛋白,抗血清效价检测用Cys C-His蛋白,多克隆抗体的抗原-抗体亲和纯化用Cys C-GST蛋白。
本实施例与实施例4的区别在于:
(1)抗血清效价检测结果:第6次免疫之后,免疫动物获得高效价的免疫效果,效价为1:64×104。在抗血清效价范围内,以直接颈部静脉放血方式获得1000ml左右的抗血清。
(2)经抗原-抗体亲和纯化后,得到的抗人胱抑素C多克隆抗体的总产量低于实施例4,但抗体活性基本无差异。
实施例7
本实施例为对比实施例,本实施例是为了比较单一重组人胱抑素C蛋白方法制备的多克隆抗体与本发明方法制备的多克隆抗体之间的差异,本实施例仅仅对单一重组人胱抑素C蛋白制备的抗体进行亲和纯化:
将实施例4中以用Cys C-His蛋白动物免疫得到的抗血清采用protein A亲和纯化方式进行纯化。
将实施例5中以用Cys C-GST蛋白动物免疫得到的抗血清采用protein A亲和纯化方式进行纯化。
将实施例6中以用Cys C-Trx蛋白动物免疫得到的抗血清采用protein A亲和纯化方式进行纯化。
本实施例与实施例4的区别在于:
(1)采用免疫用的蛋白进行抗血清效价检测,效价均偏高。
(2)经过protein A亲和纯化后,得到的多克隆抗体的总产量均高于实施例4,但抗体活性较差。
实施例8
本实施例公开了一种采用抗人胱抑素C多克隆抗体制备的胱抑素C检测试剂盒,该试剂盒由R1试剂和R2试剂组成,R1试剂和R2试剂的使用配比为4:1,采用胶体金颗粒增强免疫比浊法,可在多种全自动生化分析仪上使用。
如图3所示,本实施例是利用实施例4制备的抗人胱抑素C多克隆抗体作为重要生物原料,用于胱抑素C检测试剂中R2试剂的配制,具体过程如下:
(1)R1试剂的制备:
向800ml的0.1mol/L的Tris-HCl缓冲液(pH 8.4)中加入10g的PEG20000、0.2ml的Proclin 300和0.2ml的Tween20,充分溶解后用蒸馏水补充至1000ml,用0.22um的滤膜过滤除菌后,制成R1试剂。最终R1试剂的缓冲液组成为0.1mol/L Tris-HCl(pH 8.4),1%PEG20000(W/V),0.02%Proclin 300(V/V),0.02%Tween20(V/V)。由此制得本发明中的R1试剂。
(2)R2试剂的制备:
2.1胶体金溶液的制备:
配置0.01%的氯金酸(HAuCl4)水溶液1000ml,加热至沸腾。在玻璃棒的搅动下加入准确加入1%的柠檬酸三钠(Na3C6H5O7)水溶液8.5ml。继续加热煮沸15分钟,观察到淡黄色的氯金酸水溶液在加入柠檬酸三钠水溶液后先变黑,再逐渐变深红并稳定的过程。该过程持续大约2~3分钟。冷却至室温后,补充蒸馏水至1000ml。此方法制成的胶体金溶液,其粒径大约在55~60nm,λmax约为528~532nm。
2.2胶体金标记抗体:
2.2.1胶体金与多克隆抗体用量比的确定:用0.2mol/L的K2CO3调节胶体金溶液的pH值至8.4,分装10管,每管分装1ml胶体金溶液。将待标记的多克隆抗体用0.02mol/L的Tris缓冲液(pH8.4)稀释至浓度为10ug/ml、15ug/ml、20ug/ml、25ug/ml、30ug/ml、35ug/ml、40ug/ml、50ug/ml、60ug/ml、70ug/ml,分别取0.1ml分别加入上述1ml胶体金溶液中,充分混匀。对照管加0.1ml的Tris缓冲液。5分钟后,在上述各管中加入0.1ml 10%的NaCl溶液,充分混合后静置待观察。
观察结果:未加抗体的对照管和加入抗体量不足的管均会出现由红变紫的聚集沉淀现象,而加入抗体量超过最低稳定量的各管均保持红色不变。以稳定1ml胶体金溶液红色不变的最低抗体量作为最低标记量,在进行大量标记时可将抗体量适当增加20%以保证抗体过量。
2.2.2根据2.2.1的方法,确定抗胱抑素C多克隆抗体的最低标记浓度为60ug/ml。用0.02mol/L的Tris缓冲液(pH8.4)将抗体稀释至70ug/ml,1000ml胶体金溶液和100ml稀释后的抗体溶液用0.1mol/L的K2CO3调节胶体金溶液的pH值至8.4。在电磁搅拌器搅拌胶体金溶液,缓慢将抗体溶液加入到胶体金溶液中并继续搅拌,同时加入22g的BSA和1.1g的PEG20000。
2.2.3标记好的胶体金溶液用15000g离心30分钟,弃上清,沉淀物用含2%BSA、0.1%PEG20000的0.02mol/L Tris缓冲液(pH 8.4)重悬,恢复原体积后再次离心。如此重复两次以除去未结合的抗体。最后弃上清,沉淀物用110ml的0.02mol/L Tris-HCl(pH 8.4),2%BSA(W/V),0.1%PEG20000(W/V),0.02%Proclin 300(V/V)缓冲液重悬,制备成本发明的R2试剂,颜色为紫红色。
