CN113186172A - 共表达Apoptin与MEL基因的重组腺病毒及构建方法和应用 - Google Patents
共表达Apoptin与MEL基因的重组腺病毒及构建方法和应用 Download PDFInfo
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Abstract
本发明属于构建重组腺病毒领域,具体涉及一种共表达Apoptin与MEL基因的重组腺病毒及构建方法和应用。构建方法包括下述步骤:1)TAT‑Apoptin、RGD‑MEL、TAT‑Apoptin+UBI‑RG‑MEL基因片段的扩增与克隆;2)构建重组腺病毒穿梭质粒GV‑TAT‑Apoptin+UBI‑RGD‑MEL;3)在HEK293A细胞中重组和包装病毒Ad‑TAT‑Apoptin+UBI‑RGD‑MEL;本申请将TAT‑Apoptin和RGD‑MEL基因与腺病毒载体结合起来,构建出表达上述基因的Ad‑TAT‑Apoptin+UBI‑RGD‑MEL,藉此研制针对动物肿瘤的广谱疫苗。
Description
技术领域
本发明属于构建重组腺病毒领域,具体涉及一种共表达Apoptin与MEL基因的重组腺病毒及构建方法和应用。
背景技术
肝细胞癌(Hepatocellular carcinoma,HCC)是全球第五大常见癌症,美国兽医医学协会(AVMA)的数据显示,癌症导致10岁以上的宠物中将近50%的死亡率,其他动物中肝癌已见于牛、山羊、绵羊、熊、猴、猪、鸭、火鸡、鸽、犬、马和鱼,近15年来动物肝癌的发病率逐年增多,严重影响着动物的健康与福祉。手术、放射疗法、化学疗法和免疫疗法为肝癌的常规治疗方式,但存在各式各样的局限,本研究构建的重组腺病毒选择凋亡素Apoptin与蜂毒肽MEL作为靶向治疗基因,借助腺病毒Ad5作为载体,高效传递至靶细胞,在Apoptin以线粒体凋亡和自噬特异性诱导杀伤肿瘤细胞的同时,结合MEL溶解癌细胞的细胞膜、破坏细胞的完整结构的特性,实现双基因协调治疗动物肿瘤的目的。
发明内容
本发明的目的,在于提供一种共表达Apoptin与MEL基因的重组腺病毒及构建方法和应用。将穿膜肽TAT与Apoptin融合表达,促进裂解细胞中Apoptin蛋白二次内化进相邻癌细胞;以靶向肽RGD靶向血管中的整合素受体,使其与MEL融合表达,引导MEL特异性杀伤肿瘤细胞,减轻对正常组织的非特异性损伤。构建出能够表达上述基因的重组腺病毒,多种体外试验验证双基因共表达的重组腺病毒在体外的细胞抑制效果以及对裸鼠皮下异位瘤的抑制作用。
为实现上述目的,本发明采用的技术方案为:
一种共表达Apoptin与MEL基因的重组腺病毒的构建方法,包括下述步骤:
1)TAT-Apoptin、RGD-MEL、TAT-Apoptin+UBI-RG-MEL基因片段的扩增与克隆;
2)构建重组腺病毒穿梭质粒GV-TAT-Apoptin+UBI-RGD-MEL;
3)在HEK293A细胞中重组和包装病毒Ad-TAT-Apoptin+UBI-RGD-MEL;
具体包括步骤如下:
1)以PMD-TAT-Apoptin为模板,TAT-Apoptin F1 SEQ ID NO.1和TAT-Apoptin R1SEQ ID NO.2为引物,扩增TAT-Apoptin基因;以PMD-UBI-RGD-MEL为模板,UBI-RGD-MEL F2SEQ ID NO.3和UBI-RGD-MEL R2 SEQ ID NO.4为引物,扩增RGD-MEL基因;以PMD-TAT-Apoptin+UBI-RGD-MEL为模板,TAT-Apoptin+UBI-RGD F3 SEQ ID NO.5和TAT-Apoptin+UBI-RGD R3 SEQ ID NO.6为引物,扩增TAT-Apoptin+RGD-MEL基因。
2)将上述基因无缝克隆连接至腺病毒穿梭载体GV314,获得重组腺病毒穿梭载体GV-TAT-Apoptin、GV-UBI-RGD-MEL、GV-TAT-Apoptin+UBI-RGD-MEL。
3)将重组腺病毒穿梭载体质粒与腺病毒大骨架质粒pBHGlox(delta)E1,3Cre共转染HEK293A细胞,获得重组腺病毒Ad-TAT-Apoptin、Ad-UBI-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL。
本发明还包括一种所述的构建方法得到的重组腺病毒。
本发明还包括一种所述的重组腺病毒的应用,应用于制备动物癌症。
与现有技术相比,本发明的有益效果是:
将TAT-Apoptin和RGD-MEL基因与高效稳定表达外源基因的腺病毒载体结合起来,构建出表达上述基因的重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL,藉此研制针对动物肿瘤的广谱疫苗。CCK8结果显示,重组腺病毒对L-02无显著抑制作用,且对SMMC-7721与Huh 7细胞抑制作用呈现时间和剂量依赖性;1000MOI重组腺病毒侵染SMMC-7721细胞36h后,早期和晚期凋亡率均极显著升高,且极显著抑制SMMC-7721细胞的迁移(P<0.0001);该重组腺病毒可延缓模型老鼠的体重下降趋势,抑制肿瘤生长速度。本发明最终研制出一种针对动物肿瘤性疾病的广谱疫苗。
附图说明
