CN116732078A - 以pET28a作为载体制备肿瘤相关抗原NY-ESO-1的方法及应用 - Google Patents
以pET28a作为载体制备肿瘤相关抗原NY-ESO-1的方法及应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域,提供了以pET28a作为载体制备肿瘤相关抗原NY‑ESO‑1的方法及应用。将NY‑ESO‑1基因与pET28a载体重组后转染感受态细胞,依次经种子获取及扩大培养、IPTG诱导后破菌收集上清、纯化后得到毫克级别的NY‑ESO‑1融合蛋白。本发明选择pET28a载体作为承载载体,并在后续表达纯化过程对各项工艺过程及条件进行优化,使得目标蛋白在可溶性上清中出现,避免了现有包涵体制备方式中需要对包涵体进行复性的步骤,简化了分离纯化流程及操作难度。得率方面,5L菌液经过本发明方法进行表达、纯化后,获得的NY‑ESO‑1蛋白约为10mg,相较于现有微克级别的得率有大幅提升。
Description
技术领域
本发明属于生物医药领域,具体涉及肿瘤相关抗原NY-ESO-1的制备方法及其相关应用。
背景技术
肿瘤相关抗原(tumor associated antigen,TAA)是指在肿瘤细胞或正常细胞上都存在的一类抗原分子,常用于临床肿瘤的诊断。它并非肿瘤细胞所特有,正常细胞可微量合成,并且在肿瘤细胞增殖时高度表达。在实体瘤治疗中,TAA靶点是特异性抗肿瘤免疫治疗的首选,针对TAA的抗体不仅可以通过ADCC效应直接杀死肿瘤细胞,也可以作为诊断标记物或者创新性的增加传统癌症疗法的靶向性。以TAA为基础的肿瘤治疗性疫苗可以激活机体的免疫系统,诱导产生特异性的免疫细胞,对肿瘤细胞产生杀伤,还可以TAA为靶点,设计相关的TCR/CAR,制备出TAA特异性的T细胞,用以开展细胞治疗。
NY-ESO-1(New York Esophageal Squamous Cell Carcinoma)抗原首先是由ChenYT等[1]通过SEREX技术从食管癌组织中筛选得到。此后,大量文献相继报道,在滑膜肉瘤(80%)、恶性黑素瘤(45%)、卵巢癌(43%)等肿瘤组织中表达较高,在肝癌(43%)、尿路上皮癌(35%)、多发性骨髓瘤(26%)、肺癌(18.2%)中也有不同程度的表达[2-5],为将来免疫治疗的应用提供了基础。NY-ESO-1在正常组织中也有表达,但是由于血睾屏障的存在,使得这些正常组织并不会遭受免疫毒性,说明NY-ESO-1具有肿瘤特异性,展现了良好的临床应用前景。研究发现,在NY-ESO-1mRNA阳性的恶性黑素瘤患者中,50%患者血清中存在自发性特异性抗体[6];对1374名乳腺癌患者进行血液检测后发现,1%的患者体内可以检测到自发性的NY-ESO-1特异性抗体[7];此外,在肺癌、多发性骨髓瘤等患者中也有类似发现[8,9],说明NY-ESO-1具有较强的激发特异性体液免疫应答的能力。不仅如此,NY-ESO-1还能诱导自发性特异性细胞免疫反应,主要是激活CD4+和CD8+T淋巴细胞。Jager团队[10]首先发现NY-ESO-1能诱导特异性的细胞免疫应答,随后Chen、Zeng等[11,12]也相继发现NY-ESO-1抗原肽能被CD4+或CD8+T淋巴细胞所识别,并与HLA-I类或HLA-II类分子结合,产生特异性的细胞免疫应答,而且90%以上血清NY-ESO-1抗体(+)的患者体内存在特异性CTL。因此,NY-ESO-1被认为是理想的肿瘤免疫治疗靶标。
现有NY-ESO-1抗原的制备主要通过包涵体的方法,不仅需要复性等复杂的操作,而且也会影响得率,仅能得到微克级别的蛋白。与此同时,商品化的NY-ESO-1蛋白价格极其昂贵,如某品牌通过大肠杆菌重组表达的NY-ESO-1蛋白价格为:¥3220/2μg、
¥4945/5μg、¥8970/10μg。因此亟需对现有制备方法进行优化,以期优化NY-ESO-1蛋白的价格。
发明内容