最终R2试剂的缓冲液组成为:0.02mol/L Tris-HCl(pH 8.4);2%BSA(W/V);0.1%PEG20000(W/V);0.02%Proclin 300(V/V);标记抗人胱抑素C多克隆抗体的均相胶体金颗粒,55~60nm。
(3)试剂盒临床检测性能
本实施例中R1试剂和R2试剂的用量为200ul和50ul,样本用量为2ul。采用终点法进行测定,200ul R1试剂加入2ul样本后于37℃孵育5分钟,然后加入50ul R2试剂,开始读点,反应5分钟后再次读点,达到吸光度的差值。采用日立7080(Hitachi)型全自动生化分析检测,检测主波长540nm,副波长660nm。对不同胱抑素浓度的样本溶液进行检测,检测的胱抑素浓度与其检测出的Δ吸光值如表3所示。
准确度和精密度的检测:利用重庆中原生物技术有限公司的胶体金免疫比浊试剂盒及配套校准品,用本发明制备的试剂对校准品进行检测,各校准点(S1~S6)的Δ吸光值的偏差均小于10%,标准曲线梯度良好,符合诊断试剂性能要求。结果如表3所示。
表3
用本发明制备的胱抑素检测试剂与对照试剂平行检测20个临床样本,然后进行比较。本实施例中该对照试剂采用重庆中原生物技术有限公司的胶体金免疫比浊试剂盒,结果显示出良好的一致性。结果如表4和图1所示。
表4
本发明方法制备出的抗人胱抑素C多克隆抗体具有特异性强,灵敏度高的特点,可作为重要生物原料应用于胱抑素C检测试剂的制备,试剂性能符合产品要求。
实施例9
本实施例是分别利用实施例4、实施例5、实施例6及实施例7制备的多种抗人胱抑素C多克隆抗体作为生物原料,用于胱抑素C检测试剂中R2试剂的配制。
本实施例试剂的配置方法与实施例4相同。
用本实施例制备的试剂对重庆中原生物技术有限公司的校准品进行检测,各校准点(S1~S6)的Δ吸光值的偏差结果如表5和图2所示。
表5
结果显示本发明实施例4(组合四)、实施例5(组合九)、实施例6(组合十二)制备的抗人胱抑素C多克隆抗体均可作为重要生物原料应用于检测试剂的制备,各校准点(S1~S6)的曲线符合诊断试剂要求;而实施例7利用单一抗原制备的抗人胱抑素C多克隆抗体,其制备的试剂在各校准点(S1~S6)的Δ吸光值显著偏低,均未达到诊断试剂要求。由此可见,本发明制备出的抗人胱抑素C多克隆抗体与单一抗原制备出的抗人胱抑素C多克隆抗体相比较具有特异性强、灵敏度高的特点,可作为重要生物原料应用于胱抑素C检测试剂的制备,试剂性能符合诊断要求。
上述实施例仅为本发明的优选实施例,除去组合四、组合九和组合十二以外的其他组合方式,虽然实施例中未进行详细阐述,但其他组合方式依然能很好地达到本发明的效果,因而本实施例也并非对本发明保护范围的限制,但凡采用本发明的设计原理,以及在此基础上进行非创造性劳动而作出的变化,均应属于本发明的保护范围之内。
SEQUENCE LISTING
<110> 周珂
<120> 一种组合蛋白制备抗人胱抑素C多克隆抗体的方法和应用
<130> 2017
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 360
<212> DNA
<213> 人工序列
<400> 1
TCCAGTCCCG GCAAGCCGCC GCGCCTGGTG GGAGGCCCCA TGGACGCCAG CGTGGAGGAG 60
GAGGGTGTGC GGCGTGCACT GGACTTTGCC GTCGGCGAGT ACAACAAAGC CAGCAACGAC 120
ATGTACCACA GCCGCGCGCT GCAGGTGGTG CGCGCCCGCA AGCAGATCGT AGCTGGGGTG 180
AACTACTTCT TGGACGTGGA GCTGGGCCGA ACCACGTGTA CCAAGACCCA GCCCAACTTG 240
GACAACTGCC CCTTCCATGA CCAGCCACAT CTGAAAAGGA AAGCATTCTG CTCTTTCCAG 300
ATCTACGCTG TGCCTTGGCA GGGCACAATG ACCTTGTCGA AATCCACCTG TCATGACGCC 360
<210> 2
<211> 120
<212> PRT
<213> 人工序列
<400> 2
Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu
5 10 15 20
