CN102702324B - 人降钙素原b细胞表位肽段及其单克隆抗体的应用 - Google Patents
人降钙素原b细胞表位肽段及其单克隆抗体的应用 Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
本发明属于医学中的免疫学领域,特别涉及降钙素原B细胞表位肽及其单克隆抗体的应用,所述表位肽段如SEQ ID NO:3所示,其可用于制备杂交瘤细胞并分泌相应的单克隆抗体;该单克隆抗体可用于制备检测降钙素原的诊断试剂;本单克隆抗体具有纯度高>95%,效价高达1:256000,且特异性好、可大批量制备等优点;通过本发明制备的单克隆抗体、多克隆抗体可用于病人血液降钙素原含量的检测,如可采用双抗体夹心ELISA反应模式,即酶标记人抗PCT单抗、酶标板包被抗PCT多抗和被测样品PCT抗原形成“双抗体夹心”结构进行测定。
Description
技术领域
本发明涉及免疫分析医学领域,特别涉及细胞表位肽段抗原及其单克隆抗体的应用。
背景技术
细菌性感染只要能够早期发现、早期诊断、针对性的早期治疗,大多预后良好。否则,可能发展成为重度菌血症或/和严重脓毒症。根据美国CDC的报告,目前脓毒症已经成为非心脏ICU死亡的主要原因(N Engl J Med.2003;348: 1546-1554),因此感染性炎症的早期诊断极为重要。另外,随着细菌耐药性、重症感染患者的增加,如何指导临床用药也显得愈发重要。
降钙素原(procalcitonin,PCT)是上世纪九十年代才发现的细菌、真菌性感染的特异性标志物,是一种无激素活性的降钙素前肽,有116个氨基酸组成,1~57为 N-残端,60~91为降钙素,96~116为降钙蛋白。分子量为13kD,由位于11号染色体 (11p1514) 上的CALC-1基因编码,半衰期为25~30小时,在体内外稳定性好,不会降解为降钙素,亦不受体内激素水平的影响,实验室检测快速简便,有利于临床特别是急诊广泛开展应用。
在严重全身性细菌、真菌、寄生虫、急性疟疾感染、系统炎性反应综合症(SIRS)、多器官功能衰竭综合症(MODS)的诊断方面,PCT是一个具有高灵敏度、特异性的新指标。PCT主要是在细菌毒素和炎性细胞因子的刺激下产生,而在非感染性炎症状态下血清PCT一般不升高,感染性炎症过程中,PCT的生成非常快,对内毒素刺激反应2~6小时内即升高,严重全身感染者血PCT在24 h内可升高达1000倍。PCT作为一种新的感染性炎性标志物目前已被广泛认可。不仅能早期鉴别细菌与非细菌感染,更是败血症的预警和诊断指标,且PCT与细菌感染的程度成正相关,它的上升或下降直接反应疾病恶化或好转的趋势,因此,PCT的检测在细菌性感染、败血症、MODS的鉴别诊断、预后判断、疗效观察及合理指导应用抗菌药等均有重要参考价值。
现有技术对PCT的制备方法多直接取自组织细胞,如甲状旁腺组织,成本高,制备过程复杂,获得产品量低,制约了对PCT的研究及应用开发。利用基因工程方法制备PCT重组蛋白是一种简单、经济、可靠的方法,可大量制备PCT蛋白,并且在此基础上可进一步制备抗PCT抗体,以进行PCT免疫检测。免疫检测的首要关键问题是如何获得特异性针对PCT的抗体,而且最好获得针对不同抗原决定簇的特异性抗体,所以进行预测选择PCT的B细胞免疫表位尤为重要。目前还没有相应的研究。
发明内容
本发明的目的之一在于提供降钙素原B细胞表位肽段及其衍生物,其能作为降钙素原单克隆抗体的抗原。本发明的目的之二在于提供一种杂交瘤细胞,其能特异性的分泌降钙素原单克隆抗体。同时,本发明还提供所述一种单克隆抗体,其可作为加检测降钙素原的试剂。
为实现上述目的,本发明的技术方案为:
1、人降钙素原B细胞表位肽段,人降钙素原B细胞表位肽段的氨基酸序列如SEQ ID NO:3所示。
2、含有1所述的人降钙素原B细胞表位肽段的衍生物,所述的人降钙素原B细胞表位肽段的衍生物为所述人降钙素原B细胞表位肽段偶联有载体蛋白BSA或KLH。
3、根据2所述的人降钙素原B细胞表位肽段的衍生物,所述的人降钙素原B细胞表位肽段在N末端引入半胱氨酸。
4、1-3任一项所述的人降钙素原B细胞表位肽段制备的单克隆抗体的杂交瘤细胞。
