CN115028741A - 一种肿瘤抗原抗体复合物及制备方法和应用 - Google Patents
一种肿瘤抗原抗体复合物及制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种肿瘤抗原抗体复合物及制备方法和应用,该肿瘤抗原抗体复合物包括肿瘤抗原和抗体,肿瘤抗原抗体复合物通过将肿瘤抗原与抗体结合得到,其为抗体偶联蛋白3G9‑NY‑ESO‑1。本发明中,利用断裂内含肽GP41‑1反式剪接方法实现肿瘤抗原与抗体的连接,二者之间利用肽键连接,其稳定性更高,特异性结合更强,反应条件温和,效率高,工艺简单,且未加入有毒有害物质,也不会产生副产物等,安全性高,克服了现有技术中利用化学偶联方法等实现抗原抗体连接时需要加入化学试剂、有一定毒性、化学偶联不稳定、偶联键易断裂等问题,肿瘤抗原抗体复合物能作为一种新型的肿瘤疫苗使用,为新型肿瘤疫苗的研发提供技术支撑。
Description
技术领域
本发明属于生物技术领域,具体涉及一种肿瘤抗原抗体复合物及制备方法和应用。
背景技术
肿瘤抗原主要包括肿瘤特异性抗原和肿瘤相关抗原。机体产生肿瘤抗原的可能机制有:基因突变;细胞原本不表达的基因被激活;胚胎时期抗原或分化抗原的异常、异位表达;外源性基因(如病毒基因)表达等。纽约食管鳞状上皮癌抗原1(NY-ESO-1)是肿瘤-睾丸抗原(CTA)亚家族之一,NY-ESO-1在细胞内经加工产生抗原肽,与白细胞抗原(HLA)分子结合形成复合物,经CD4+T淋巴细胞及细胞毒性T淋巴细胞(CTL)识别,可诱导机体产生针对相应肿瘤细胞的较强的特异性免疫应答(国际检验医学杂志.36(21),3138-3141,2015)。
内含肽是其宿主蛋白质自切除并催化侧翼序列通过肽键连接的内部蛋白质元件。断裂内含肽在蛋白质内含子中间特定位点发生断裂形成N端片段和C端片段,分别由两个相距较远的两个基因编码,两个片段通过非共价键相互识别,重建催化活性中心,介导蛋白质反式剪接(中国生物化学与分子生物学报.24(6),512-521,2008)。断裂内含肽GP41-1是文献报导中剪接最稳定和最快速的(J Biol Chem.287,28686-28696,2012)。Wang Han等利用断裂内含肽GP41-1成功将一个低温酵母株的DNA结合域(DBD)与合成VP64激活域偶联,构建成基于GAL4的双向表达***(Pnas.115(15),3900-3905,2018)。Yao Zhong等利用断裂内含肽GP41-1开发了断裂内含肽介导的蛋白质连接(SINPL)的活细胞方法,可应用于哺乳动物细胞系(Nature Communications.11:2440,2020)。
3G9是由专利CN 101888856 B中提供的抗人树突状细胞(DC细胞)上的DEC205的抗体。DEC205作为DC细胞表面的模式识别受体,能够特异性的捕获抗原分子,加工递呈给机体免疫***,诱导免疫反应。抗DEC205单克隆抗体可以作为靶向性载体将药物特异性的传输至DC细胞,并能够有效的增强这一过程。体外实验表明,经过DEC205靶向抗体辅助的抗原递呈效率能够增强1000倍(张顶.DEC-205靶向抗体的制备及其人源化改造[D].北京协和医学院(清华大学医学部)&中国医学科学院,2013)。
随着免疫治疗领域的发展,NY-ESO-1过继性T细胞疗法、联合检查点抑制剂治疗等方法的局限性突显,DEC205单克隆抗体作为载体在肿瘤免疫治疗中有巨大的应用前景。
发明内容