Glu Gly Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys Ala Ser Asn Asp
25 30 35 40
Met Tyr His Ser Arg Ala Leu Gln Val Val Arg Ala Arg Lys Gln Ile Val Ala Gly Val
45 50 55 60
Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Tyr Gln Pro Asn Leu
65 70 75 80
Asp Asn Cys Pro Phe His Asp Gln Pro His Leu Lys Arg Lys Ala Phe Cys Ser Phe Gln
85 90 95 100
Ile Tyr Ala Val Pro Trp Gln Gly Thr Met Thr Leu Ser Lys Ser Thr Cys His Asp Ala
105 110 115 120
<210> 3
<211> 468
<212> DNA
<213> 人工序列
<400> 3
ATGGGCAGCA GCCATCATCA TCATCATCAC AGCAGCGGCC TGGTGCCGCG CGGCAGCCAT 60
ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCGAATTCTC CAGTCCCGGC 120
AAGCCGCCGC GCCTGGTGGG AGGCCCCATG GACGCCAGCG TGGAGGAGGA GGGTGTGCGG 180
CGTGCACTGG ACTTTGCCGT CGGCGAGTAC AACAAAGCCA GCAACGACAT GTACCACAGC 240
CGCGCGCTGC AGGTGGTGCG CGCCCGCAAG CAGATCGTAG CTGGGGTGAA CTACTTCTTG 300
GACGTGGAGC TGGGCCGAAC CACGTGTACC AAGACCCAGC CCAACTTGGA CAACTGCCCC 360
TTCCATGACC AGCCACATCT GAAAAGGAAA GCATTCTGCT CTTTCCAGAT CTACGCTGTG 420
CCTTGGCAGG GCACAATGAC CTTGTCGAAA TCCACCTGTC ATGACGCC 468
<210> 4
<211> 156
<212> PRT
<213> 人工序列
<400> 4
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser His
5 10 15 20
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Glu Phe Ser Ser Pro Gly
25 30 35 40
Lys Pro Pro Arg Leu Val Gly Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly Val Arg
45 50 55 60
Arg Ala Leu Asp Phe Ala Val Gly Glu Tyr Asn Lys Ala Ser Asn Asp Met Tyr His Ser
65 70 75 80
Arg Ala Leu Gln Val Val Arg Ala Arg Lys Gln Ile Val Ala Gly Val Asn Tyr Phe Leu
85 90 95 100
Asp Val Glu Leu Gly Arg Thr Thr Cys Thr Lys Tyr Gln Pro Asn Leu Asp Asn Cys Pro
105 110 115 120
Phe His Asp Gln Pro His Leu Lys Arg Lys Ala Phe Cys Ser Phe Gln Ile Tyr Ala Val
125 130 135 140
Pro Trp Gln Gly Thr Met Thr Leu Ser Lys Ser Thr Cys His Asp Ala
145 150 155
<210> 5
<211> 1047
<212> DNA
<213> 人工序列
<400> 5
ATGTCCCCTA TACTAGGTTA TTGGAAAATT AAGGGCCTTG TGCAACCCAC TCGACTTCTT 60
TTGGAATATC TTGAAGAAAA ATATGAAGAG CATTTGTATG AGCGCGATGA AGGTGATAAA 120