5、根据4所述的杂交瘤细胞,杂交瘤细胞的生物保藏号为CCTCC NO:C201137。
6、5所述的杂交瘤细胞分泌的单克隆抗体。
本发明的目的还在于提供上述单克隆抗体的制备方法,该方法操作简单,适用于大规模生产该单克隆抗体。
为实现上述目的,本发明的技术方案在于:
所述的单克隆抗体的制备方法:用如SEQ ID NO:3所示氨基酸序列的人降钙素原B细胞表位肽段为抗原,免疫所得的血清效价大于1:3200的动物的脾细胞与SP2/0骨髓瘤细胞融合,经筛选克隆获得分泌单克隆抗体的杂交瘤细胞株,所述杂交瘤细胞株分泌,得单克隆抗体。
本发明的另一目的在于提供所述单克隆抗体的应用,该应用为检测降钙素原提供了新思路。
为实现上述目的,本发明的技术方案在于:
所述的单克隆抗体在制备用于检测降钙素原的诊断试剂中的应用。
进一步,所述单克隆抗体与PCT重组蛋白联合用于制备检测降钙素原的诊断试剂中的应用。
进一步,所述PCT重组蛋白为如SEQ ID NO:1所示的核苷酸序列所编码的蛋白。
本发明的有益效果在于:本发明制备的人降钙素原是高纯度的PCT重组蛋白(rhPCT),该蛋白可用于抗体制备的免疫原及筛选原,同时可以作为建立PCT定量检测时的校准品;
通过PCT蛋白序列的B细胞表位肽段免疫小鼠制备的单克隆抗体具有纯度高(SDS-PAGE检测纯度>95%)、效价高(ELISA效价达1:256000)、特异性好、可大批量制备等优点;
人PCT蛋白中B细胞表位肽段化学合成时在其N端引入半胱氨酸,提高了其与KLH或BSA交联率(>50%),可获得优质免疫原;
通过本发明制备的单克隆抗体、多克隆抗体可用于病人血液PCT含量的检测,如可采用“双抗体夹心”ELISA反应模式,即酶标记人抗PCT单抗、酶标板包被抗PCT多抗和被测样品PCT抗原形成“双抗体夹心”结构进行测定。
附图说明
图1为抗PCT单克隆抗体的纯化色谱图,其中UV:紫外吸收光谱;cond:电导率。
培养物保藏
本发明中杂交瘤细胞株3-PCT 4-D12 送中国典型培养物保藏中心(武汉大学保藏中心)保藏,保藏编号为CCTCC NO:C201137,地址位于中国武汉武汉大学,保存日期为2011年7月3日,保藏的培养物为小鼠杂交瘤细胞。
具体实施方式
下面结合实施例进一步阐述本发明。应理解,这些实施例仅用于说明本发明,而非限制本发明的范围。下列实施例中未注明具体条件的试验方法及未说明配方的试剂均为按照常规条件如分子克隆:实验手册(New York: Cold Spring Harbor Laboratory Press,1989)和现代免疫学实验技术(沈关心 周汝麟主编)中所述的条件或制造商建议的条件进行或配置,未注明来源的产品均可通过市场途径获得。
实施例1 PCT重组蛋白的制备
从GENBANK获取PCT 编码基因的基础上,进行密码子偏嗜性改造,化学合成编码基因片段,克隆入原核表达载体经DNA测序鉴定后诱导蛋白表达,经性质鉴定后,大量纯化制备PCT重组蛋白,该蛋白可用于抗体制备的免疫原及筛选原,同时可以作为后续实验中建立PCT定量检测时的校准品,具体的:
一 PCT重组蛋白基因克隆:
从GENBANK获得人PCT蛋白的基因序列(登录号为NM_004102),将其递交于Graphical codon usage analyzer (http://guca.schoedl.del),分析其密码子偏性情况;具体的,将人PCT基因密码子在大肠杆菌中使用率<10%的同义置换为大肠杆菌偏爱的密码子,使其更加容易在大肠杆菌中表达,优化后的核苷酸序列为如SEQ ID NO:1所示:
gcaccattca ggtctgccct ggagagcagc ccagcagacc cggccacgct cagtgaggac gaagcgcgcc tcctgctggc tgcactggtg caggactatg tgcagatgaa ggccagtgag ctggagcagg agcaagagag agagggctcc agcctggaca gccccagatc taagcggtgc ggtaatctga gtacttgcat gctgggcaca tacacgcagg acttcaacaa gtttcacacg ttcccccaaa ctgcaattgg ggttggagca cctggaaaga aaagggatat gtccagcgac ttggagagag accatcgccc tcatgttagc atgccccaga atgccaacta atag
