CN113461805B - 一种嵌入抗原表位肽的荧光素酶及其构建方法和应用 - Google Patents
一种嵌入抗原表位肽的荧光素酶及其构建方法和应用 Download PDFInfo
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Abstract
本发明涉及生物技术领域,具体公开了一种嵌入抗原表位肽的荧光素酶及其构建方法和应用。所述抗原表位肽为:至少由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的一种氨基酸序列组成。本发明构建出免疫性强的抗原表位肽或抗原组合表位肽,将其插入至荧光素酶中,使得可以构建得到灵敏度高(可调节),并可以借助活体成像技术进行追踪的肿瘤免疫研究模型。
Description
技术领域
本发明涉及生物技术领域,尤其是涉及到一种嵌入抗原表位肽的荧光素酶及其构建方法和应用。
背景技术
同系移植小鼠模型(syngeneic model)是将同种背景来源的肿瘤细胞系接种至免疫健全的近交系小鼠(比如常用的C57BL/6 或BALB/c 品系)体内,接种部位常为皮下(方便肿瘤的观察测量),原位(模拟肿瘤微环境),尾静脉注射(监测肿瘤的转移)。
肿瘤免疫疗法是通过恢复或增强机体抗肿瘤免疫应答来清除或控制肿瘤的新型肿瘤防治手段,包括肿瘤免疫检查点抑制剂(如CTL-4抑制剂、PD-1/PD-L1抑制剂)、过继性细胞免疫疗法(如CAR-T)等已经在临床上显示出卓著疗效的治疗方法,另外治疗性肿瘤疫苗、不同肿瘤免疫疗法的联用以及肿瘤免疫疗法和放化疗的联用等各种创新疗法也在不断涌现。同系移植小鼠模型是研究肿瘤免疫疗法常用动物模型,但同系移植小鼠模型的原始肿瘤细胞系通常免疫原性较弱,且抗原表位肽往往尚不明确,用于评价肿瘤免疫疗法的灵敏度不够。引入免疫原性强的外源抗原表位肽改造肿瘤细胞系可用于构建灵敏度更高的肿瘤免疫研究模型(阳性对照)。比如E.G7-OVA细胞系就引入鸡卵清蛋白(OVA)的抗原表位肽,通过分析针对OVA抗原表位肽的免疫应答,可以更灵敏地评估肿瘤免疫疗法的治疗潜力,探索优化方向。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种嵌入抗原表位肽的荧光素酶及其构建方法和应用,将嵌入免疫原性强的抗原表位肽的荧光素酶(CombiEpi-LUC)转染相关肿瘤细胞系,可以构建得到灵敏度高(可调节),并可以借助活体成像技术进行追踪的肿瘤免疫研究模型。
第一方面,本发明提供了一种抗原表位肽,所述抗原表位肽为:至少由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的一种氨基酸序列组成。
在本发明的技术方案中,抗原表位肽通过分子生物学技术在核心抗原表位肽核心氨基酸加上前后6个氨基酸构成本发明的抗原表位肽。SEQ ID NO:1中核心氨基酸序列为SIINFEKL,参见UniProt蛋白数据库accession number: A0A2H4Y842,被2-Kb呈递;SEQ IDNO:2中核心氨基酸序列为ASNENMETM,参见UniProt蛋白数据库accession number:P03466,被2-Db呈递;SEQ ID NO:3中核心氨基酸序列为IYSTVASSL,参见UniProt蛋白数据库accession number: P03452,被2-Dd呈递;SEQ ID NO:4中核心氨基酸序列为氨基酸序列为SYIGSINNI,参见UniProt蛋白数据库accession number: P04545,被2-Kd呈递。
本发明引入上述抗原表位肽嵌入荧光素酶,可实现组合式增强肿瘤免疫原性,可以从多档模式中选择评估肿瘤免疫疗法的治疗潜力的最优灵敏度。
第二方面,本发明提供了一种抗原组合表位肽,其包括由SEQ ID NO:1和SEQ IDNO:2所示的氨基酸序列构成,或者由SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列构成。
作为本发明所述抗原组合表位肽的优选实施方式,SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列之间采用接头序列连接,SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列之间采用接头序列连接。
第三方面,本发明提供了一种荧光素酶,其含有上述的抗原表位肽或上述的荧光素酶抗原组合表位肽。
在荧光素酶中插入上述免疫原性强的抗原表位肽或上述的抗原组合表位肽,其插入的抗原表位肽或上述的抗原组合表位肽的序列更短,对原始肿瘤细胞进一步转染可实现双重目的。
作为本发明所述荧光素酶的优选实施方式,所述荧光素酶如上述的抗原表位肽或抗原组合表位肽插入到荧光素酶的N端或C端得到。