TGGCGAAACA AAAAGTTTGA ATTGGGTTTG GAGTTTCCCA ATCTTCCTTA TTATATTGAT 180
GGTGATGTTA AATTAACACA GTCTATGGCC ATCATACGTT ATATAGCTGA CAAGCACAAC 240
ATGTTGGGTG GTTGTCCAAA AGAGCGTGCA GAGATTTCAA TGCTTGAAGG AGCGGTTTTG 300
GATATTAGAT ACGGTGTTTC GAGAATTGCA TATAGTAAAG ACTTTGAAAC TCTCAAAGTT 360
GATTTTCTTA GCAAGCTACC TGAAATGCTG AAAATGTTCG AAGATCGTTT ATGTCATAAA 420
ACATATTTAA ATGGTGATCA TGTAACCCAT CCTGACTTCA TGTTGTATGA CGCTCTTGAT 480
GTTGTTTTAT ACATGGACCC AATGTGCCTG GATGCGTTCC CAAAATTAGT TTGTTTTAAA 540
AAACGTATTG AAGCTATCCC ACAAATTGAT AAGTACTTGA AATCCAGCAA GTATATAGCA 600
TGGCCTTTGC AGGGCTGGCA AGCCACGTTT GGTGGTGGCG ACCATCCTCC AAAATCGGAT 660
CTGGTTCCGC GTGGATCCCC GGAATTCTCC AGTCCCGGCA AGCCGCCGCG CCTGGTGGGA 720
GGCCCCATGG ACGCCAGCGT GGAGGAGGAG GGTGTGCGGC GTGCACTGGA CTTTGCCGTC 780
GGCGAGTACA ACAAAGCCAG CAACGACATG TACCACAGCC GCGCGCTGCA GGTGGTGCGC 840
GCCCGCAAGC AGATCGTAGC TGGGGTGAAC TACTTCTTGG ACGTGGAGCT GGGCCGAACC 900
ACGTGTACCA AGACCCAGCC CAACTTGGAC AACTGCCCCT TCCATGACCA GCCACATCTG 960
AAAAGGAAAG CATTCTGCTC TTTCCAGATC TACGCTGTGC CTTGGCAGGG CACAATGACC 1020
TTGTCGAAAT CCACCTGTCA TGACGCC 1047
<210> 6
<211> 349
<212> PRT
<213> 人工序列
<400> 6
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu
5 10 15 20
Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp Lys
25 30 35 40
Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp
45 50 55 60
Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu
85 90 95 100
Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys Val
105 110 115 120
Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys
125 130 135 140
Thr Tyr Leu Asn Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys
165 170 175 180
Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile Ala
185 190 195 200
Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp
205 210 215 220
Leu Val Pro Arg Gly Ser Pro Glu Phe Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gly
225 230 235 240
Gly Pro Met Asp Ala Ser Val Glu Glu Glu Gly Val Arg Arg Ala Leu Asp Phe Ala Val