相应的多肽序列如SEQ ID NO:2所示,具体为:
Ala Pro Phe Arg Ser Ala Leu Glu Ser Ser Pro Ala Asp Pro Ala Thr Leu Ser Glu Asp Glu Ala Arg Leu Leu Leu Ala Ala Leu Val Gln Asp Tyr Val Gln Met Lys Ala Ser Glu Leu Glu Gln Glu Gln Glu Arg Glu Gly Ser Ser Leu Asp Ser Pro Arg Ser Lys Arg Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala Pro Gly Lys Lys Arg Asp Met Ser Ser Asp Leu Glu Arg Asp His Arg Pro His Val Ser Met Pro Gln Asn Ala Asn
上述胺基酸的中英文对照如下:
谷酰胺gln Q;甘氨酸 gly G;丝氨酸 ser S;丙氨酸 ala A;苏氨酸 thr T;缬氨酸 val V;异亮氨酸 ile I;亮氨酸 leu L;酪氨酸 tyr Y;苯丙氨酸 phe F;组氨酸 his H;脯氨酸 pro P;天冬酰胺 asn N;甲硫氨酸 met M;谷氨酸 glu E;色氨酸 trp W;赖氨酸 lys K;半胱氨酸 cys C;精氨酸 arg R; 天冬氨酸Asp D
为进行有效表达及纯化,在该序列的5′-及3′-端分别添加SalⅠ、BamHⅠ酶切位点,其中BamHⅠ位点后加入GATGATGATGATAAG序列,其编码Asp-Asp-Asp-Asp-Lys多肽序列为EK酶的识别位点。然后委托上海英骏生物工程有限公司进行全基因合成。合成的过程为常规基技术,可参考分子克隆:实验手册一书。
二 pET42a-PCT重组质粒构建:
将pET42a载体与合成基因经SalⅠ、BamHⅠ酶切4小时后用T4DNA连接酶4℃过夜连接。将制备好的感受态DH5α菌200μl,冰浴,吸取1μl连接产物加入管中,转化DH5α菌,轻拍混匀,冰浴30分钟,42℃水浴90秒,取出离心管再冰浴2分钟,加入800μl室温的2×YT培养液混匀,37℃摇床220rpm振荡培养1小时,分别将50μl、200μl及剩下的全部转化菌涂于3个含卡那霉素抗性的2×YT培养板上,37℃恒温培养箱过夜培养,次日挑去白素菌落接种于LB培养基扩大培养,用碱裂解法提取质粒,取质粒用SalⅠ、BamHⅠ双酶切4小时,酶切体系为:pET42a-PCT质粒DNA 10μl,SalⅠ 1μl,BamHⅠ1μl,10×缓冲液K 2μl,ddH2O 6μl,并取酶切产物10μl行1.5%琼脂糖凝胶电泳鉴定,可见与设计值378bp一致的片段。
三 PCT重组蛋白诱导表达
将转化pET42a-PCT的细菌扩大培养及诱导表达,测细菌OD值达0.6-0.8时加入IPTG(至终浓度1 mmol/L)诱导表达6 h。培养后每克湿菌以10倍体积的PBS缓冲液(pH 7.3)重悬,混匀后超声破菌,破菌完全后于4 ℃ 10,000 rpm离心15 min,上清以0.45 μm微孔滤膜过滤。分别取少量上清与沉淀SDS-PAGE电泳鉴定目的蛋白可溶性,经鉴定表达蛋白破菌后几乎都存在上清中,为可溶性表达。
四 PCT重组蛋白纯化
上清经过滤后用Amersham的AKTAprime上柱纯化,Binding buffer平衡后用Elution buffer线性洗脱,收集主洗脱峰,用His-Binding buffer稀释10倍后进行HisTrap HP再次纯化,纯化后的产物用PBS平衡过的分子筛,收集蛋白。将获得的蛋白用HiTrap Desalting 置换缓冲体系为EK切割buffer(50 mmol/L Tris-HCl ,PH8.0),按EK酶与蛋白1:1000的质量比加入EK酶,4℃摇床60rpm切割24h。切割后用His- Binding buffer稀释后HisTrap HP纯化,15% SDS-PAGE电泳鉴定,PCT重组蛋白分子量为13Kda左右。IPTG未诱导的重组菌作阴性对照,诱导6h的重组菌为阳性对照,纯化后目的蛋白纯度达到95%以上。