为解决现有技术中存在的技术问题,本发明的目的在于提供一种肿瘤抗原抗体复合物及制备方法和应用。
为实现上述目的,达到上述技术效果,本发明采用的技术方案为:
一种肿瘤抗原抗体复合物,所述肿瘤抗原抗体复合物包括肿瘤抗原和抗体,所述肿瘤抗原抗体复合物的重链氨基酸序列如SEQ ID NO:10所示,轻链氨基酸序列如SEQ IDNO:6所示,轻链不参与反应。
进一步的,所述肿瘤抗原抗体复合物通过将肿瘤抗原与抗体结合得到,所述肿瘤抗原抗体复合物为抗体偶联蛋白3G9-NY-ESO-1,所述肿瘤抗原为NY-ESO-1,所述抗体为DEC205的单克隆抗体3G9。
进一步的,所述肿瘤抗原抗体复合物通过断裂内含肽GP41-1反式剪接将肿瘤抗原与抗体结合得到。
一种肿瘤抗原抗体复合物的制备方法,包括以下步骤:
步骤(1):抗体融合蛋白3G9-IntN重链表达载体构建
步骤(2):抗体融合蛋白3G9-IntN制备
步骤(3):融合蛋白GB1-IntC-NY-ESO-1表达载体构建
步骤(4):融合蛋白GB1-IntC-NY-ESO-1制备
步骤(5):抗体偶联蛋白3G9-NY-ESO-1制备。
进一步的,步骤(1)中,抗体融合蛋白3G9-IntN重链表达载体构建的步骤包括:以质粒pUC57-Kan-GP41IN为模板,设计碱基序列如SEQ ID NO:3所示的Fc-IntN-F和碱基序列如SEQ ID NO:4所示的Fc-IntN-R,通过PCR扩增Fc-IntN基因片段,然后凝胶回收相应片段,克隆至phIgG1-3G9载体上的多克隆位点BamH I与HindⅢ之间,构建成可表达抗体融合蛋白3G9-IntN的重链表达载体phIgG1-3G9-IntN。
进一步的,步骤(2)中,抗体融合蛋白3G9-IntN制备的步骤包括:将步骤(1)构建的抗体融合蛋白3G9-IntN重链表达载体和抗体3G9轻链表达载体phIgK-3G9以1:1的质量比共同转染293T细胞,表达抗体融合蛋白3G9-IntN,蛋白重链氨基酸序列如SEQ ID NO:5所示,轻链氨基酸序列如SEQ ID NO:6所示。
进一步的,步骤(3)中,融合蛋白GB1-IntC-NY-ESO-1表达载体构建的步骤包括:
通过使用BamH I和NdeI双酶切质粒pUC57-Kan-GB1-GP41IC和pET-30a(+)-NY-ESO-1,分别得到GB1-IntC片段和线性化载体pET-30a(+)-NY-ESO-1,然后使用T4连接酶,用T4连接方法构建成融合蛋白GB1-IntC-NY-ESO-1表达载体pET-30a(+)-GB1-IntC-NY-ESO-1。
进一步的,步骤(4)中,融合蛋白GB1-IntC-NY-ESO-1制备的步骤包括:
将步骤(3)构建的融合蛋白GB1-IntC-NY-ESO-1表达载体pET-30a(+)-GB1-IntC-NY-ESO-1转化至BL21(DE3)感受态细胞中,,根据《pET***手册》制备融合蛋白GB1-IntC-NY-ESO-1,蛋白氨基酸序列如SEQ ID NO:9所示,使用HisTrapTM HP纯化目的蛋白。
进一步的,步骤(5)中,抗体偶联蛋白3G9-NY-ESO-1制备的步骤包括:
将步骤(2)所得抗体融合蛋白3G9-IntN和步骤(4)所得融合蛋白GB1-IntC-NY-ESO-1以一定的摩尔比混合,加终浓度10微摩每升的DTT诱导催化断裂内含肽GP41-1反式剪接反应,剪接反应后,IntN剩余TRSGY五个氨基酸残基与IntC的五个氨基酸残基SSSDV以肽键连接,得到所需抗体偶联蛋白3G9-NY-ESO-1,蛋白重链氨基酸序列如SEQ ID NO:10所示,轻链氨基酸序列如SEQ ID NO:6所示,轻链不参与反应。