245 250 255 260
Gly Glu Tyr Asn Lys Ala Ser Asn Asp Met Tyr His Ser Arg Ala Leu Gln Val Val Arg
265 270 275 280
Ala Arg Lys Gln Ile Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg Thr
285 290 295 300
Thr Cys Thr Lys Tyr Gln Pro Asn Leu Asp Asn Cys Pro Phe His Asp Gln Pro His Leu
305 310 315 320
Lys Arg Lys Ala Phe Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gln Gly Thr Met Thr
325 330 335 340
Leu Ser Lys Ser Thr Cys His Asp Ala
345
<210> 7
<211> 861
<212> DNA
<213> 人工序列
<400> 7
ATGAGCGATA AAATTATTCA CCTGACTGAC GACAGTTTTG ACACGGATGT ACTCAAAGCG 60
GACGGGGCGA TCCTCGTCGA TTTCTGGGCA GAGTGGTGCG GTCCGTGCAA AATGATCGCC 120
CCGATTCTGG ATGAAATCGC TGACGAATAT CAGGGCAAAC TGACCGTTGC AAAACTGAAC 180
ATCGATCAAA ACCCTGGCAC TGCGCCGAAA TATGGCATCC GTGGTATCCC GACTCTGCTG 240
CTGTTCAAAA ACGGTGAAGT GGCGGCAACC AAAGTGGGTG CACTGTCTAA AGGTCAGTTG 300
AAAGAGTTCC TCGACGCTAA CCTGGCCGGT TCTGGTTCTG GCCATATGCA CCATCATCAT 360
CATCATTCTT CTGGTCTGGT GCCACGCGGT TCTGGTATGA AAGAAACCGC TGCTGCTAAA 420
TTCGAACGCC AGCACATGGA CAGCCCAGAT CTGGGTACCG ACGACGACGA CAAGGCCATG 480
GCTGATATCG GATCCGAATT CTCCAGTCCC GGCAAGCCGC CGCGCCTGGT GGGAGGCCCC 540
ATGGACGCCA GCGTGGAGGA GGAGGGTGTG CGGCGTGCAC TGGACTTTGC CGTCGGCGAG 600
TACAACAAAG CCAGCAACGA CATGTACCAC AGCCGCGCGC TGCAGGTGGT GCGCGCCCGC 660
AAGCAGATCG TAGCTGGGGT GAACTACTTC TTGGACGTGG AGCTGGGCCG AACCACGTGT 720
ACCAAGACCC AGCCCAACTT GGACAACTGC CCCTTCCATG ACCAGCCACA TCTGAAAAGG 780
AAAGCATTCT GCTCTTTCCA GATCTACGCT GTGCCTTGGC AGGGCACAAT GACCTTGTCG 840
AAATCCACCT GTCATGACGC C 861
<210> 8
<211> 287
<212> PRT
<213> 人工序列
<400> 8
Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala
5 10 15 20
Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala
25 30 35 40
Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn
45 50 55 60
Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu
85 90 95 100
Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly Ser Gly His Met His His His His
105 110 115 120
His His Ser Ser Gly Leu Val Pro Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys
125 130 135 140
Phe Glu Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met
145 150 155 160
Ala Asp Ile Gly Ser Glu Phe Ser Ser Pro Gly Lys Pro Pro Arg Leu Val Gly Gly Pro
165 170 175 180
Met Asp Ala Ser Val Glu Glu Glu Gly Val Arg Arg Ala Leu Asp Phe Ala Val Gly Glu
185 190 195 200
Tyr Asn Lys Ala Ser Asn Asp Met Tyr His Ser Arg Ala Leu Gln Val Val Arg Ala Arg
205 210 215 220
Lys Gln Ile Val Ala Gly Val Asn Tyr Phe Leu Asp Val Glu Leu Gly Arg Thr Thr Cys
225 230 235 240
Thr Lys Tyr Gln Pro Asn Leu Asp Asn Cys Pro Phe His Asp Gln Pro His Leu Lys Arg
245 250 255 260
Lys Ala Phe Cys Ser Phe Gln Ile Tyr Ala Val Pro Trp Gln Gly Thr Met Thr Leu Ser
265 270 275 280
Lys Ser Thr Cys His Asp Ala
285
Claims (8)
1.一种组合蛋白制备抗人胱抑素C多克隆抗体的方法,其特征在于,在动物免疫、抗血清效价检测、多克隆抗体的抗原-抗体亲和纯化三个步骤中均采用了重组人胱抑素C蛋白;动物免疫中采用一种重组人胱抑素C蛋白用于动物免疫;抗血清效价检测中采用一种重组人胱抑素C蛋白用于酶联免疫检测中酶标板的包被;多克隆抗体的抗原-抗体亲和纯化中采用一种重组人胱抑素C蛋白用于偶联填料并纯化抗体;
所述重组人胱抑素C蛋白为Cys C-His蛋白、Cys C-GST蛋白和Cys C-Trx蛋白中的任意一种;其中,多克隆抗体的抗原-抗体亲和纯化步骤以及抗血清效价检测步骤中采用的重组人胱抑素C蛋白均与动物免疫步骤中采用的重组人胱抑素C蛋白不同;
所述人胱抑素C蛋白的氨基酸序列如SEQ ID NO:2所示。
2.根据权利要求1所述的一种组合蛋白制备抗人胱抑素C多克隆抗体的方法,其特征在于,所述Cys C-His蛋白的氨基酸序列如SEQ ID NO:4所示,编码构成Cys C-His蛋白的CysC-His基因的核苷酸序列如SEQ ID NO:3所示;所述Cys C-GST蛋白的氨基酸序列如SEQ IDNO:6所示,编码构成Cys C-GST蛋白的Cys C-GST基因的核苷酸序列如SEQ ID NO:5所示;所述Cys C-Trx蛋白的氨基酸序列如SEQ ID NO:8所示,编码构成Cys C-Trx蛋白的Cys C-Trx基因的核苷酸序列如SEQ ID NO:7所示。
3.根据权利要求1所述的一种组合蛋白制备抗人胱抑素C多克隆抗体的方法,其特征在于,动物免疫后抗血清效价达1:16×104~1:1024×104。
4.如权利要求1~3任一项所述的方法制备出的抗人胱抑素C多克隆抗体。
5.一种如权利要求1~3任一项所述的方法制备出的抗人胱抑素C多克隆抗体在胶体金颗粒增强免疫比浊法中的应用。
6.一种如权利要求1~3任一项所述的方法制备出的抗人胱抑素C多克隆抗体制成的检测试剂。
7.根据权利要求6所述的一种检测试剂,其特征在于:该试剂由配比为4:1的R1试剂和R2试剂组成;
R1试剂的缓冲液组成为: pH 7.5~8.5的Tris-HCl,0.05~0.1 mol/L;PEG20000,0.5~2%(W/V);Tween20,0.01~0.05%(V/V);Proclin 300,0.02~0.05%(V/V);
R2试剂的缓冲液组成为:pH 7.5~8.5的Tris-HCl,0.01-0.05 mol/L ;BSA,0.5~2%(W/V);PEG20000,0.1~0.5%(W/V);Proclin 300,0.02~0.05%(V/V);标记抗人胱抑素C多克隆抗体的均相胶体金颗粒。
8.根据权利要求7所述的一种检测试剂,其特征在于:所述均相胶体金颗粒的粒径大小为30~60nm。
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