五 PCT重组蛋白浓度、纯度测定:
1 Lowry法测定PCT蛋白含量
制备标准曲线
Lowry法测定的试剂盒购于上海美季生物技术有限公司,
试剂A:1) 10克 Na2CO3,2克 NaOH和0.25克酒石酸钾钠 (KNaC4H4O6·4H2O)。溶解于500毫升蒸馏水中;2) 0.5克硫酸铜(CuSO4·5H2O)溶解于100毫升蒸馏水中,每次使用前,将50份(A)与1份(B)混合,即为试剂甲。
试剂B:在2升磨口回流瓶中,加入100克钨酸钠(Na2WO4·2H2O),25克钼酸钠(Na2MoO4·2H2O)及700毫升蒸馏水,再加50毫升85%磷酸,100毫升浓盐酸,充分混合,接上回流管,以小火回流10小时,回流结束时,加入150克 硫 酸 锂(Li2SO4),50毫升蒸馏水及数滴液体溴,开口继续沸腾15分钟,以便驱除过量的溴。冷却后溶液呈黄色(如仍呈绿色,须再重复滴加液体溴的步骤)。稀释至1升,过滤,滤液置于棕色试剂瓶中保存。使用时用标准NaOH滴定,酚酞作指示剂,然后适当稀释,约加水1倍,使最终的酸浓度为1N左右。
标准蛋白质溶液:精确称取结晶牛血清清蛋白或 g—球蛋白,溶于蒸馏水,浓度为250 mg/ml左右。牛血清清蛋白溶于水若混浊,可改用0.9 % NaCl溶液。
在650nm波长下,以空白管为对照调零,分别测定各管的吸光度,以蛋白浓度为横坐标,吸光度为纵坐标,制作标准曲线。将待测蛋白稀释后,紫外分光光度计测定A260值与A280值。根据公式,蛋白浓度C=(1.45×A280-0.75×A260)×稀释倍数,计算出待测蛋白的粗略浓度,然后将蛋白样品用蒸馏水稀释至 25~150μg范围,按照上表的操作程序反应,测定处650nm吸光度值,然后在标准曲线上查出相应的浓度,再乘以稀释倍数即为待测蛋白的浓度,多管计算平均值,测得浓度为1.075g/ml。
2 高效液相色谱法(HPLC)进行纯度测定和PCT获得量计算
将纯化的蛋白用HPLC分析其纯度,可知经SP Sepharose Fast Flow阳离子交换柱初纯化后PCT蛋白的纯度达93.25%以上,再次过分子筛纯化后PCT纯度可达98%。经计算,每升诱导菌可获得36.5mg该融合蛋白,经切割后可以获得9.6mg左右的PCT重组蛋白。
六Western blot对PCT重组蛋白进行免疫反应特异性鉴定
取最后纯化的降钙素原蛋白行15% SDS-PAGE鉴定,用Abcam公司生产的抗PCT单克隆抗体对其进行Western Blot分析,选用Millipore Immobilon Western Chemiluminescent HRP Subscrate系统显色,结果显示抗PCT单抗结合PCT蛋白处可见清洗条带。
七 稳定性研究
采用冻干保存的方式在4℃的条件下保存180天,其检测值降低<5%,而用校准品稀释液保存一个月即降低20%以上,6个月减少70%以上,说明长期保存宜采用冻干的形式。此外,在冻干品用校准品稀释液溶解后,7天内降低<10%,因此在这期间可用于定量检测。
实施例2降钙素原B细胞表位肽段的制备
所述的化学合成的B细胞表位肽段的序列来自生物信息学分析结果,综合分析PCT蛋白的二级结构、抗原性、亲疏水性、可及性及柔韧性,对各区段进行分析评分,选取得分高的区域作为B细胞表位区域。具体的,利用Chou & Fasman预测β转角、Emini法预测抗原表面可及性、Karplus& Schulz法预测蛋白柔韧性、Kolaskar & Tongaonkar蛋白质抗原性分析、Parker法蛋白质疏水分析和Bepipred线性抗原表位预测等技术参数,获得人PCT蛋白中潜在的B细胞表位肽段;
所述的筛选B细胞表位肽段,其中化学合成的表位肽段为:N-- DMSSDLERDHRPHV—C(SEQ ID NO:3),所述的B细胞表位肽段化学合成中在其N端引入半胱氨酸,以提高其与BSA的交联能力,接着与BSA交联。交联率(>50%),得降钙素原B细胞表位肽段抗原。