一种肿瘤抗原抗体复合物在制备肿瘤疫苗中的应用。
与现有技术相比,本发明的有益效果为:
本发明公开了一种肿瘤抗原抗体复合物及制备方法和应用,该肿瘤抗原抗体复合物包括肿瘤抗原和抗体,肿瘤抗原为NY-ESO-1,抗体为DEC205的单克隆抗体3G9,肿瘤抗原抗体复合物通过将肿瘤抗原与抗体结合得到,肿瘤抗原抗体复合物为抗体偶联蛋白3G9-NY-ESO-1,重链氨基酸序列如SEQ ID NO:10所示,轻链氨基酸序列如SEQ ID NO:6所示,轻链不参与反应。本发明中,利用断裂内含肽GP41-1反式剪接方法实现肿瘤抗原与抗体的连接,二者之间利用肽键连接,其稳定性更高,特异性结合更强,反应条件温和,效率高,工艺简单,且未加入有毒有害物质,也不会产生副产物等,安全性高,克服了现有技术中利用化学偶联方法等实现抗原抗体连接时需要加入化学试剂、有一定毒性、化学偶联不稳定、偶联键易断裂等问题,肿瘤抗原抗体复合物能够作为一种新型的肿瘤疫苗使用,为新型肿瘤疫苗的研发提供技术支撑和新思路。
附图说明
图1是本发明的抗体融合蛋白3G9-IntN的重链表达载体phIgG1-3G9-IntN的质粒图谱;
图2是本发明的抗体融合蛋白3G9-IntN的SDS-PAGE考染鉴定图;
图3是本发明的融合蛋白GB1-IntC-NY-ESO-1的表达载体pET-30a(+)-GB1-IntC-NY-ESO-1的质粒图谱;
图4是本发明的融合蛋白GB1-IntC-NY-ESO-1的SDS-PAGE考染鉴定图;
图5是本发明的断裂内含肽反式剪接制备抗体偶联蛋白3G9-NY-ESO-1的SDS-PAGE考染鉴定图。
具体实施方式
下面对本发明进行详细阐述,以使本发明的优点和特征能更易于被本领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定。
以下给出一个或多个方面的简要概述以提供对这些方面的基本理解。此概述不是所有构想到的方面的详尽综览,并且既非旨在指认出所有方面的关键性或决定性要素亦非试图界定任何或所有方面的范围。其唯一的目的是要以简化形式给出一个或多个方面的一些概念以为稍后给出的更加详细的描述之序。
一种肿瘤抗原抗体复合物,该肿瘤抗原抗体复合物包括肿瘤抗原和抗体,肿瘤抗原为NY-ESO-1,抗体为中国发明专利公开号CN 101888856 B中提供的抗人树突状细胞(DC细胞)上的DEC205的单克隆抗体3G9,该肿瘤抗原抗体复合物通过断裂内含肽GP41-1反式剪接将肿瘤抗原与抗体结合得到,肿瘤抗原抗体复合物为抗体偶联蛋白3G9-NY-ESO-1,肿瘤抗原抗体复合物的重链氨基酸序列如SEQ ID NO:10所示,轻链氨基酸序列如SEQ ID NO:6所示,轻链不参与反应。
一种肿瘤抗原抗体复合物的制备方法,包括以下步骤:
肿瘤抗原NY-ESO-1与GB1-GP41-1-InteinC(C端外显肽)融合表达制备融合蛋白GB1-IntC-NY-ESO-1,GB1是常见蛋白助溶标签,完成剪接反应后会与IntC一起被切掉;
抗体3G9与GP41-1-InteinN(C端外显肽)融合表达制备抗体融合蛋白3G9-IntN;
融合蛋白GB1-IntC-NY-ESO-1与抗体融合蛋白3G9-IntN混合,通过断裂内含肽GP41-1反式剪接,生成抗体偶联蛋白3G9-NY-ESO-1。融合蛋白GB1-IntC-NY-ESO-1与抗体融合蛋白3G9-IntN的制备步骤无相互顺序。
