CN108948209A - 一种cd105单链抗体-es融合蛋白的制备方法和用途 - Google Patents
一种cd105单链抗体-es融合蛋白的制备方法和用途 Download PDFInfo
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Abstract
本发明涉及靶向抗体内皮抑素融合蛋白制备技术领域,具体涉及本技术方案提供一种CD105单链抗体‑ES融合蛋白,融合蛋白由CD105单链抗体和ES组成,CD105单链抗体‑ES融合蛋白。CD105单链抗体和ES氨基酸序列分别如序列表中1和2所示。有益效果:本技术方案以肿瘤组织中的CD105为靶点,将抗CD105单链抗体与ES构成融合蛋白scFV(CD105)‑ES,使ES获得靶向递送特性,能够一定程度上解决用药量大和半衰期短的问题,提高ES的疗效。
Description
技术领域
本发明属于靶向抗体内皮抑素融合蛋白制备技术领域,具体涉及一种CD105单链抗体-ES融合蛋白的制备方法和用途。
背景技术
血管内皮抑制素(Endostatin,简称内皮抑素或ES)是1997年O′Reilly等从培养的小鼠内皮细胞瘤(EOMA)上清中分离纯化的一种内源性血管生成抑制剂,为20kd分子量蛋白质。氨基酸序列分析显示:内皮抑素(ES)是胶原18的降解产物,具体为胶原18分子C端部分,共184个氨基酸。进一步晶体结构分析发现:ES结构表面有一个由11个精氨酸残基组成的碱性区域,为肝素结合位点,这解释了ES对肝素的高亲和力特性,也可能是通过该区域与血管生成因子竞争结合肝素,起到抑制血管生成作用。但也有研究表明ES与血管壁的结合不依赖于肝素结合位点,且与FGF-2无竞争性抑制作用。通过基因修饰方法去除锌离子结合位点的研究表明,ES抑制内皮细胞的迁移及肿瘤的生长并不依赖锌离子结合位点。
通过多个研究和实验显示,ES对血管内皮细胞、血管生成和肿瘤生长和转移都有抑制作用,且无耐药性和明显毒副作用,为肿瘤的临床治疗开辟了一条新路。随着ES作用机制的完全阐明,如何延长ES在患者体内半衰期,选择安全高效的合适载体来进行基因治疗等这问题将是接下来该领域的研究热点。
发明内容
本发明所要解决的技术问题是:提供的一种CD105单链抗体-ES融合蛋白,及其制备方法和用途。
为解决上述技术问题,本技术方案提供一种CD105单链抗体-ES融合蛋白,融合蛋白由CD105单链抗体和ES组成。
进一步地,CD105单链抗体-ES融合蛋白,其特征在于:所述融合蛋白具有如下氨基酸序列:
CD105单链抗体和ES氨基酸序列分别如序列表中1和2所示;或与序列表中1和2所示的氨基酸序列同源性在80%-100%编码相同功能蛋白质的氨基酸序列;或序列表中1和2所示的氨基酸序列经增加、缺失或者替换一个或多个氨基酸具有同等活性的衍生的蛋白。
进一步地,CD105单链抗体-ES融合蛋白的制备方法,包括以下步骤:
1)CD105单链抗体-ES融合蛋白表达载体pET28a-antiCD105-ES的构建;
2)将1)中构建表达载体转化至菌株BL21 DE3,获得转基因重组菌;
3)CD105单链抗体-ES融合蛋白的诱导表达及纯化:对CD105单链抗体-ES融合蛋白用1mM IPTG诱导4小时,收集菌液进行超声破碎,离心后收集沉淀,用8M尿素变性剂溶解沉淀,并依次在6M、4M、2M、0M尿素溶液中透析复性,CD105单链抗体-ES融合蛋白经AKTAPurifier 900 FPLC系统,镍离子亲和层析柱纯化:用SDS-PAGE方法检测目的蛋白。
进一步地,表达CD105单链抗体-ES融合蛋白转的基因重组菌,包含pET28a-antiCD105-ES表达载体。
进一步地,纯化分离得到的CD105单链抗体-ES融合蛋白可作为抗肿瘤药物的应用。
采用了上述技术方案后,本发明的有益效果是:
靶向给药能有效克服ES在临床治疗中存在用药量大和体内半衰期短等缺点。CD105是新生血管标志物,在血管丰富的实体瘤中高表达,在癌旁正常组织几乎不表达,具有较好的肿瘤组织特异性。单链抗体(scFV)具有非特异性反应低,分子小易于渗透,可大量生产等优点;抗CD105单链抗体具有较好的肿瘤组织靶向性。本技术方案以肿瘤组织中的CD105为靶点,将抗CD105单链抗体与ES构成融合蛋白CD105单链抗体-ES,使ES获得靶向递送特性,能够一定程度上解决用药量大和半衰期短的问题,提高ES的疗效。
构建融合蛋白的表达载体,转化后得到表达融合蛋白的工程菌中。纯化融合蛋白后,给药鸡胚,比较实验组CD105单链抗体-ES融合蛋白与空白对照组结果显示,CD105单链抗体-ES融合蛋白对鸡胚血管形成有显著抑制效果。相关研究结果将为该靶向治疗方法应用于临床奠定基础。
附图说明
图1表示CD105-ES阳性菌株全菌蛋白SDS-PAGE电泳图。