另外,如SEQ ID NO:3所示的表位肽段也可选择同KLH进行交联。
实施例3 单克隆抗体的制备与鉴定
一 降钙素原B细胞表位肽段衍生物免疫Balb/c小鼠
将实施例2获得降钙素原B细胞表位肽段抗原从-80℃冰箱取出作抗原,溶解后过滤。
选取6周龄、体重约20g的雌性Balb/c小鼠免疫。抗原乳化选用双注射器互推法。首次免疫时,将如SEQ ID NO:3所示的降钙素原B细胞表位肽段抗原与等体积的弗氏完全佐剂乳化混合,得抗原混合物,每只小鼠按100μg 的量皮内多点加腹腔注射。第14和第28天分别进行第二次第三次免疫,佐剂改用不完全弗氏佐剂,抗原量、注射体积和途径不变,第3次免疫后间接ELISA法测定效价。融合前3天进行加强免疫,每只小鼠腹腔注射不加佐剂的100μg PCT,3天后细胞融合。
二 被免疫Balb/c小鼠血清效价测定
第3次免疫后10天从小鼠尾静脉取血检测血清抗体效价。将新购酶标板用双蒸水浸泡过夜,晾干后备用;用包被液(0.05mol/L碳酸钠缓冲液:0.16g Na2CO3,0.293g NaHCO3,0.02g NaN3,加去离子水溶解定容100ml)将实施例1所得PCT重组蛋白抗原稀释为最佳工作浓度5μg/ml,每孔加100μl抗原稀释液,37℃温育1小时后,以胶带封口,于4℃过夜,倒尽板孔中液体,吸干孔内残余反应液,加满洗涤液过一次,再注满洗涤液缓缓晃动2min,倾去,反复五次,最后将反应板倒置在吸水纸上,使孔中洗涤液流尽。自然干燥后以胶带封口,此为PCT重组蛋白抗原包被的酶标板,加封闭液300μl,37℃孵育1.5小时,洗涤5次;采血及稀释血清:捏住鼠尾,75%酒精消毒后用剪刀在尾静脉剪一缺口,取血20μl,2000rpm离心30min,取上清1μl加入999μl抗体稀释液混匀,并进行体积倍比稀释,从1:100至1:3200,将稀释的被检血清每孔加入100μl,同时取小鼠免疫前血清1:100稀释做阴性对照,抗体稀释液做空白对照。37℃孵育1~1.5小时,洗涤5次;将辣根过氧化物酶羊抗小鼠IgG(上海亿欣生物科技有限公司)稀释到1:10000, 每孔加100μl,37℃孵育1.5小时,洗涤5次;加邻苯二胺溶液100μl /孔,室温暗处15分钟,每孔加终止液100μl观察结果,OPD氧化后的产物呈橙红色,用酶联免疫检测仪记录492nm读数,以空白对照孔调零后测各孔OD值大于阴性对照OD值的2.1倍,即为阳性。血清效价达到1:3200,可用于细胞融合。
三 小鼠脾细胞悬液和SP2/0细胞悬液的制备
取免疫好的Balb/c小鼠,摘除小鼠眼球放血处死,眼血离心后收集血清作ELISA的阳性对照,无菌操作取出脾脏,放入盛有10ml 不完全培养基的玻璃皿中,洗涤,小心剥去周围的结缔组织和脂肪组织,换一玻璃皿,将脾脏捞出,置于200目不锈钢网中,用注射器的内芯研磨,不时用不完全培养基冲洗,使脾细胞穿过网孔进入溶液中,将脾细胞移至10ml玻璃离心管中,800rpm水平离心10min,去上清。同法用不完全培养基10ml洗涤细胞1次,离心收集沉淀的细胞,将细胞用10ml不完全培养基重悬混匀,细胞计数约为1×108个细胞。
将SP2/0细胞从液氮中取出,迅速放入37℃水浴中,不断摇晃,直至细胞溶液完全溶解,将细胞转移到10ml离心管中,800rpm水平离心10min,弃上清,10ml完全培养液重悬沉淀,把细胞悬液转移到50ml培养瓶中,置37℃、5%CO2培养箱中培养。待细胞生长良好后用含8-AG的选择培养基筛选细胞一周;融合前2天,将1瓶细胞传至4瓶,则融合当天细胞正处于对数生长期,活力正好,细胞大小均匀,圆而透亮,融合当天,用弯头滴管将SP2/0细胞从管壁上轻轻吹下,收集于离心管中,离心,弃上清,沉淀用不完全培养基洗涤后,10ml不完全培养基重悬,细胞计数,约为5×107。
四 滋养细胞的制备
取一只未免疫的Balb/c小鼠,摘眼球放血处死,75%乙醇浸泡消毒5min,剪开小鼠皮肤,用镊子提起腹膜,用剪刀剪一小口,弯头滴管吸取预冷的不完全培养基冲洗腹腔,将洗液吸至50ml离心管中。同法用不完全培养基冲洗腹腔3次,收集洗液,室温下1000rpm水平离心10min,去上清,10ml不完全培养基重悬细胞并计数。