图1是TAT-Apoptin+UBI-RGD-MEL、TAT-Apoptin和UBI-RGD-MEL基因PCR扩增结果图(M:DL 2501 DNA Ladder;1:TAT-Apoptin+UBI-RGD-MEL基因;2.TAT-Apoptin基因;3:UBI-RGD-MEL基因)。
图2是TAT-Apoptin+UBI-RGD-MEL基因PCR扩增结果图和质粒图谱(M:DL 2501 DNALadder;1:TAT-Apoptin+UBI-RGD-MEL基因)。
图3是TAT-Apoptin基因PCR扩增结果图和质粒图谱(M:DL 2501 DNA Ladder;1.TAT-Apoptin基因)。
图4是UBI-RGD-MEL基因PCR扩增结果图和质粒图谱(M:DL 2501 DNA Ladder;1:UBI-RGD-MEL基因)。
图5是TAT-Apoptin基因测序峰图。
图6是RGD-MEL基因测序峰图。
图7是重组腺病毒包装过程细胞状态图。
图8是各重组腺病毒侵染SMMC-7721细胞后与DAPI染色定位结果图。
图9是Western Blotting检测SMMC-7721细胞中目的基因表达结果图。
图10是间接免疫荧光检测SMMC-7721细胞中目的基因表达结果图。
图11是在不同剂量和时间下各重组腺病毒对L-02细胞的抑制效果图。
图12是在不同剂量和时间下各重组腺病毒对SMMC-7721细胞的抑制效果图。
图13是在不同剂量和时间下各重组腺病毒对Huh 7细胞的抑制效果图。
图14是流式细胞术检测和重组腺病毒诱导SMMC-7721细胞凋亡结果图。
图15是细胞划痕试验检测各重组腺病毒抑制SMMC-7721细胞的迁移结果图。
图16是荷瘤裸鼠体重变化曲线结果图。
图17是荷瘤裸鼠肿瘤生长趋势图。
图18是各治疗组裸鼠脏器图。
图19是荷瘤裸鼠脏重比结果图。
具体实施方式
为了使本技术领域的技术人员更好地理解本发明的技术方案,下面结合附图和最佳实施例对本发明作进一步的详细说明。
肝细胞癌(HCC)是引起原发性肝癌病例的主要原因,发病率逐年增加,癌症影响着人类和动物的健康和福祉,美国兽医医学协会(AVMA)的数据显示,癌症导致10岁以上的宠物中将近50%的死亡,手术、放射疗法、化学疗法和免疫疗法为常规治疗方式,但存在各式各样的局限,本研究构建的重组腺病毒中Apoptin可以杀死P53蛋白功能缺失或突变的肿瘤细胞,在肿瘤细胞中苏氨酸的第108个残基处磷酸化激活,导致Apoptin发生细胞核定位,激活PI3K/Akt信号通路诱导肿瘤细胞凋亡。同时,选择TAT穿膜肽帮助Apoptin蛋白自裂解肿瘤细胞中释放后,内化到其他肿瘤细胞发生二次杀伤作用。MEL通过破坏磷脂双层,诱导细胞膜通透性增加和孔形成,但其细胞溶解作用是非特异性的。肿瘤归巢肽RGD是研究最深入的整联蛋白结合域,可特异性识别肿瘤新生血管的ανβ3和ανβ5受体,RGD与MEL融合表达,以此降低MEL对正常细胞的杀伤作用,实现MEL特异性靶向肝癌细胞的研究目的。因此本发明构建了共表达TAT-Apoptin与RGD-MEL基因的重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL,藉此研制一种针对动物肿瘤性疾病的广谱疫苗。
本发明涉及利用Apoptin基因与MEL基因构建的重组腺病毒,构建步骤如下:
1)TAT-Apoptin、RGD-MEL、TAT-Apoptin+UBI-RG-MEL基因片段的扩增与克隆;
2)构建重组腺病毒穿梭质粒GV-TAT-Apoptin+UBI-RGD-MEL;
3)在HEK293A细胞中重组和包装病毒Ad-TAT-Apoptin+UBI-RGD-MEL;
具体包括步骤如下:
1)以PMD-TAT-Apoptin为模板,TAT-Apoptin F1 SEQ ID NO.1和TAT-Apoptin R1SEQ ID NO.2为引物,扩增TAT-Apoptin基因;以PMD-UBI-RGD-MEL为模板,UBI-RGD-MEL F2SEQ ID NO.3和UBI-RGD-MEL R2 SEQ ID NO.4为引物,扩增RGD-MEL基因;以PMD-TAT-Apoptin+UBI-RGD-MEL为模板,TAT-Apoptin+UBI-RGD F3 SEQ ID NO.5和TAT-Apoptin+UBI-RGD R3 SEQ ID NO.6为引物,扩增TAT-Apoptin+RGD-MEL基因。
2)将上述基因无缝克隆连接至腺病毒穿梭载体GV314,获得重组腺病毒穿梭载体GV-TAT-Apoptin、GV-UBI-RGD-MEL、GV-TAT-Apoptin+UBI-RGD-MEL。
3)将重组腺病毒穿梭载体质粒与腺病毒大骨架质粒pBHGlox(delta)E1,3Cre共转染HEK293A细胞,获得重组腺病毒Ad-TAT-Apoptin、Ad-UBI-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL。
