CN108379596A - 水蛭素靶向定位释放基因给药系统及其制备方法和用途 - Google Patents
水蛭素靶向定位释放基因给药系统及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种水蛭素靶向定位释放基因给药系统,该给药系统为纳米基因给药系统,包括给药载体以及分泌表达质粒,所述分泌表达质粒由真核表达分泌型质粒载体pSecTag2A和重组水蛭素基因组成,所述重组水蛭素基因是在水蛭素蛋白的编码基因的N端依次连接凝血因子活性形式Xa(FXa)识别序列和精氨酰‑甘氨酰‑天冬氨酸(RGD)序列,水蛭素蛋白的编码基因的C端依次连接凝血因子活性形式Xa(FXa)识别序列和增强绿色荧光蛋白(EGFP)的编码基因而得到。本发明是重组水蛭素融合蛋白血栓靶向和血栓部分定点激活的长效缓释双重靶向定位释放的纳米DNA制剂。
Description
技术领域
本发明属于生物治疗与生物医药技术领域,特别是涉及水蛭素靶向定位释放基因给药系统及其制备方法和用途。
背景技术
水蛭素的结构和活性研究显示,它的N末端延伸会使水蛭素丧失抗凝血活性的特点,而C末端的延伸对其活性影响不大。如果在水蛭素蛋白链的N端接上血栓靶向肽,可暂时封闭水蛭素的活性,使它能够在特定部位激活后恢复活性,减少出血风险;在水蛭素蛋白链的C端接上荧光蛋白,可在保持水蛭素活性的条件下,便于体内跟踪检测。
血栓形成过程涉及到血小板血栓形成、体内凝血系统及纤维蛋白溶解系统激活等多个方面,血小板聚集形成血小板血栓在整个血栓形成过程中起到了核心作用,而纤维蛋白原(Fibrinogen,Fg)和活化血小板膜糖蛋白(Glycoprotein,GP)IIb/IIIa受体的特异性结合是引起血小板聚集的最终共同通道之一。血小板膜糖蛋白IIb/IIIa受体即GP IIb/IIIa受体,又名整合素αIIbβ3受体,是整合素(integrin)家族受体中的一员。GP IIb/IIIa受体是一种膜结合蛋白,是血小板膜上最丰富的受体,正常情况下,此受体空间构象稳定。当血小板受外界刺激活化时,该受体空间构象发生改变,暴露Fg的识别部位,介导Fg和它的结合,完成血小板聚集过程。Fg是一种粘附蛋白,Fg中的精氨酰-甘氨酰-天冬氨酸(Arg-Gly-Asp,RGD)序列是Fg和活化的GPIIb/IIIa受体结合的活性位点,存在于其两条Aα链中(RGDF 95-98,RGDS 572-575)。含有RGD序列的物质均可与活化的GPIIb/IIIa受体结合,竞争性地抑制Fg和GPIIb/IIIa受体的结合,从而阻断血小板聚集和血栓的形成。只有血栓部位才存在活化的GPIIb/IIIa受体,因此RGD序列具有血栓靶向性和抗血小板聚集的作用。
凝血因子活性形式Xa(FXa)属于丝氨酸蛋白水解酶,它能激活凝血酶原变成有活性的凝血酶,是凝血过程中的一个关键因子,同时它也能特异性识别并切割Ile-Glu-Gly-Arg序列。FXa在血栓部位特异性的集聚并有强大的活性,而在其它部位浓度很低且几无活性。因为FXa特异质切割Ile-Glu-Gly-Arg序列的特点,可让药物在血栓部分定点释放。