实施例1
抗体融合蛋白3G9-IntN重链表达载体构建
GP41-1-InteinN多肽段的氨基酸序列(SEQ ID NO:1)的哺乳动物密码子优化碱基序列(SEQ ID NO:2)从质粒pUC57-Kan-GP41IN(由苏州工业园区唯可达生物科技有限公司提供)上获得,抗体3G9重链表达载体phIgG1-3G9和轻链表达载体phIgK-3G9由苏州工业园区唯可达生物科技有限公司提供。构建方法如下:
以质粒pUC57-Kan-GP41IN(由苏州工业园区唯可达生物科技有限公司提供)为模板,设计表1的引物,通过PCR扩增Fc-IntN基因片段,然后凝胶回收相应片段(DNA凝胶回收试剂盒,碧云天生物技术研究所,货号D0056),克隆至phIgG1-3G9载体上的多克隆位点BamHI与HindⅢ之间,构建成可表达抗体融合蛋白3G9-IntN的重链表达载体phIgG1-3G9-IntN,质粒图谱如图1所示,经测序鉴定正确后入库。
PCR扩增条件如下:
PCR扩增程序:98℃10min,98℃40s,56℃30s,72℃1min,72℃10min,10℃10min,PCR产物跑1%琼脂糖凝胶电泳。
表1
实施例2
抗体融合蛋白3G9-IntN制备
将实施例1中构建的抗体融合蛋白3G9-IntN重链表达载体和抗体3G9(抗人DEC205单克隆抗体)轻链表达载体phIgK-3G9(由苏州工业园区唯可达生物科技有限公司提供)以质量比1:1共同转染293T细胞,表达3G9-IntN蛋白(蛋白重链氨基酸序列见SEQ ID NO:5,轻链氨基酸序列见SEQ ID NO:6)。具体方法如下:
第1天,在10cm细胞培养皿(JET,TCP-010-100)铺293T细胞,5×106/皿,于37℃二氧化碳细胞培养箱中过夜孵育。第二天,准备蛋白的表达载体/转染试剂复合物。转染试剂为Turbofect(Thermo Fisher Scientific,R0531),转染剂量与复合方法可参见转染试剂说明书。复合体系完成后,将293T细胞上清换为10mL/皿的含20%Ultraculture(含1%Glu,Lonza,BEBP12-725F)的DMEM维持培养基,然后加入表达载体/转染试剂复合物。转染5天后,取上清使用HiTrapTM rProtein A FF(GE Healthcare,17-5080-01)纯化目的蛋白,纯化方法可参见纯化柱说明书。经BCA法(碧云天生物技术研究所,货号P0009)检测所制备的融合蛋白浓度,检测方法参看检测试剂盒说明书所制蛋白分装后,-80℃保存。取80微升3G9-IntN蛋白加20微升5×SDS-PAGE上样缓冲液(由苏州工业园区唯可达生物科技有限公司提供),沸水浴10分钟制样,SDS-PAGE,考染,鉴定蛋白。SDS-PAGE的方法为本领域人员熟知,简述如下。按照蛋白标记(Thermo,货号26616)、3G9-IntN蛋白的顺序依次点样,电泳80V 30分钟后再调电压至120V至指示剂到底,用考马斯亮蓝染色液(由苏州工业园区唯可达生物科技有限公司提供)室温染色1小时后,沸水浴30分钟脱色,结果如图2所示。
实施例3
融合蛋白GB1-IntC-NY-ESO-1表达载体构建
GB1-GP41-1-InteinC多肽段的氨基酸序列(SEQ ID NO:7)的大肠杆菌密码子优化碱基序列(SEQ ID NO:8)从质粒pUC57-Kan-GB1-GP41IC(由苏州工业园区唯可达生物科技有限公司提供)上获得。