也就是说,将TAT-Apoptin与RGD-MEL基因进行密码子优化,构建至pMD18-T载体,线性化腺病毒穿梭载体,并将目的基因无缝克隆连接到腺病毒穿梭载体上,获得GV-TAT-Apoptin+UBI-RGD-MEL、GV-TAT-Apoptin、GV-RGD-MEL,并与大骨架质粒AdGloxdelE13cre共转染至HEK293A细胞,获得重组腺病毒Ad-TAT-Apoptin、Ad-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL,并继续扩增、纯化、测定滴度。用上述构建好的重组腺病毒侵染SMMC-7721细胞,通过Western blotting与间接免疫荧光检测外源基因的蛋白表达情况,采用CCK8、细胞划痕、流式细胞术检测重组腺病毒对SMMC-7721细胞的凋亡诱导作用,构建皮下异位瘤模型,检测重组腺病毒对裸鼠的体重以及肿瘤大小的影响。评价共表达TAT-Apoptin+UBI-RGD-MEL重组腺病毒对肝癌的抑制效果。
下面结合具体实例对本发明作进一步详细说明:
1材料与方法
根据细胞穿膜肽TAT(ADK70247.1)第47-57氨基酸序列(YGRKKRRQRRR)和凋亡素Apoptin基因(AY171617.1),设计TAT-Apoptin;肿瘤靶向肽RGD基因序列(1FUL_A)和蜂毒肽MEL基因序列(AY745248.1)设计RGD-MEL,以及TAT-Apoptin+UBI-RGD-MEL;所述的基因由吉凯有限公司优化并合成。
1.1TAT-Apoptin基因、RGD-MEL基因、TAT-Apoptin+UBI-RGD-MEL基因的扩增:以PMD-TAT-Apoptin为模板,TAT-Apoptin F1 SEQ ID NO.1和TAT-ApoptinR1 SEQ ID NO.2为引物,扩增TAT-Apoptin基因;以PMD-UBI-RGD-MEL为模板,UBI-RGD-MEL F2 SEQ ID NO.3和UBI-RGD-MEL R2 SEQ ID NO.4为引物,扩增RGD-MEL基因;以PMD-TAT-Apoptin+UBI-RGD-MEL为模板,TAT-Apoptin+UBI-RGDF3 SEQ ID NO.5和TAT-Apoptin+UBI-RGD R3 SEQ IDNO.6为引物,扩增TAT-Apoptin+RGD-MEL基因。
引物序列见表1;其PCR反应体系见表2。
表1
表2
注:反应程序:95℃,2min;{95℃,20s;55℃,20s;72℃,15s}35个循环,72℃延伸5min,4℃保存备用。
1.2腺病毒穿梭载体的构建
将PCR的产物进行胶回收,获得TAT-Apoptin基因、UBI-RGD-MEL基因、TAT-Apoptin+UBI-RGD-MEL基因与线性化的腺病毒穿梭载体GV314产物,用ClonExpressⅡOne StepCloning Kit进行连接,37℃反应30min,4℃或置于冰上冷却(反应体系见表3)。
表3
1.3连接产物转化DH5α感受态细胞
冰浴解冻-80℃超低温冷冻冰箱中的DH5α感受态细胞100μL,预冷PE管中加入5μL连接产物和DH5α感受态细胞50μL,混匀冰浴30min;放入42℃水浴锅热激90s后,迅速冰浴2min;加入800μL LB液体培养基,37℃170rpm/min摇菌培养1h;500×g,离心5min,弃掉上清液,重悬菌液,均匀涂布于含50mg/mL卡那霉素(+)的LB固体培养基上,37℃培养24-36h;挑单菌落接种于含有卡那霉素的LB液体培养基中,37℃170rpm摇床过夜培养,PCR验证质粒转化成功与否。
1.4重组质粒的鉴定
将重组质粒GV-TAT-Apoptin、GV-UBI-RGD-MEL和GV-TAT-Apoptin+UBI-RGD-MEL菌液进行PCR鉴定。取上述过夜培养的菌液,分别利用表2-1中引物F1/R1、F2/R2、F3/R3进行PCR鉴定,反应体系见表4。
表4
反应程序:95℃,预变性5min;{95℃,30s;60℃,30s;72℃,1min}35个循环,72℃延伸15min。4℃保存备用。反应结束后,琼脂糖凝胶电泳检测检测目的片段。
1.5重组腺病毒包装
提取腺病毒穿梭质粒GV-TAT-Apoptin、GV-RGD-MEL和GV-TAT-Apoptin+UBI-RGD-MEL质粒,将腺病毒穿梭载体质粒与AdGloxdelE13cre大骨架质粒按照1:2比例共转染至HEK293A细胞中,每天镜下观察细胞,培养14-21d,镜检观察荧光情况,维持细胞汇合度在70%-80%左右。待HEK293A细胞大部分出现变圆、呈现葡萄串状聚集,荧光显微镜下呈现大面积绿色荧光时,将细胞液收集置于-80℃冰箱保存备用。
1.6各重组腺病毒扩增与纯化
分别取包装好的重组腺病毒50μL接种到汇合度为80%的正常HEK293A细胞中,置于37℃,5%CO2培养箱中培养,每12h观察一次细胞生长状况及荧光状况;待大部分HEK293A细胞变圆从瓶底脱落,荧光显微镜下观察有大量绿色荧光时,收集细胞液,冻存于-80℃冰箱备用;按上述步骤对各重组腺病毒进行大量扩增,赛多利斯腺病毒纯化试剂盒纯化各重组腺病毒后进行滴度测定。
1.7重组腺病毒侵染SMMC-7721细胞。
将各重组腺病毒感染汇合度为80%的SMMC-7721细胞,观察各重组腺病毒感染效率,并利用Western blotting和间接免疫荧光检测目的蛋白的表达情况。
1.8重组腺病毒介导肝癌SMC-7721细胞凋亡的体外试验
为评价重组腺病毒对肿瘤细胞的抑制作用,本研究选择SMMC-7721细胞为研究对象,将各重组腺病毒侵染SMMC-7721细胞,观察重组腺病毒诱导SMMC-7721细胞的凋亡效果。