五 骨髓瘤细胞和脾脏B淋巴细胞融合
融合前将PEG1500置于37℃培养箱中预温,吸取1×107个骨髓瘤细胞悬液和1×108个脾脏B淋巴细胞悬液(细胞数1:10)至一个50ml离心管中,补加30ml不完全培养基,充分混匀,1000rpm离心10min,弃上清,轻弹管底,使细胞团松散成糊状,将离心管37℃水浴,用滴管吸取0.8ml预温的50% PEG1500溶液,在离管底约2cm处沿管壁慢慢加入细胞中,边加边转动离心管,在1min左右加完,然后静置90s,逐滴加入37℃预温的不完全培养基30ml终止融合,3min之内加完,速度先慢后快,动作轻柔,将离心管在37℃培养箱中静置5min,取出离心管,1000rpm离心5min,弃去上清,加入10ml HAT培养基重悬细胞,轻轻吹打,混匀,将融合细胞接种至已铺有滋养细胞的96孔细胞培养板,按100μl/孔,每块培养板留6孔接种SP2/0细胞,作为HAT选择的阴性对照,置37℃,5%CO2培养箱中培养。
六 融合细胞的选择性培养及杂交瘤细胞的筛选
融合后第5天即可在倒置显微镜下观察细胞的生长情况,并补加HAT培养基100μl,第14天换HT培养基培养。融合后10~14天,待细胞长到满培养孔的1/2孔底时,采用间接ELISA法检测培养上清,筛选阳性克隆;以实施例1制备的纯化后PCT重组蛋白包被酶标板(0.5μg/孔),4℃过夜,洗涤缓冲液洗涤5次,每次5min,拍干液体,每孔加入封闭液300μl,37℃孵育2h,加入100μl细胞培养上清,阳性对照选小鼠的免疫血清,阴性对照选SP2/0培上清,空白对照用洗涤液,37℃孵育2h;洗涤酶标板:每孔加入100μl 1:10000稀释的HRP标记的羊抗小鼠IG抗体,37℃孵育2h;洗涤,拍干液体,加新鲜配制的邻苯二胺溶液100μl /孔,室温暗处反应10~15分钟,加终止液每孔100μl终止反应,酶标仪检测450nm吸光度值。结果为以PCT表位肽段为抗原,免疫 BALB/C小鼠,融合成功后,经克隆化和ELISA筛选后,得到分泌PCT单克隆抗体的杂交瘤细胞株,其细胞培养上清效价达到1:6400。这株杂交瘤细胞经数次冻存,体外传代培养3个月以上仍能稳定分泌抗体。
七 阳性杂交瘤细胞的克隆化
筛选出阳性克隆后,立即采用有限稀释法将阳性杂交瘤细胞进行克隆化培养,制备饲养细胞,用10ml不完全培养基重悬,收集阳性克隆细胞并计数,用不完全培养基将阳性克隆细胞稀释到100个/20ml,取一块预先已加有滋养细胞的96孔细胞培养板,加入200μl细胞悬液,将剩下的阳性克隆细胞转移到24孔板中扩大培养,收集细胞液氮冻存,同时将培养板在37℃,5%CO2培养箱培养,第3天后显微镜下观察细胞生长情况,10天后用ELISA法检测效价,并将最强的阳性克隆再次克隆化,直至细胞阳性率达100%,即可定株;测取定株的杂交瘤细胞株培养上清的效价后再将定株的杂交瘤细胞扩大培养,并送中国典型培养物保藏中心(武汉大学保藏中心)保藏,保藏号为CCTCC NO:C201137,其可稳定分泌所述单克隆抗体。
八 小鼠腹水制备、抗体纯化及效价测定
选用健康雌性Balb/c小鼠10只,腹腔注射0.5ml灭菌石蜡油/鼠,1-2周后每只小鼠腹腔注射0.5×106~1×106个杂交瘤细胞,同时注射0.25ml等量混合的液体石蜡与不完全弗氏佐剂的混合物。小鼠明显产生腹水后拉颈处死,用吸管从腹腔取出腹水,4℃离心15min,分离收集中段的澄清腹水液。选用HiTrap rProtein A HP柱接入AKTA Explorer纯化抗体,详见图1,经SDS-PAGE检测纯度大于95%。纯化的抗体用间接ELISA法检测效价达1:256000,初步说明获得的单克隆抗体对PCT分子有较高结合能力,分装冷冻保存,待实施例5使用。
九 抗PCT单克隆抗体的亲和力测定