第四方面,本发明提供了一种基因,所述基因编码上述的荧光素酶。
第五方面,本发明提供了一种重组质粒,所述重组质粒携带上述的基因。
第六方面,本发明提供了一种宿主细胞,所述宿主细胞携带上述的基因或上述的重组质粒。
第七方面,本发明提供了一种多肽疫苗,包括上述抗原组合表位肽和药学上可用的载体。优选地,药学上可用的载体包括佐剂,所述佐剂包括CpG-ODN 1826。
第八方面,本发明提供了上述疫苗组合物在制备抗肿瘤制剂中的应用。
第九方面,本发明提供了一种同系移植瘤模型的构建方法,将上述的重组质粒转染肿瘤细胞,筛选细胞株,将细胞株接种至小鼠。优选地,肿瘤细胞包括MC38、4T1、CT26,肿瘤细胞还可以包括其他种类。
另外当采用原位接种、尾静脉注射时,可借助活体成像技术,通过追踪体内荧光素酶的活性分布推断肿瘤细胞的分布。
第十方面,本发明提供了上述荧光素酶在构建同系移植瘤模型中的应用。
与现有技术相比,本发明具有以下有益效果:
1)本发明构建出免疫性强的抗原表位肽或抗原组合表位肽,将其插入至荧光素酶中,使得可以构建得到灵敏度高(可调节),并可以借助活体成像技术进行追踪的肿瘤免疫研究模型;
2)抗原表位肽或抗原组合表位肽具有较强地诱导抗肿瘤免疫应答的效果;
3)本发明将免疫性强的抗原表位肽或抗原组合表位肽与荧光素酶标记技术相结合,插入序列更短,对原始肿瘤细胞一步转染即可实现双重目的。
附图说明
图1为改造前的质粒图谱;
图2为本发明改造后的重组质粒图谱;
图3为实施例2中PBS组活体成像图I;
图4为实施例2中PBS组活体成像图II;
图5为实施例2中PBS组活体成像图III;
图6为实施例2中PADRE+CpG 组活体成像图I;
图7为实施例2中PADRE+CpG组活体成像图II;
图8为实施例2中PADRE+CpG组活体成像图III;
图9为实施例2中A+PADRE+CpG组活体成像图I;
图10为实施例2中A+PADRE+CpG组活体成像图II;
图11为实施例2中A+PADRE+CpG组活体成像图III;
图12为实施例2中A+B+PADRE+CpG组活体成像图I;
图13为实施例2中A+B+PADRE+CpG组活体成像图II;
图14为实施例2中A+B+PADRE+CpG组活体成像图III;
图15为实施例2中接种各组试剂的小鼠的肺的解剖图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图和具体实施例对本发明作进一步说明。
在以下实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、嵌入抗原表位肽的荧光素酶的构建
根据需要改造的鼠源肿瘤细胞系和其来源(或组织相容)的小鼠品系,选择外源抗原表位肽并构建组合表位序列(称为CombiEpi)。B16和MC38细胞系来源于C57小鼠,选择OVA257-264(氨基酸序列为SIINFEKL,参见UniProt蛋白数据库accession number:A0A2H4Y842,被2-Kb呈递)和influzenza A NP 366-374(氨基酸序列为ASNENMETM,参见UniProt蛋白数据库accession number: P03466,被2-Db呈递),4T1和CT26细胞系来源于BALB/c小鼠,可以选择influzenza A HA 532-540(氨基酸序列为IYSTVASSL,参见UniProt蛋白数据库accession number: P03452,被2-Dd呈递)和respiratory syncytial virusM2 82–90(氨基酸序列为SYIGSINNI,参见UniProt蛋白数据库accession number: P04545,被2-Kd呈递)。
将OVA 257-264、influzenza A NP 366-374、influzenza A HA 532-540和respiratory syncytial virus M2 82–90中的核心氨基酸前后各加上6个氨基酸,分别形成SEQ ID NO:1~SEQ ID NO:4,其序列分别GLEQLESIINFEKLTEWTSS、TRGVQIASNENMETMESSTLE、IYQILAIYSTVASSLVLLVSL和VVGVLESYIGSINNITKQSAC。将由SEQ IDNO:1和SEQ ID NO:2所示的氨基酸序列构成或者由SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列构成得到荧光素酶抗原组合表位肽(分别为荧光素酶抗原组合表位肽I和荧光素酶抗原组合表位肽II),SEQ ID NO:1和SEQ ID NO:2所示的氨基酸序列之间采用接头序列连接,SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列之间采用接头序列连接,在本实施例中,接头序列可采用MWQW、MWRW等。