泳道M:蛋白分子量标准(116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa);
泳道1:宿主菌全菌蛋白;
泳道2:IPTG诱导全菌蛋白(箭头所指为重组蛋白)。
图2表示CD105-ES阳性菌株破碎上清及沉淀SDS-PAGE电泳图。
泳道M:蛋白分子量标准(116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa);
泳道1:37℃诱导表达破碎后的上清蛋白;
泳道2:37℃诱导表达破碎后的不溶蛋白(箭头所指为重组蛋白)。
图3表示CD105-ES阳性菌株破碎上清及沉淀分别在16℃和30℃SDS-PAGE电泳图。
泳道M:蛋白分子量标准(116.0/66.2/45.0/35.0/25.0/18.4/14.4kDa);
泳道1:16℃诱导表达破碎后的上清蛋白;
泳道2:16℃诱导表达破碎后的不溶蛋白;
泳道3:30℃诱导表达破碎后的上清蛋白;
泳道4:30℃诱导表达破碎后的不溶蛋白(箭头所指为重组蛋白)。
图4表示CD105单链抗体-ES融合蛋白抑制鸡胚血管生成实验结果图(A:PBS处理,空白对照组)。
图5表示CD105单链抗体-ES融合蛋白抑制鸡胚血管生成实验结果图(50mM ScFV-ES融合蛋白处理)。
具体实施方式
下面结合附图1至5和具体实施例对本发明进行详细描述,但不作为对本发明的限定。
实施例一
本技术方案提供一种CD105单链抗体-ES融合蛋白,融合蛋白由CD105单链抗体和ES连接组成。
CD105单链抗体-ES融合蛋白,所述融合蛋白具有如下氨基酸序列:
CD105单链抗体和ES的氨基酸序列分别如序列表中1和2所示;或与序列表中1和2所示的氨基酸序列同源性在80%-100%编码相同功能蛋白质的氨基酸序列;或序列表中1和2所示的氨基酸序列经增加、缺失或者替换一个或多个氨基酸具有同等活性的衍生的蛋白。
实施例二
在实施例一的基础上,CD105单链抗体-ES融合蛋白的表达和纯化过程如下:
1.表达载体pET28a-antiCD105-ES的构建:
ES基因两端加上Nco I、HindIII酶切位点序列,并委托由上海吉玛制药技术有限公司进行化学合成并克隆至pGLV-H1-GFP+Puro,得到克隆载体pGLV-H1-GFP+Puro-ES。
pET28a-antiCD105(广西医科大学基础医学院细胞生物学与遗传学教研室保存)进行Nco I和HindIII核酸酶酶切后进行核酸电泳,切胶纯化得到含有CD105单链抗体基因的线性载体;
pGLV-H1-GFP+Puro-ES通过进行Nco I和HindIII核酸酶酶切后进行核酸电泳,切胶纯化得ES基因;
pET28a-antiCD105和pGLV-H1-GFP+Puro-ES的切胶纯化产物进行连接,中得到的连接产物转化到大肠杆菌TOP10感受态细胞中,涂布Kan抗性平板进行筛选,通过对菌液PCR和测序筛选序列符合要求的质粒,即获得CD105单链抗体-ES融合蛋白基因克隆载体pET28a-antiCD105-ES。
2.将表达载体pET28a-antiCD105-ES转化至表达菌株BL21 DE3:
取已经确认的100ng/μL表达载体pET28a-antiCD105-ES 0.1μL至5μL转化入氯化钙制备的表达菌株BL21DE3感受态细胞中,涂布到特定卡那抗性的LB平板上。挑选阳性克隆低温冰箱内甘油保存该菌种。
3.CD105单链抗体-ES融合蛋白的诱导表达及纯化:
1)CD105单链抗体-ES融合蛋白IPTG诱导表达:
对CD105单链抗体-ES融合蛋白用1mMIPTG诱导表达,诱导4小时后,SDS-PAGE全蛋白电泳,如附图1所示,在58KD左右可以看到清晰特异性蛋白条带,在无IPTG诱导和存在IPTG诱导对比显示,CD105单链抗体-ES融合蛋白已经表达成功。
全蛋白IPTG诱导表达,实验步骤如下:
a)取3μL菌液(表达菌株甘油保存菌)加入3mL LB(卡那抗性)培养基,220rpm 37℃过夜摇菌;
b)测OD600至0.5时;
c)加入浓度为1mM的IPTG;
d)220rpm 37℃摇菌4小时;
e)取出菌液,12000g 10min离心去上清;
2)CD105单链抗体-ES融合蛋白用8M尿素变性,透析法复性,并经AKTA Purifier900 FPLC系统,镍离子亲和层析柱纯化:
收集1000mL经过IPTG诱导表达后的BL21 DE3菌液,经4200r·min-1离心10min,收集菌体。沉淀重悬于PBS(pH 7.4)、5mM咪唑80mL中进行超声裂解,超声条件为350W、工作3s、间歇3s、总时间为45min。超声裂解物于4℃、12000r·min-1离心20min。收集沉淀于含8M尿素的蛋白变性液中充分溶解,并依次在6M、4M、2M、0M尿素溶液中透析复性,于AKTAPurifier 900 FPLC系统纯化。镍柱用5倍柱体积PBS(pH7.4)、5mmol 1·L-1咪唑平衡后上样,经PBS(pH 7.4)、40mmol·L-1咪唑洗杂后,目的蛋白用PBS(pH 7.4)、500mmol·L-1咪唑洗脱。洗脱后的纯化蛋白在PBS(pH 7.4)中透析。
4.用SDS-PAGE方法检测目的蛋白:
选取未诱导表达的表达菌株BL21 DE3 pET28a-antiCD105-ES菌液1mL、加入IPTG分别在16℃、30℃、37℃诱导表达4小时后,各取菌液1mL进行12000r·min-1离心1min;上清分别作为跑胶样品收集;沉淀用100μLddH2O重悬,超声波破碎;超声波处里后的菌液100μL进行12000r·min-1离心1min,上清移至新的EP管,沉淀用100μLddH2O分别作为跑胶样品。跑胶结果如附图2、附图3所示,表达的目标蛋白可见主要出现在上清中。
实施例三
通过鸡胚绒毛尿囊膜(chick chorioallantoic membrane,CAM)模型观察纯化后CD105单链抗体-ES融合蛋白在活体中抑制血管生成的能力。
1.鸡胚孵育选择0d白皮种蛋,质量相近(60±5)g,消毒后,温度37.5℃、湿度60%条件下孵育,每天照蛋转蛋,去死胚,至第8天,在照卵灯下观测,选择发育良好的鸡胚并标记气室。
2.分组情况随机将发育良好的40枚鸡胚平均分为4组作为对照组及实验组。
3.给药消毒后,以移液枪透过小洞分别于鸡胚绒毛尿囊膜上加入50mM纯化后CD105单链抗体-ES融合蛋白及PBS(空白对照)溶液50μL,封口,续保温孵育。孵育48h后,沿蛋壳长轴剪开鸡胚,小心去掉内容物,操作过程中保证加药端鸡胚绒毛尿囊膜贴壁,以加药口为中心拍照取材、分别统计两组血管受抑制的百分比并描述血管长势。
4.观察指标的确立鸡胚剪开后立即观察以加药点为中心的血管生长情况,观察有无血管向心生长情况。若无向心生长情况,并出现血管网稀疏,苍白,血管长势不自然,加药口及附近三、四级血管数量显著减少,或出现较明显的无血管区域,说明可能存在抑制血管生长作用。
5.结论及分析在CAM实验中,如附图4所示,空白对照组血管网内血管均匀分布,呈现明显树枝状,开窗口处具有丰富的三四级血管(但个别假阳性个体也可见加药口处三四级血管抑制情况)。如附图5所示,加药物组血管网内血管分布不均,加药口处血管扭曲破坏,三四级血管生长受到抑制,新生血管不明显;空白对照组只有10%血管抑制现象。50mM加药组相对空白对照组出现明显血管抑制现象。
SEQUENCE LISTING
<110> 广西医科大学基础医学院细胞生物学与遗传学教研室
<120> 一种CD105单链抗体-ES融合蛋白的制备方法和用途
<130> 2018.5.14
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 252
<212> PRT
<213> Homo sapiens
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Claims (5)
1.一种CD105单链抗体-ES融合蛋白,其特征在于:所述融合蛋白包括CD105单链抗体和ES。
2.根据权利要求1所述的CD105单链抗体-ES融合蛋白,其特征在于:所述融合蛋白具有如下氨基酸序列:
CD105单链抗体和ES氨基酸序列分别如序列表中1和2所示;或与序列表中1和2所示的氨基酸序列同源性在80%-100%编码相同功能蛋白质的氨基酸序列;或序列表中1和2所示的氨基酸序列经增加、缺失或者替换一个或多个氨基酸具有同等活性的衍生的蛋白。
3.一种权利要求1所述CD105单链抗体-ES融合蛋白的制备方法,其特征在于,包括以下步骤:
1)CD105单链抗体-ES融合蛋白表达载体pET28a-antiCD105-ES的构建;
2)将1)中构建表达载体转化至菌株BL21 DE3,获得转基因重组菌;
3)CD105单链抗体-ES融合蛋白的诱导表达及纯化:对CD105单链抗体-ES融合蛋白用1mM IPTG诱导4小时,收集菌液进行超声破碎,离心后收集沉淀,用8M尿素变性剂溶解沉淀,并依次在6M、4M、2M、0M尿素溶液中透析复性,CD105单链抗体-ES融合蛋白经AKTA Purifier900 FPLC系统,镍离子亲和层析柱纯化:用SDS-PAGE方法检测目的蛋白。
4.一种权利要求3所述的转基因重组菌,其特征在于:包含权利要求3所述的表达载体。
5.根据权利要求1所述的CD105单链抗体-ES融合蛋白经纯化分离后可作为抗肿瘤药物的应用。
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