为检验抗PCT单克隆抗体的对PCT抗原的结合能力,运用基于抗原/抗体竞争结合原理的单抗亲和力常数(Kd)检测方法对所获单抗进行亲和力测定。将纯化好的PCT抗原溶解在0.05mol/l的碳酸缓冲液(pH9.5)中,调整PCT的终浓度为1μg/ml,于ELISA板孔中每孔加100μl,胶带封闭条板4℃过夜。次日拍干孔中液体,以含1%BSA的PBS溶液对各孔封闭2h,洗板并干燥后4℃保存备用。根据测定原理及方法建立抗原抗体反应系统,抗PCT单克隆抗体初始反应浓度稀释至40ng/ml, PCT抗原初始浓度稀释至360mg/ml。抗原浓度倍比稀释按30、15、7.5、3.75、1.875、0.938、0.469、0.235(单位为10-12mol/l)进行,计算PCT单抗的亲和力常数。结果显示其具有高亲和力, Kd=5.3×10-8mol/L。
本实施例的实验步骤详见《现代免疫学实验技术》(沈关心 周汝麟主编)。
实施例4 抗PCT兔多克隆抗体(PCT重组蛋白的特异性多克隆抗体)的制备
一 免疫动物
以实施例1所得纯化PCT重组蛋白为抗原,采用背部皮下及四肢多点注射免疫新西兰大白兔。免疫程序:基础免疫前耳缘静脉取血5ml分离血清作为阴性对照。每只用抗原500μg与等体积完全弗氏佐剂充分乳化后进行注射,首次免疫后3天用等量抗原配以完全弗氏佐剂加强免疫,第28天用等量抗原配以不完全弗氏佐剂第3次免疫。第3次免疫后7天,耳缘静脉取血5ml分离血清,用间接ELISA检测抗血清的效价。效价达1:64000时颈动脉插管收集全血,4℃放置过夜,4000rpm离心收集血清,-70℃保存。效价达不到要求可再加强免疫1次。
二 特异性亲和纯化抗体
将待纯化的血清用上样缓冲液(0.1M磷酸钠,0.1M柠檬酸三钠,pH7.0)适当稀释后加入到Protein A柱中,用洗脱缓冲液(0.1M磷酸钠,0.1M柠檬酸钠,pH3.0)洗脱柱子,收集单峰。收集的纯化产物再经抗原抗体特异性亲和纯化,得到纯化的抗PCT多克隆抗体,即PCT重组蛋白的特异性多克隆抗体。
所述的抗原抗体特异性亲和纯化方法:PCT抗原用buffer A(0.1mol/L碳酸氢钠,0.5mol/L氯化钠,pH 8.0),按照0.5:1(buffer: sample)处理,将填料NHS-activated Sepharose 4FAST Flow装柱,室温下将PCT重组蛋白偶联在柱上,经过纯化过量的抗原决定簇及大量清洗柱体后,备用。反复上样,保证特异性的抗PCT抗体充分与柱结合,用100mol/L甘氨酸,pH 2.5洗脱,特异性抗体蛋白直接被洗脱收集入1mol/L Tris,pH 9.0,-20℃保存备用。
本实施例的实验步骤详见《现代免疫学实验技术》(沈关心 周汝麟主编)。
实施例5 单克隆抗体在制备用于检测降钙素原的诊断试剂中的应用
一 双抗体夹心ELISA的建立
用0.05 mol/L碳酸盐缓冲液(pH 9.6)稀释所述PCT单克隆抗体包被酶标板,所述单克隆抗体由保藏号为CCTCC NO:C201137的杂交瘤细胞分泌或体内诱生法所得,100μl/孔,4℃过夜,用洗液(0.05% PBST,pH 7.4)洗3次;5% BSA封闭,200μl/孔,37℃孵育2h后洗3次;加入PCT标准品(即实施例1所 得纯化的PCT重组蛋白)和待测血清样本,100μl/孔,标准品做倍比稀释。37℃孵育1h后 洗3次;加入抗PCT多抗(即实施例4所得抗PCT多抗),100μl/孔,37℃孵育1h后洗3次;再加入HRP标记的羊抗兔IgG(上海亿欣生物科技有限公司),37℃孵育45min后用洗涤液(0.1% PBST,pH 7.4)洗6次;加TMB底物,100μl/孔,37℃显色10min,以2mol/L硫酸终止反应,在酶标板450nm处测吸光度值(OD450)。
2 ELISA最佳反应条件的确定
用棋盘滴定法确定各抗体的最佳工作浓度,将所述PCT单克隆抗体按照1:2000、1:10000、1:50000稀释3个浓度包被酶标板,阳性对照(0.5μg/ml PCT重组蛋白为标准品)和阴性对照(PBS)为样品,多抗按1:2000、1:5000、1:10000稀释3个浓度,HRP标记羊抗兔IgG按实际说明书推荐稀释度,按照上述实验步骤操作,确定抗体的最佳工作浓度。然后将HRP标记羊抗兔IgG倍比稀释,再次做棋盘滴定。去阳性对照OD450值在1.5左右,阴性对照OD450值小于0.1的条件下为最佳,初次结果不理想,可进一步缩小或扩大稀释度以取得抗原抗体的最佳反应浓度。结果:在捕获抗体(所述PCT单克隆抗体)稀释度为1:10000、夹心抗体(为实施例4所得抗PCT兔多克隆抗体)的稀释度为1:5000和所述HRP标记的羊抗兔IgG稀释度为1:5000条件下该ELISA检测方法的性价比最高。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离所附权利要求书所限定的本发明的精神和范围。