用不同MOI的重组腺病毒Ad-TAT-Apoptin、Ad-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL感染SMMC-7721细胞、Huh7细胞和L-02细胞,在24h、48h、72h时用CCK-8法检测细胞存活率。
将SMMC-7721细胞铺板,使其汇合度为80%,加入重组腺病毒Ad-TAT-Apoptin、Ad-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL,流式细胞术检测重组腺病毒对SMMC-7721细胞的凋亡作用,细胞划痕试验检测重组腺病毒的迁移能力。
1.9重组腺病毒抑制裸鼠异位瘤生长速度的体内试验
右腋皮下接种107个SMMC-7721细胞,构建肝癌皮下异位瘤模型,从成瘤开始对小鼠体重、肿瘤大小及死亡情况进行记录,通过游标卡尺测量肿瘤的长和宽。
待小鼠长出肉眼可见肿瘤时,对小鼠进行治疗,各组分别瘤内注射各重组腺病毒,每只注射2×108PFU/dose的病毒量、顺铂(60μg/只)和PBS(100μL/只),每2天注射一次,共6次;
2结果与分析
2.1目的基因特异性扩增
以pMD-TAT-Apoptin、pMD-RGD-MEL、pMD-TAT-Apoptin+UBI-RGD-MEL质粒为模板,分别以F1/R1、F2/R2、F3/R3为引物扩增目的片段。PCR产物经凝胶电泳检测,获得大小分别为492bp、1424bp、1875bp的预期片段,无非特异性条带,经测序和序列比对,目的基因序列无碱基缺失、增添和突变,可进行胶回收。其中,图1是TAT-Apoptin+UBI-RGD-MEL、TAT-Apoptin和UBI-RGD-MEL基因PCR扩增结果图(M:DL 2501 DNA Ladder;1:TAT-Apoptin+UBI-RGD-MEL基因;2.TAT-Apoptin基因;3:UBI-RGD-MEL基因)。图2是TAT-Apoptin+UBI-RGD-MEL基因PCR扩增结果图和质粒图谱(M:DL 2501 DNA Ladder;1:TAT-Apoptin+UBI-RGD-MEL基因)。图3是TAT-Apoptin基因PCR扩增结果图和质粒图谱(M:DL 2501 DNA Ladder;1.TAT-Apoptin基因)。图4是UBI-RGD-MEL基因PCR扩增结果图和质粒图谱(M:DL 2501 DNALadder;1:UBI-RGD-MEL基因)。图5是TAT-Apoptin基因测序峰图。图6是RGD-MEL基因测序峰图。
2.2重组腺病毒穿梭载体的验证
经PCP及测序鉴定,结果显示获得1875bp、492bp、1424bp的片段,与预期结果相符。表明腺病毒穿梭质粒GV-TAT-Apoptin+UBI-RGD-MEL、GV-TAT-Apoptin、GV-RGD-MEL构建成功,可用于后续重组腺病毒包装。
2.3各重组腺病毒包装及滴度测定
各基因的重组质粒与腺病毒大骨架按照1:2的比例转染HEK293A细胞,培养14d后,显微镜下细胞有明显绿色荧光,细胞出现CPE,呈现葡萄串状并从底壁脱落(图7)。利用TCID50方法检测重组病毒滴度,结果显示,Ad-TAT-Apoptin滴度为1010.75pfu/mL,Ad-RGD-MEL的滴度为1010.35pfu/mL,TAT-Apoptin+UBI-RGD-MEL的滴度为1010.25pfu/mL,Ad-EGFP的滴度为1011pfu/mL,可满足后续实验对滴度的要求。
2.4重组腺病毒侵染SMMC-7721细胞
将各重组腺病毒以200MOI感染SMMC-7721细胞48h后,SMMC-7721细胞出现病毒载体EGFP标签的绿色荧光(图8),利用DAPI染核,定位结果显示各重组腺病毒在细胞质、细胞核中均有表达,感染效率较高,可进行后续试验。
2.5Western blotting检测SMMC-7721细胞中目的基因表达情况
Western Blotting检测经重组腺病毒感染的SMMC-7721细胞中目的基因表达情况,重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL处理组中约在7KDa、16KDa处出现特异性条带;Ad-TAT-Apoptin处理组中约在16KDa处出现特异性条带;重组腺病毒Ad-RGD-MEL处理组中约在7KDa处出现特异性条带,均与预期目标蛋白分子质量相当(图9)。表明重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin、Ad-RGD-MEL中目的蛋白成功表达。
2.6间接免疫荧光检测SMMC-7721细胞中目的基因表达
利用间接免疫荧光法,检测经重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin、Ad-RGD-MEL感染SMMC-7721细胞后目的蛋白的表达情况。结果显示,成功检测到flag和myc蛋白表达,表明重组腺病毒目的基因TAT-Apoptin-myc、RGD-MEL-flag在SMMC-7721中成功表达(图10)。
2.7各重组腺病毒对L-02细胞增殖影响