通过使用BamH I(宝生物工程(大连)有限公司,货号1010A)和NdeI(宝生物工程(大连)有限公司,货号1161A)双酶切质粒pUC57-Kan-GB1-GP41IC和pET-30a(+)-NY-ESO-1(由苏州工业园区唯可达生物科技有限公司提供),分别得到GB1-IntC片段和线性化载体pET-30a(+)-NY-ESO-1,然后使用T4连接酶(宝生物工程(大连)有限公司,货号2050A),用本领域熟知的T4连接方法构建成融合蛋白GB1-IntC-NY-ESO-1表达载体pET-30a(+)-GB1-IntC-NY-ESO-1,质粒图谱如图3所示,经测序鉴定正确后入库。
实施例4
融合蛋白GB1-IntC-NY-ESO-1制备
将实施例3中构建的融合蛋白GB1-IntC-NY-ESO-1表达载体pET-30a(+)-GB1-IntC-NY-ESO-1转化至BL21(DE3)感受态细胞(康为世纪,CW0809S)中,转化方法可参见感受态细胞说明书。根据《pET***手册》(TB055 8th Edition02/99,Novagen)制备融合蛋白GB1-IntC-NY-ESO-1(蛋白氨基酸序列见SEQ ID NO:9)。具体方法如下:
转化BL21(DE3)感受态细胞后,涂卡那霉素抗性板,挑单克隆至1毫升LB液体培养基(由苏州工业园区唯可达生物科技有限公司提供)中,37℃,200rpm,培养8h,保甘油菌。取10微升甘油菌接至5毫升LB液体培养基中,37℃,200rpm,过夜培养。再将5毫升菌液扩大培养至500毫升LB液体培养基中,37℃,200rpm,培养2-3h,菌液OD600到0.8h左右,加终浓度1毫摩每升的IPTG(Solarbio,货号I8070)诱导表达,诱导条件:25℃,180rpm,诱导20h。诱导后10000g离心5min收集菌体,使用60毫升包涵体洗涤缓冲液(由苏州工业园区唯可达生物科技有限公司提供)重悬,加终浓度100微克每毫升的溶菌酶(Sigma-Aldrich,货号L6876)30℃孵育15min,然后在冰上用超声波细胞粉粹机超声,超声条件为:400瓦,5秒超声/5秒间隔,共30min,超声结果为菌液不粘稠。细胞裂解后10000g离心10min,收集包涵体。去上清,用100毫升包涵体洗涤缓冲液重悬洗涤包涵体沉淀,重复洗涤包涵体至少3次,直至包涵体清洗干净。加50毫升包涵体溶解缓冲液(由苏州工业园区唯可达生物科技有限公司提供)溶解包涵体,在磁力搅拌器上室温溶解2-3h,包涵体基本完全溶解。20000g,4℃离心30min,弃沉淀,上清用0.22微米过滤后使用脱盐柱(GE Healthcare,17-5087-01)脱盐,脱盐方法可参见脱盐柱说明书。置换为脱盐缓冲液(由苏州工业园区唯可达生物科技有限公司提供)后,使用HisTrapTM HP(GE Healthcare,17-5248-01)纯化目的蛋白,纯化方法可参见纯化柱说明书。经BCA法(碧云天生物技术研究所,货号P0009)检测所制备的重组蛋白浓度,检测方法参看检测试剂盒说明书。所制蛋白分装后,-80℃保存。取80微升GB1-IntC-NY-ESO-1蛋白加20微升5×SDS-PAGE上样缓冲液(由苏州工业园区唯可达生物科技有限公司提供),沸水浴10分钟制样,SDS-PAGE,考染,鉴定蛋白。SDS-PAGE的方法为本领域人员熟知,简述如下。按照蛋白标记(Thermo,货号26616)、GB1-IntC-NY-ESO-1蛋白的顺序依次点样,电泳80V30分钟后再调电压至120V至指示剂到底,用考马斯亮蓝染色液(由苏州工业园区唯可达生物科技有限公司提供)室温染色1小时后,沸水浴30分钟脱色,结果如图4所示。