本发明基于上述研究进行,对现有NY-ESO-1蛋白的制备进行改进,使得蛋白主要在上清中,避免包涵体复性步骤,制备流程更加简便,同时一次得率可达mg级别,得率更高。
本发明的第一方面,提供了一种肿瘤相关抗原NY-ESO-1的制备方法,主体步骤如下:将NY-ESO-1基因与pET28a载体重组后转染感受态细胞,依次经种子获取及扩大培养、IPTG诱导后破菌收集上清、纯化后得到毫克级别的NY-ESO-1融合蛋白。
其中,NY-ESO-1的基因序列如SEQ ID NO.1所示。
进一步,本发明肿瘤相关抗原NY-ESO-1的制备方法具体包括如下步骤:
A、pET28a/NY-ESO-1重组载体构建
采用EcoR I和Xho I双酶切位点,将pET28a载体进行切开,与NY-ESO-1基因序列进行拼接,所得的pET28a/NY-ESO-1重组载体经测序验证后,用于后续扩增、表达;
其中,NY-ESO-1的BamHI酶切位点引物序列如SEQ ID NO.2所示,XhoI酶切位点的引物序列如SEQ ID NO.3所示。
B、转染
将pET28a/NY-ESO-1重组载体转入Rosetta或BL-21/DE3感受态细胞,扩增后涂到氨苄抗性的LB培养板上(氨苄浓度20μg/mL),挑菌;
C、种子获取及扩大培养
用枪头挑出菌落,放入氨苄抗性的LB培养基内,37℃,200rpm进行扩增;而后取菌液加入到250倍体积的氨苄抗性LB培养基中37℃,200rpm继续扩增,作为接种的种子;取多份种子液分别加入至多瓶200倍体积的氨苄抗性LB培养基中37℃,200rpm摇床扩大培养,过程中监测OD值,当OD值为0.6-0.7时,停止摇菌;
D、IPTG诱导后破菌收集上清
向步骤C的每瓶培养瓶中加入终浓度为0.1~10mM(优选0.1mM)的IPTG,20℃、180rpm诱导过夜(优选24h);而后将所有菌液转到离心瓶中,配平,5000rpm,15min离心后弃上清;向菌体中加入破菌缓冲液并充分打散,采用超声264W/m2、0.5Hz破菌12min后,将破好的菌液转到离心瓶中,配平,12000rpm,离心30min,收集上清;
其中,破菌缓冲液的配方组成如下:50mM Tris/HCl、1mM EDTA、0.5M NaCl,调节pH为8.0。
E、纯化
采用HisTrap柱子纯化,通过AKTA explorer蛋白纯化系统进行纯化并收集流穿液;上样完毕后,采用60%或80%的咪唑洗脱液进行洗脱,并收集洗脱液保存,洗脱液经透析处理后得到NY-ESO-1蛋白。
其中,洗脱液透析方法如下:配制体积为洗脱液100倍的透析液,4h后换液一次,换液后透析过夜,透析液的配方组成如下:20m Tris/HCl、1mM EDTA、0.15M NaCl。
本发明的第二方面,提供了采用上述方法制备得到的NY-ESO-1融合蛋白的应用,如在制备NY-ESO-1抗体检测试剂盒、肿瘤治疗性疫苗、NY-ESO-1单克隆抗体或NY-ESO-1特异性T细胞中的应用。
具体的,NY-ESO-1融合蛋白作为抗原制备ELISA检测试剂盒,检测患者体内是否存在抗NY-ESO-1抗体及其滴度;作为抗原制备成肿瘤治疗性疫苗;作为抗原免疫小鼠等动物,筛选和制备可以产生抗NY-ESO-1抗体的杂交细胞瘤,制备大量单克隆抗体;NY-ESO-1为基础的治疗性疫苗免疫小鼠等动物后,获取NY-ESO-1特异性T细胞,克隆其TCR序列,可用于后续NY-ESO-1特异性TCR-T细胞治疗。
发明的作用与效果
本发明选择pET28a载体作为承载载体,并在后续表达纯化过程对各项工艺过程及条件进行优化,使得目标蛋白在可溶性上清中出现,避免了现有包涵体制备方式中需要对包涵体进行复性的步骤,简化了分离纯化流程及操作难度。得率方面,5L菌液经过本发明方法进行表达、纯化后,一次获得的NY-ESO-1蛋白约为10mg,相较于现有微克级别的得率有大幅提升。因此,本发明为NY-ESO-1蛋白的制备提供了新的思路。
此外,本发明载体的标签为His标签(6个组氨酸),相对于其他标签,His标签分子量小,更有助于后续研究。
附图说明
图1是pET28a载体质粒图谱信息;