CCK-8法检测各重组腺病毒对L-02正常肝细胞的抑制作用。0MOI、100MOI、200MOI、500MOI、1 000MOI的重组腺病毒Ad-EGFP、Ad-TAT-Apoptin、Ad-UBI-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL分别作用24h、48h、72h后,结果显示,各重组腺病毒Ad-TAT-Apoptin、Ad-UBI-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL在不同时间点,不同浓度下与对照组Ad-EGFP相比对正常肝细胞无明显杀伤作用(图11,P>0.05)。
2.8各重组腺病毒抑制SMMC-7721、Huh7细胞增殖
各重组腺病毒对SMMC-7721细胞的抑制能力呈现时间与剂量依赖性,其中,共表达组Ad-TAT-Apoptin+UBI-RGD-MEL对SMMC-7721细胞的抑制效果最佳,200MOI重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL处理组与对照组Ad-EGFP相比在24h后细胞存活率开始出现显著降低(P<0.05),48h与72h的细胞细胞存活率极显著降低(P<0.01或P<0.001),1 000MOI Ad-TAT-Apoptin+UBI-RGD-MEL处理72h时细胞存活率最低(P<0.0001);500MOI重组腺病毒Ad-TAT-Apoptin与对照组Ad-EGFP存活率相比,自48h后细胞存活率显著降低(P<0.05,P<0.01或P<0.001);重组腺病毒Ad-RGD-MEL处理组与对照组Ad-EGFP细胞存活率相比,在48h细胞存活率极显著降低(P<0.01),抑制效果与剂量和时间相关(图11)
各重组腺病毒对Huh7细胞的抑制能力与时间与剂量相关,1 000MOI各重组腺病毒Ad-TAT-Apoptin、Ad-UBI-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL作用24h后与对照组Ad-EGFP相比,细胞存活率极显著降低(P<0.01),具有时间和剂量依赖性;Ad-TAT-Apoptin、Ad-RGD-MEL与处理组Ad-EGFP细胞存活率相比,随剂量增加变化不大,但与作用时间相关;重组腺病毒Ad-TAT-Apoptin与Ad-TAT-Apoptin+UBI-RGD-MEL相比细胞存活率无显著差异,Ad-RGD-MEL抑制作用稍弱(图12)。
2.9流式细胞术检测SMMC-7721细胞凋亡结果
重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin、Ad-UBI-RGD-MEL以及PBS处理SMMC-7721细胞24h、36h,利用流式细胞仪检测SMMC-7721细胞的凋亡情况(图4-5)。流式结果显示,各重组腺病毒侵染SMMC-7721肝癌细胞24h后,活细胞数与凋亡细胞数间无显著差异(P>0.05);各重组腺病毒侵染SMMC-7721细胞36h后,各重组腺病毒的活细胞率以及总凋亡细胞率与对照组相比均存在极显著差异(P<0.01或P<0.001);共表达组重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL处理SMMC-7721细胞36h后,早期与晚期凋亡率与对照组Control组相比差异极显著(图13,P<0.001)。
2.10细胞划痕试验结果
用重组腺病毒Ad-EGFP、Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin、Ad-RGD-MEL侵染SMMC-7721细胞,对比分析不同时间点的划痕面积变化(图14)。结果显示,与对照组相比,重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin、Ad-RGD-MEL处理SMMC-7721细胞自12h后极显著抑制细胞迁移(P<0.0001);重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin处理组与其他所有处理组相比,创伤愈合程度低,极显著抑制SMMC-7721细胞的迁移(图15,P<0.0001);
2.11荷瘤裸鼠体重变化曲线
成功构建肝癌皮下异位瘤模型后,重组腺病毒治疗组瘤内注射108pfu/只,每2d给一次药,共给药6次。各治疗组间裸鼠体重无显著差异(P>0.05),但与PBS和顺铂组相比,重组腺病毒治疗组体重下降趋势趋于减缓(图16),肿瘤生长速度收到抑制(图17)。
试验结束后处死裸鼠,摘取肝脏、脾脏、心脏、肺脏、肾脏以及肿瘤,PBS对照组肿瘤较大。其他组织肉眼观察无明显损伤(图18)。
对各组裸鼠器官称重并利用统计学分析脏重比,结果显示(图19),顺铂组、各重组腺病毒治疗组与PBS相比,肿瘤与体重比值极显著下降(P<0.0001),但组间没有显著差异;除肾脏和心脏外,顺铂治疗组的脏重比较其他治疗组低,说明顺铂治疗对正常组织器官杀伤力较大;肝脏与体重的比值结果显示,PBS和顺铂治疗组与Control正常鼠及重组腺病毒治疗组相比,其肝重比显著降低(P<0.05或P<0.0001),重组腺病毒治疗组延缓了肝重比的下降趋势。
3.结论
3.1成功构建出表达TAT-Apoptin+UBI-RGD-MEL、TAT-Apoptin和RGD-MEL基因的重组腺病毒穿梭载体,GV-TAT-Apoptin、GV-UBI-RGD-MEL和GV-TAT-Apoptin+UBI-RGD-MEL。