包涵体洗涤缓冲液:20mM Tris-HCl pH 7.5,10mM EDTA,1%Triton X-100。
包涵体溶解缓冲液:20mM Tris-HCl pH 8.5,6M盐酸胍。
脱盐缓冲液:20mM Tris-HCl pH 8.5,150mM氯化钠。
实施例5
抗体偶联蛋白3G9-NY-ESO-1制备
将实施例2中制备的抗体融合蛋白3G9-IntN和实施例4中制备的融合蛋白GB1-IntC-NY-ESO-1以不同摩尔比(3G9-IntN:GB1-IntC-NY-ESO-1的摩尔比分别是3:1、1.5:1、1:1、1:1.5和1:3)混合,加终浓度10微摩每升的DTT(Solarbio,D8220-25g)诱导催化断裂内含肽GP41-1反式剪接反应,剪接反应后,IntN剩余TRSGY五个氨基酸残基与IntC的五个氨基酸残基SSSDV以肽键连接,制备得到肿瘤疫苗3G9-NY-ESO-1蛋白(蛋白重链氨基酸序列见SEQ ID NO:10和轻链氨基酸序列见SEQ ID NO:6,轻链不参与反应)。反应缓冲液:pH值8.5,20毫摩每升Tris盐酸,含150毫摩每升氯化钠(由苏州工业园区唯可达生物科技有限公司提供),反应条件:37℃反应2小时。取80微升反应后产物加20微升5×SDS-PAGE上样缓冲液(由苏州工业园区唯可达生物科技有限公司提供),沸水浴10分钟制样,SDS-PAGE,考染,鉴定蛋白。SDS-PAGE的方法为本领域人员熟知,简述如下。按照蛋白标记(Thermo,货号26616)、3G9-IntN蛋白、GB1-IntC-NY-ESO-1蛋白、3G9-IntN:GB1-IntC-NY-ESO-1的摩尔比分别是3:1、1.5:1、1:1、1:1.5和1:3的顺序依次点样,电泳80V 30分钟后再调电压至120V至指示剂到底,用考马斯亮蓝染色液(由苏州工业园区唯可达生物科技有限公司提供)室温染色1小时后,沸水浴30分钟脱色,结果如图5所示,确定3G9-IntN:GB1-IntC-NY-ESO-1的最佳摩尔比为1.5:1。
本发明未具体描述的部分或结构采用现有技术或现有产品即可,在此不做赘述。
以上所述仅为本发明的实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书内容所作的等效结构或等效流程变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
Claims (10)
1.一种肿瘤抗原抗体复合物,其特征在于,所述肿瘤抗原抗体复合物包括肿瘤抗原和抗体,所述肿瘤抗原抗体复合物的重链氨基酸序列如SEQ ID NO:10所示,轻链氨基酸序列如SEQ ID NO:6所示,轻链不参与反应。
2.根据权利要求1所述的一种肿瘤抗原抗体复合物,其特征在于,所述肿瘤抗原抗体复合物通过将肿瘤抗原与抗体结合得到,所述肿瘤抗原抗体复合物为抗体偶联蛋白3G9-NY-ESO-1,所述肿瘤抗原为NY-ESO-1,所述抗体为DEC205的单克隆抗体3G9。
3.根据权利要求2所述的一种肿瘤抗原抗体复合物,其特征在于,所述肿瘤抗原抗体复合物通过断裂内含肽GP41-1反式剪接将肿瘤抗原与抗体结合得到。
4.根据权利要求1-3任一所述的一种肿瘤抗原抗体复合物的制备方法,其特征在于,包括以下步骤:
步骤(1):抗体融合蛋白3G9-IntN重链表达载体构建
步骤(2):抗体融合蛋白3G9-IntN制备
步骤(3):融合蛋白GB1-IntC-NY-ESO-1表达载体构建
步骤(4):融合蛋白GB1-IntC-NY-ESO-1制备
步骤(5):抗体偶联蛋白3G9-NY-ESO-1制备。
5.根据权利要求4所述的一种肿瘤抗原抗体复合物的制备方法,其特征在于,步骤(1)中,抗体融合蛋白3G9-IntN重链表达载体构建的步骤包括:以质粒pUC57-Kan-GP41IN为模板,设计碱基序列如SEQ ID NO:3所示的Fc-IntN-F和碱基序列如SEQ ID NO:4所示的Fc-IntN-R,通过PCR扩增Fc-IntN基因片段,然后凝胶回收相应片段,克隆至phIgG1-3G9载体上的多克隆位点BamH I与HindⅢ之间,构建成可表达抗体融合蛋白3G9-IntN的重链表达载体phIgG1-3G9-IntN。
6.根据权利要求4所述的一种肿瘤抗原抗体复合物的制备方法,其特征在于,步骤(2)中,抗体融合蛋白3G9-IntN制备的步骤包括:将步骤(1)构建的抗体融合蛋白3G9-IntN重链表达载体和抗体3G9轻链表达载体phIgK-3G9以1:1的质量比共同转染293T细胞,表达抗体融合蛋白3G9-IntN,蛋白重链氨基酸序列如SEQ ID NO:5所示,轻链氨基酸序列如SEQ IDNO:6所示。
7.根据权利要求4所述的一种肿瘤抗原抗体复合物的制备方法,其特征在于,步骤(3)中,融合蛋白GB1-IntC-NY-ESO-1表达载体构建的步骤包括:
通过使用BamH I和NdeI双酶切质粒pUC57-Kan-GB1-GP41IC和pET-30a(+)-NY-ESO-1,分别得到GB1-IntC片段和线性化载体pET-30a(+)-NY-ESO-1,然后使用T4连接酶,用T4连接方法构建成融合蛋白GB1-IntC-NY-ESO-1表达载体pET-30a(+)-GB1-IntC-NY-ESO-1。
8.根据权利要求4所述的一种肿瘤抗原抗体复合物的制备方法,其特征在于,步骤(4)中,融合蛋白GB1-IntC-NY-ESO-1制备的步骤包括:
将步骤(3)构建的融合蛋白GB1-IntC-NY-ESO-1表达载体pET-30a(+)-GB1-IntC-NY-ESO-1转化至BL21(DE3)感受态细胞中,,根据《pET***手册》制备融合蛋白GB1-IntC-NY-ESO-1,蛋白氨基酸序列如SEQ ID NO:9所示,使用HisTrapTM HP纯化目的蛋白。
9.根据权利要求4所述的一种肿瘤抗原抗体复合物的制备方法,其特征在于,步骤(5)中,抗体偶联蛋白3G9-NY-ESO-1制备的步骤包括:
将步骤(2)所得抗体融合蛋白3G9-IntN和步骤(4)所得融合蛋白GB1-IntC-NY-ESO-1以一定的摩尔比混合,加终浓度10微摩每升的DTT诱导催化断裂内含肽GP41-1反式剪接反应,剪接反应后,IntN剩余TRSGY五个氨基酸残基与IntC的五个氨基酸残基SSSDV以肽键连接,得到所需抗体偶联蛋白3G9-NY-ESO-1,蛋白重链氨基酸序列如SEQ ID NO:10所示,轻链氨基酸序列如SEQ ID NO:6所示,轻链不参与反应。
10.根据权利要求1-4任一所述的一种肿瘤抗原抗体复合物在制备肿瘤疫苗中的应用。
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