<110> 重庆业为基生物科技有限公司
<120> 人降钙素原B细胞表位肽段及其单克隆抗体的应用
<160> 3
<210> 1
<211> 354
<212> DNA
<213> 人工序列
<220>
<223> PCT重组蛋白基因
<400> 1
gcaccattca ggtctgccct ggagagcagc ccagcagacc cggccacgct cagtgaggac 60
gaagcgcgcc tcctgctggc tgcactggtg caggactatg tgcagatgaa ggccagtgag 120
ctggagcagg agcaagagag agagggctcc agcctggaca gccccagatc taagcggtgc 180
ggtaatctga gtacttgcat gctgggcaca tacacgcagg acttcaacaa gtttcacacg 240
ttcccccaaa ctgcaattgg ggttggagca cctggaaaga aaagggatat gtccagcgac 300
ttggagagag accatcgccc tcatgttagc atgccccaga atgccaacta atag 354
<210> 2
<211> 116
<212> PRT
<213> 人工序列
<220>
<223> PCT重组蛋白
<400> 2
Ala Pro Phe Arg Ser Ala Leu Glu Ser Ser Pro Ala Asp Pro Ala
1 5 10 15
Thr Leu Ser Glu Asp Glu Ala Arg Leu Leu Leu Ala Ala Leu Val
20 25 30
Gln Asp Tyr Val Gln Met Lys Ala Ser Glu Leu Glu Gln Glu Gln
35 40 45
Glu Arg Glu Gly Ser Ser Leu Asp Ser Pro Arg Ser Lys Arg Cys
50 55 60
Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe
65 70 75
Asn Lys Phe His Thr Phe Pro Gln Thr Ala Ile Gly Val Gly Ala
80 85 90
Pro Gly Lys Lys Arg Asp Met Ser Ser Asp Leu Glu Arg Asp His
95 100 105
Arg Pro His Val Ser Met Pro Gln Asn Ala Asn
110 115
<210> 3
<211> 14
<212> PRT
<213> 智人(homo sapiens)
<220>
<223> 人降钙素原B细胞表位肽段
<400> 3
Asp Met Ser Ser Asp Leu Glu Arg Asp His Arg Pro His Val
1 5 10
Claims (5)
1.人降钙素原B细胞表位肽段制备的单克隆抗体杂交瘤细胞,其特征在于,人降钙素原B细胞表位肽段的氨基酸序列如SEQ ID NO:3所示,所述单克隆抗体杂交瘤细胞的生物保藏号为CCTCC NO:C201137。
2.权利要求1所述的杂交瘤细胞分泌的单克隆抗体。
3.权利要求2所述的单克隆抗体在制备用于检测降钙素原的诊断试剂中的应用。
4.根据权利要求3所述的应用,其特征在于:所述单克隆抗体与PCT重组蛋白的特异性多克隆抗体联合用于制备检测降钙素原的诊断试剂中的应用。
5.根据权利要求4所述的应用,其特征在于:所述PCT重组蛋白为如SEQ ID NO:1所示的核苷酸序列所编码的蛋白。
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