3.2成功包装出Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin、Ad-RGD-MEL、Ad-EGFP重组腺病毒,病毒滴度符合后续实验要求。
3.3重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL、Ad-TAT-Apoptin、Ad-RGD-MEL能够在SMMC-7721细胞中表达目的蛋白。
3.4重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL对SMMC-7721、Huh7肝癌细胞具有时间和剂量依赖性抑制作用,且正常L-02肝细胞无显著抑制作用。
3.5重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL感染肝癌细胞后总凋亡细胞率升高,活细胞率下降。
3.6重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL可极显著抑制SMMC-7721细胞迁移。
3.7重组腺病毒抑制了皮下异位瘤的生长速度,减缓了裸鼠的体重下降趋势,使肿瘤与体重比值极显著下降,对正常组织无明显损伤作用。
本发明的创新之处在于,首次将治疗基因Apoptin与MEL与高效稳定的腺病毒载体结合,构建出双基因协同抑瘤的重组腺病毒,western blotting与间接免疫荧光证实目的基因在SMMC-7721细胞中成功表达,CCK8、流式细胞术、细胞划痕试验证实重组腺病毒Ad-TAT-Apoptin+UBI-RGD-MEL显著抑制SMMC-7721细胞的增殖和迁移能力,抑制了裸鼠皮下异位瘤的生长速度,延缓了肿瘤的生长速度,对其他组织器官无明显杀伤作用,成功研制了针对动物肿瘤性疾病的广谱疫苗。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 天津农学院
<120> 共表达Apoptin与MEL基因的重组腺病毒及构建方法和应用
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 441
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tacggccgca agaaacgccg ccagcgccgc cgcggaggtg gaggatcaat gaacgctctc 60
caagaagata ctccacccgg accatcaacg gtgttcaggc caccaacaag ttcacggccg 120
ttggaaaccc ctcactgcag agagatccgg attggtatcg ctggaattac aatcactcta 180
tcgctgtgtg gctgcgcgaa tgctcgcgct cccacgctaa gatctgcaac tgcggacaat 240
tcagaaagca ctggtttcaa gaatgtgccg gacttgagga ccgatcaacc caagcctccc 300
tcgaagaagc gatcctgcga cccctccgag tacagggtaa gcgagctaaa agaaagcttg 360
attaccacta ctcccagccg accccgaacc gcaagaaggc gtataagact ggaacaaaaa 420
ctcatctcag aagaggatct g 441
<210> 2
<211> 1381
<212> DNA
<213> 人工序列(Artificial Sequence)
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cccgtgtcgg ctccagatct ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc 60
cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc 120
cgcccggacg ctcaggacag cggcccgctg ctcataagac tcggccttag aaccccagta 180
tcagcagaag gacattttag gacgggactt gggtgactct agggcactgg ttttctttcc 240
agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg agggatctcc 300
gtggggcggt gaacgccgat gattatataa ggacgcgccg ggtgtggcac agctagttcc 360
gtcgcagccg ggatttgggt cgcggttctt gtttgtggat cgctgtgatc gtcacttggt 420
gagtagcggg ctgctgggct ggccggggct ttcgtggccg ccgggccgct cggtgggacg 480
gaagcgtgtg gagagaccgc caagggctgt agtctgggtc cgcgagcaag gttgccctga 540
actgggggtt ggggggagcg cagcaaaatg gcggctgttc ccgagtcttg aatggaagac 600