图2是pET28a载体的克隆及表达区域;
图3显示了PET28a/NY-ESO-1重组质粒在25℃下不同感受态细胞诱导表达情况;
图4显示了PET28a/NY-ESO-1重组质粒在25℃下不同感受态细胞诱导表达的蛋白WB验证结果;
图5显示了PET28a/NY-ESO-1重组质粒25℃下不同IPTG浓度诱导表达情况对比;
图6显示了PET28a/NY-ESO-1重组质粒在IPTG诱导前后、破菌后上清液以及包涵体中诱导表达情况对比;
图7显示了不同洗脱条件下His/NY-ESO-1蛋白的HisTrap纯化情况对比;
图8显示了PET28a/NY-ESO-1重组质粒诱导表达蛋白经纯化、透析后的WB验证结果。
具体实施方式
下面结合实施例和附图对本发明进行详细描述。但下列实施例不应看作对本发明范围的限制。
一、表达载体的构建
根据EMD Biosciences(Novagen)公司的pET28a载体信息,设计酶切位点和引物。采用Xho I和BamH I双酶切位点,将pET28a载体进行切开,与NY-ESO-1基因序列进行拼接。所得的pET28a/NY-ESO-1重组载体经测序验证后,用于后续扩增、表达。
其中,pET28a载体质粒图谱和多克隆位点信息如图1和图2所示,其带有His标签蛋白。NY-ESO-1的基因序列如SEQ ID NO.1所示,NY-ESO-1的BamHI酶切位点引物序列如SEQID NO.2所示,XhoI酶切位点的引物序列如SEQ ID NO.3所示。
NY-ESO-1基因序列(SEQ ID NO.1):
atgcagg ccgaaggccg gggcacaggg ggttcgacgg gcgatgctga tggcccaggaggccctggca
ttcctgatgg cccagggggc aatgctggcg gcccaggaga ggcgggtgcc acgggcggca
gaggtccccg gggcgcaggg gcagcaaggg cctcggggcc gggaggaggc gccccgcggg
gtccgcatgg cggcgcggct tcagggctga atggatgctg cagatgcggg gccagggggc
cggagagccg cctgcttgag ttctacctcg ccatgccttt cgcgacaccc atggaagcag
agctggcccg caggagcctg gcccaggatg ccccaccgct tcccgtgcca ggggtgcttc
tgaaggagtt cactgtgtcc ggcaacatac tgactatccg actgactgct gcagaccacc
gccaactgca gctctccatc agctcctgtc tccagcagct ttccctgttg atgtggatca
cgcagtgctt tctgcccgtg tttttggctc agcctccctc agggcagagg cgctaa
NY-ESO-1的BamHI酶切位点引物(SEQ ID NO.2):
5'CCGCTCGAGCAGGCCGAAGGCCGGGGCACA3';
NY-ESO-1的XhoI酶切位点引物(SEQ ID NO.3):
5'CGGATCCTTAGCGCCTCTGCCCTGAGGGAGGC 3'。
二、NY-ESO-1的表达与纯化
1、重组质粒转染:将pET28a/NY-ESO-1重组质粒转入Rosetta或BL-21/DE3感受态细胞,扩增后涂到氨苄抗性的LB培养板上,挑菌;其中,氨苄浓度为20μg/ml,下同。
2、菌落扩增:用枪头挑出菌落,放入4ml的氨苄抗性的LB培养基,37℃,200rpm进行扩增;
3、种子获取:取200μl的菌液加入50ml的氨苄抗性的LB培养基中扩增作为接种的种子,37℃,200rpm进行扩增;
4、扩大培养:配制氨苄抗性的LB培养基6瓶,每瓶400ml,加入200μL菌液,37℃,200rpm;