将上述的构建得到的荧光素酶抗原组合表位肽插入到萤火虫荧光素酶(FireflyLuciferase,简称LUC,UniProt 蛋白数据库accession number: P08659)序列的N端或C端(荧光素酶抗原组合表位肽II插入到萤火虫荧光素酶序列的N端),得到改造后的荧光素酶。
将改造后的荧光素酶的氨基酸序列转换成DNA序列并进行密码子优化,命名为CombiEpi-LUC,将CombiEpi-LUC的DNA序列(两端包含酶切位点),用内切酶XhoI和BamHI双分别双酶切质粒pLv CMV LUC和合成序列,胶回收酶切产物(质粒pLv- CMV -LUC酶切后有两条产物,LUC是较小的序列,回收片段长的产物),用T4连接酶连接,转化并挑单克隆测序筛选出最终质粒,得重组质粒pLv CMV CombiEpi-LUC(见图2),改造前的质粒图谱见图1。
将重组质粒pLv CMV CombiEpi-LUC转染至293T细胞中包装成慢病毒颗粒,再转染至4T1细胞中,筛选得到稳定表达CombiEpi-LUC的细胞株(即4T1-CombiEpi-LUC细胞株),并验证LUC活性。
实施例2、活体成像实验
实验:12只6周龄雌性BALB/c小鼠,随机分成4组(PBS组、PADRE+ CpG组、A+ PADRE+CpG组、A+B+ PADRE+ CpG组),每组3只,编号分别为#1~#12。
间隔14天皮下免疫接种3次;PBS组每只皮下注射100 μl PBS(编号#1~#3);
PADRE+CpG 组(编号#4~#6)每只皮下注射25μg CpG-ODN 1826 和50 μg CD4+T细胞(即辅助性T细胞)通用激活肽PADRE(氨基酸序列为AKFVAAWTLKAAA),混合溶解在100 μlPBS。
A+PADRE+CpG组(编号#7~#9)每只皮下注射100 μg抗原肽A(抗原表位肽,SEQ IDNO:3)、25μg CpG-ODN 1826和50μg CD4+T细胞通用激活肽PADRE,混合溶解在100 μl PBS。
A+B+ PADRE+CpG组(编号#10~#12)每只皮下注射50 μg抗原肽A(抗原表位肽,SEQID NO:3)、50 μg抗原肽B(荧光素酶抗原表位肽,SEQ ID NO:4)、25μg CpG-ODN 1826 以及50 μg CD4+T细胞通用激活肽PADRE混合溶解在100 μl PBS。
第3次免疫接种7天后每只小鼠尾静脉注射2× 105 4T1-CombiEpi-LUC细胞株,注射后12天活体成像观察是否发生肺部转移,并解剖取肺。
结果:参照图3-14,活体成像实验A+B+ PADRE+CpG组未观察到肺部荧光素酶活性,A+PADRE+CpG组相比未经荧光素酶抗原表位肽免疫的PBS组和PADRE+CpG组,其肺部荧光素酶活性也明显更低。
参照图15,解剖结果也证实抗原肽A联合抗原肽B的组合物起到了很强的抗肿瘤定植效果,抗原肽A单用也有明显抑制肿瘤效果(PBS组和PADRE+CpG 组肺部可观察到大量肿瘤结节和病变,A+ PADRE+CpG组则病变相对轻微)。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 广州吉妮欧生物科技有限公司
<120> 一种嵌入抗原表位肽的荧光素酶及其构建方法和应用
<130> 2021.07.16
<160> 4
<170> PatentIn version 3.5
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<211> 20
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<213> 人工合成
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Claims (8)
1.一种抗原表位肽的组合物,其特征在于,包括如SEQ ID NO:3所示的抗原表位肽和如SEQ ID NO:4所示的抗原表位肽。
2.一种抗原组合表位肽,其特征在于,SEQ ID NO:3和SEQ ID NO:4所示的氨基酸序列之间采用接头序列连接。
3.一种嵌入抗原表位肽的荧光素酶,其特征在于,如权利要求2所述的抗原组合表位肽插入到荧光素酶的N端或C端。
4.一种基因,其特征在于,所述基因编码权利要求3所述的嵌入抗原表位肽的荧光素酶。
5.一种重组质粒,其特征在于,所述重组质粒携带如权利要求4所述的基因。
6.一种宿主细胞,其特征在于,所述宿主细胞携带如权利要求4所述的基因或权利要求5所述的重组质粒。
7.一种多肽疫苗,其特征在于,包括如权利要求2所述的抗原组合表位肽和药学上可用的载体。
8.如权利要求7所述的多肽疫苗在制备抗肿瘤制剂中的应用。
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