gcttgtgagg cgggctgtga ggtcgttgaa acaaggtggg gggcatggtg ggcggcaaga 660
acccaaggtc ttgaggcctt cgctaatgcg ggaaagctct tattcgggtg agatgggctg 720
gggcaccatc tggggaccct gacgtgaagt ttgtcactga ctggagaact cggtttgtcg 780
tctgttgcgg gggcggcagt tatggcggtg ccgttgggca gtgcacccgt acctttggga 840
gcgcgcgccc tcgtcgtgtc gtgacgtcac ccgttctgtt ggcttataat gcagggtggg 900
gccacctgcc ggtaggtgtg cggtaggctt ttctccgtcg caggacgcag ggttcgggcc 960
tagggtaggc tctcctgaat cgacaggcgc cggacctctg gtgaggggag ggataagtga 1020
ggcgtcagtt tctttggtcg gttttatgta cctatcttct taagtagctg aagctccggt 1080
tttgaactat gcgctcgggg ttggcgagtg tgttttgtga agttttttag gcaccttttg 1140
aaatgtaatc atttgggtca atatgtaatt ttcagtgtta gactagtaaa ttgtccgcta 1200
aattctggcc gtttttggct tttttgttag acgaagcttg ggctgcaggt cgactctaga 1260
ggatcccgcc accatgtgcg actgccgcgg agactgcttc tgcggtattg gtgcggtgct 1320
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g 1381
<210> 3
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ttggaaaccc ctcactgcag agagatccgg attggtatcg ctggaattac aatcactcta 180
tcgctgtgtg gctgcgcgaa tgctcgcgct cccacgctaa gatctgcaac tgcggacaat 240
tcagaaagca ctggtttcaa gaatgtgccg gacttgagga ccgatcaacc caagcctccc 300
tcgaagaagc gatcctgcga cccctccgag tacagggtaa gcgagctaaa agaaagcttg 360
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ctcatctcag aagaggatct gtaacccgtg tcggctccag atctggcctc cgcgccgggt 480
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gcgcagcgag cgtcctgatc cttccgcccg gacgctcagg acagcggccc gctgctcata 600
agactcggcc ttagaacccc agtatcagca gaaggacatt ttaggacggg acttgggtga 660
ctctagggca ctggttttct ttccagagag cggaacaggc gaggaaaagt agtcccttct 720
cggcgattct gcggagggat ctccgtgggg cggtgaacgc cgatgattat ataaggacgc 780
gccgggtgtg gcacagctag ttccgtcgca gccgggattt gggtcgcggt tcttgtttgt 840
ggatcgctgt gatcgtcact tggtgagtag cgggctgctg ggctggccgg ggctttcgtg 900
gccgccgggc cgctcggtgg gacggaagcg tgtggagaga ccgccaaggg ctgtagtctg 960
ggtccgcgag caaggttgcc ctgaactggg ggttgggggg agcgcagcaa aatggcggct 1020
gttcccgagt cttgaatgga agacgcttgt gaggcgggct gtgaggtcgt tgaaacaagg 1080
tggggggcat ggtgggcggc aagaacccaa ggtcttgagg ccttcgctaa tgcgggaaag 1140
ctcttattcg ggtgagatgg gctggggcac catctgggga ccctgacgtg aagtttgtca 1200
ctgactggag aactcggttt gtcgtctgtt gcgggggcgg cagttatggc ggtgccgttg 1260
ggcagtgcac ccgtaccttt gggagcgcgc gccctcgtcg tgtcgtgacg tcacccgttc 1320