5、监测OD值:2小时后测OD值,然后每半小时监测一次,当OD值为0.6-0.7左右时,停止摇菌,留样1;
6、IPTG诱导:每瓶加入IPTG,终浓度为0.1mM/L,20℃,180rpm诱导过夜(具体为24h),留样2;
7、收集细菌:将所有菌液转到离心瓶中,配平,5000rpm,15min,弃上清;
8、破菌:配制破菌缓冲液(50mM Tris/HCl;1mM EDTA;0.5M NaCl;共200ml,调节pH=8.0)。将菌液冲下混匀,用搅拌子充分打散菌液30min,然后用超声264W/m2,0.5Hz破菌12min;
4、收集可溶上清:将破好的菌液转到离心瓶中,配平,12000rpm,离心30min,收集上清,留样3;同时用枪头挑一点沉淀(包涵体)加入50μl超纯水中,留样4;
5、蛋白烤染:将不同阶段留取的样本进行电泳分析,分离胶浓度12%,浓缩胶浓度5%,120V,35min,然后将凝胶用考马斯亮蓝染色,漂洗至凝胶透明后,观察目的蛋白位置,显示位于上清中;
6、蛋白纯化(HisTrap):利用GE公司的HisTrap蛋白质纯化层析柱,通过AKTAexplorer蛋白纯化系统进行纯化,收集流穿液。上样完毕后,采用浓度为60%或80%的咪唑洗脱液进行洗脱,并收集洗脱的样品保存。
7、咪唑洗脱的样品进行透析,去除盐分:洗脱液透析:配制透析液(20mM Tris/HCl;1mM EDTA;0.15M NaCl;共2L,4h后换液一次,换液后透析过夜)。
8、透析后的样本收集保存。BCA法蛋白含量检测试剂盒(ThermoFisher)测定蛋白浓度。经过多次重复,5L菌液经过本法进行表达、纯化后,获得的NY-ESO-1蛋白约为10mg。
三、不同表达与纯化条件下蛋白诱导表达情况对比
1、不同感受态细胞及诱导条件组合下蛋白表达情况
将PET28a/NY-ESO-1重组质粒于25℃下分别转染六组Rosetta感受态细胞、六组BL-21感受态细胞以及两组BL-21plus感受态细胞。其中三组Rosetta感受态细胞、三组BL-21感受态细胞以及一组BL-21plus感受态细胞在后续纯化过程中进行IPTG诱导(0.1mM/L,20℃,180rpm,24h),其余细胞不进行诱导。
结果显示,经过Rosetta和BL-21感受态细胞的表达,经IPTG诱导后在目标位置均出现大量蛋白,而未经IPTG诱导后在目标位置出现蛋白的数量明显低于诱导后细胞(图3和图3)。WB验证结果显示,目标位置的蛋白为NY-ESO-1(图4)。
2、不同IPTG诱导条件对比
将未诱导菌液作为阴性对照,分别考察20℃和37℃下0.1mM、0.5mM、1.0mM IPTG诱导结果,诱导时间均为24h。
结果如图5所示,IPTG浓度对蛋白表达影响不大,温度对蛋白表达影响较大。最适条件:20℃、0.1mM IPTG。
3、NY-ESO-1目标位置考察
将表达与纯化步骤中的留样1(诱导前菌液)、留样2(诱导后菌液)、留样3(可溶上清)、留样4(包涵体)进行蛋白烤染,结果如图6所示,目的蛋白NY-ESO-1主要存在于上清中。
4、不同咪唑洗脱浓度影响对比
将破菌后上清液作为阳性对照、流穿液作为阴性对照,考察20%、40%、60%、80%、100%咪唑洗脱后洗脱液中的蛋白浓度。如7图所示,经过HisTrap纯化后,目的蛋白得到了较好的纯化,尤其通过60%、80%的咪唑洗脱液获得的蛋白相对较纯。
5、PET28a/NY-ESO-1重组质粒诱导表达蛋白的WB验证
经过纯化、透析后的蛋白,经过WB验证,确认其为目的蛋白NY-ESO-1(图8)。
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以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。
Claims (10)
1.一种肿瘤相关抗原NY-ESO-1的制备方法,其特征在于,包括如下步骤:将NY-ESO-1基因与pET28a载体重组后转染感受态细胞,依次经种子获取及扩大培养、IPTG诱导后破菌收集上清、纯化后得到毫克级别的NY-ESO-1蛋白。