tgttggctta taatgcaggg tggggccacc tgccggtagg tgtgcggtag gcttttctcc 1380
gtcgcaggac gcagggttcg ggcctagggt aggctctcct gaatcgacag gcgccggacc 1440
tctggtgagg ggagggataa gtgaggcgtc agtttctttg gtcggtttta tgtacctatc 1500
ttcttaagta gctgaagctc cggttttgaa ctatgcgctc ggggttggcg agtgtgtttt 1560
gtgaagtttt ttaggcacct tttgaaatgt aatcatttgg gtcaatatgt aattttcagt 1620
gttagactag taaattgtcc gctaaattct ggccgttttt ggcttttttg ttagacgaag 1680
cttgggctgc aggtcgactc tagaggatcc cgccaccatg tgcgactgcc gcggagactg 1740
cttctgcggt attggtgcgg tgctgaaagt gctgaccacc ggtctgccgg cgctgattag 1800
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<210> 4
<211> 48
<212> DNA
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<400> 4
gaggatcccc gggtaccggc gccaccatgt acggccgcaa gaaacgcc 48
<210> 5
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acattccaca gttagctagt tacagatcct cttctgagat gag 43
<210> 6
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
taatatttgt ctagggccgc ggcccgtgtc ggctccagat ctgg 44
<210> 7
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atccttgtag tccataccgg tctgctgacg tttacgttta atc 43
<210> 8
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aggtcgactc tagaggatcc cgccaccatg tacggccgca agaaacgcc 49
<210> 9
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
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tccttgtagt ccataccggt ctgctgacgt ttacgtttaa tcc 43
Claims (4)
1.一种共表达Apoptin与MEL基因的重组腺病毒的构建方法,其特征在于,包括下述步骤:
1)TAT-Apoptin、RGD-MEL、TAT-Apoptin+UBI-RG-MEL基因片段的扩增与克隆;
2)构建重组腺病毒穿梭质粒GV-TAT-Apoptin+UBI-RGD-MEL;
3)在HEK293A细胞中重组和包装病毒Ad-TAT-Apoptin+UBI-RGD-MEL;
2.根据权利要求1所述的共表达Apoptin与MEL基因的重组腺病毒的构建方法,其特征在于,具体包括步骤如下:
1)以PMD-TAT-Apoptin为模板,TAT-Apoptin F1 SEQ ID NO.1和TAT-Apoptin R1 SEQID NO.2为引物,扩增TAT-Apoptin基因;以PMD-UBI-RGD-MEL为模板,UBI-RGD-MEL F2 SEQID NO.3和UBI-RGD-MEL R2 SEQ ID NO.4为引物,扩增RGD-MEL基因;以PMD-TAT-Apoptin+UBI-RGD-MEL为模板,TAT-Apoptin+UBI-RGD F3 SEQ ID NO.5和TAT-Apoptin+UBI-RGD R3SEQ ID NO.6为引物,扩增TAT-Apoptin+RGD-MEL基因。
2)将上述基因无缝克隆连接至腺病毒穿梭载体GV314,获得重组腺病毒穿梭载体GV-TAT-Apoptin、GV-UBI-RGD-MEL、GV-TAT-Apoptin+UBI-RGD-MEL。
3)将重组腺病毒穿梭载体质粒与腺病毒大骨架质粒pBHGlox(delta)E1,3Cre共转染HEK293A细胞,获得重组腺病毒Ad-TAT-Apoptin、Ad-UBI-RGD-MEL、Ad-TAT-Apoptin+UBI-RGD-MEL。
3.一种权利要求1-2任一项所述的构建方法得到的重组腺病毒。
4.一种权利要求3所述的重组腺病毒的应用,其特征在于,应用于制备动物癌症。
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