2.根据权利要求1所述的肿瘤相关抗原NY-ESO-1的制备方法,其特征在于:
其中,NY-ESO-1的基因序列如SEQ ID NO.1所示。
3.根据权利要求1所述的肿瘤相关抗原NY-ESO-1的制备方法,其特征在于:
其中,所述感受态细胞为Rosetta或BL-21/DE3感受态细胞。
4.根据权利要求1所述的肿瘤相关抗原NY-ESO-1的制备方法,其特征在于:
其中,所述肿瘤相关抗原NY-ESO-1的制备方法具体包括如下步骤:
A、pET28a/NY-ESO-1重组载体构建
采用EcoR I和Xho I双酶切位点,将pET28a载体进行切开,与NY-ESO-1基因序列进行拼接,所得的pET28a/NY-ESO-1重组载体经测序验证后,用于后续扩增、表达;
B、转染
将pET28a/NY-ESO-1重组载体转入Rosetta或BL-21/DE3感受态细胞,扩增后涂到氨苄抗性的LB培养板上,挑菌;
C、种子获取及扩大培养
用枪头挑出菌落,放入氨苄抗性的LB培养基内,37℃,200rpm进行扩增;而后取菌液加入到250倍体积的氨苄抗性LB培养基中继续扩增,作为接种的种子;取多份种子液分别加入至多瓶200倍体积的氨苄抗性LB培养基中摇床扩大培养,当OD值为0.6-0.7时,停止摇菌;
D、IPTG诱导后破菌收集上清
向步骤C的每瓶培养瓶中加入终浓度为0.1~10mM的IPTG,20℃、180rpm诱导过夜;而后将所有菌液转到离心瓶中,配平,5000rpm,15min离心后弃上清;向菌体中加入破菌缓冲液并充分打散,采用超声264W/m2、0.5Hz破菌后收集可溶上清;
E、纯化
采用HisTrap柱子纯化,通过AKTAexplorer蛋白纯化系统进行纯化并收集流穿液;上样完毕后,采用60%或80%的咪唑洗脱液进行洗脱,并收集洗脱液保存,洗脱液经透析处理后得到NY-ESO-1蛋白。
5.根据权利要求4所述的肿瘤相关抗原NY-ESO-1的制备方法,其特征在于:
其中,步骤A中,NY-ESO-1的BamHI酶切位点引物序列如SEQ ID NO.2所示,XhoI酶切位点的引物序列如SEQ ID NO.3所示。
6.根据权利要求4所述的肿瘤相关抗原NY-ESO-1的制备方法,其特征在于:
其中,步骤B和步骤C中,氨苄浓度均为20μg/mL,
步骤C中,种子获取与扩大培养的条件与菌落扩增条件相同。
7.根据权利要求4所述的肿瘤相关抗原NY-ESO-1的制备方法,其特征在于:
其中,步骤D中,IPTG的浓度为0.1mM,诱导时间为24h;
破菌缓冲液的配方组成如下:50mM Tris/HCl、1mM EDTA、0.5M NaCl,调节pH为8.0;破菌时,将破菌缓冲液冲下混匀,用搅拌子充分打散菌液30min,然后用超声264W/m2,0.5Hz破菌12min;
收集可溶上清的步骤如下:将破好的菌液转到离心瓶中,配平,12000rpm,离心30min,收集上清。
8.根据权利要求4所述的肿瘤相关抗原NY-ESO-1的制备方法,其特征在于:
其中,步骤E中,洗脱液透析方法如下:配制体积为洗脱液100倍的透析液,4h后换液一次,换液后透析过夜,透析液的配方组成如下:20m Tris/HCl、1mM EDTA、0.15M NaCl。
9.权利要求1~8任一项所述的方法制备得到的NY-ESO-1蛋白在制备NY-ESO-1抗体检测试剂盒、肿瘤治疗性疫苗、NY-ESO-1单克隆抗体或NY-ESO-1特异性T细胞中的应用。
10.根据权利要求9所述的应用,其特征在于,NY-ESO-1单克隆抗体或NY-ESO-1特异性T细胞制备时,先将NY-ESO-1作为抗原免疫动物,然后筛选和制备可以产生抗NY-ESO-1抗体的杂交细胞瘤,制备大量单克隆抗体;或者从免疫动物体内获取NY-ESO-1特异性T细胞,克隆其TCR序列,用于后续NY-ESO-1特异性TCR-T细胞治疗。
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