绿色荧光蛋白(GFP)是一种来源于发光水母生物的发光蛋白,由238个氨基酸组成,分子量为27kb,由11个β-折叠围绕着一个α-螺旋而围成圆桶状结构,α-螺旋包含着由3个氨基酸(Ser65、Tyr66、Gly67)经过环化而成的发光基团,在钙离子和紫外光激发下会产生绿色荧光,可以用倒置荧光显微镜下观察到。GFP发出的荧光较稳定,抗光漂白能力强,在pH7-12范围内都能正常发光。GFP可以在不同种属原核、真核细胞中表达,无需复杂的翻译后加工过程就会自动折叠催化并开始发光,既可以作为荧光标记物监测活体细胞中的基因表达,也可以作为体外实验中有用的工具。GFP是基因递送靶向载体的理想报告基因,将GFP编码基因连着目的基因上,根据荧光强度,在体外能评价基因递送载体的细胞转染效率,在体内则能跟踪目的蛋白的转运、分布和靶向性研究。
基因治疗是通过基因载体,将具有治疗作用的特定基因递送至病人靶细胞内,从而达到治疗目的的一种新兴的治疗方法。目前应用于基因治疗的转染细胞的载体主要有病毒载体(viral vector)和非病毒载体(non-viral vector)两种。到目前为止,研究者们已设计了多种类型且各有特点的非病毒基因载体,主要包括阳离子脂质体(cationicliposome)及阳离子聚合物(cationic polymer,如多聚赖氨酸、聚乙烯亚胺、壳聚糖、树状大分子等)。
基于以上理论,本发明开发出一种水蛭素双重靶向定位释放基因给药系统。
发明内容
本发明的目的在于提供一种水蛭素靶向定位释放基因给药系统及其制备方法和用途。
为了达到上述的目的,本发明提供以下技术方案:
一种水蛭素靶向定位释放基因给药系统,该给药系统为纳米基因给药系统,包括给药载体以及分泌表达质粒,所述分泌表达质粒由真核表达分泌型质粒载体pSecTag2A和重组水蛭素基因组成,所述重组水蛭素基因是在水蛭素蛋白的编码基因的N端依次连接凝血因子活性形式Xa(FXa)识别序列和精氨酰-甘氨酰-天冬氨酸(RGD)序列,水蛭素蛋白的编码基因的C端依次连接凝血因子活性形式Xa(FXa)识别序列和增强绿色荧光蛋白(EGFP)的编码基因而得到。
进一步地,所述给药载体为聚酰胺-胺型树枝状高分子(PAMAM),优选5代PAMAM。
进一步地,所述分泌表达质粒为在真核表达分泌型质粒载体pSecTag2A的多克隆位点上插入重组水蛭素。
进一步地,所述重组水蛭素基因序列为SEQ 1D NO.1。
进一步地,所述给药载体包裹分泌表达质粒,并且给药载体表面通过RGD肽和PEG进行修饰。
本发明还提供另一个技术方案:
一种水蛭素靶向定位释放基因给药系统的制备方法,包括如下步骤:
(1)合成重组水蛭素基因片段,在水蛭素蛋白的编码基因的N端依次连接凝血因子活性形式Xa(FXa)识别序列和精氨酰-甘氨酰-天冬氨酸(RGD)序列,水蛭素蛋白的编码基因的C端依次连接凝血因子活性形式Xa(FXa)识别序列和增强绿色荧光蛋白(EGFP)的编码基因;
(2)构建分泌表达质粒,在真核表达分泌型质粒载体pSecTag2A的多克隆位点上插入重组水蛭素;
(3)在PAMAM表面用RGD肽和PEG进行修饰,然后包裹步骤(2)得到的分泌表达质粒,最后得到水蛭素靶向定位释放基因给药系统。
本发明还提供另一个技术方案:
一种水蛭素靶向定位释放基因给药系统的应用,包括在抗凝、抗血栓药物中的应用。
本发明具有以下技术特点:
本发明采取PAMAM树状大分子,其是阳离子聚合物的一种,携带基因转染细胞的能力较效率高。
本发明将水蛭素蛋白的编码基因的N端连上RDG肽和FXa识别序列的编码基因,在水蛭素蛋白的编码基因的C端连上FXa识别序列和EGFP的编码基因,构建pSecTag2A-RGD-FXa-Hirudin-FXa-EGFP分泌表达质粒后,制备成PAMAM/DNA纳米粒,并在纳米粒上连上血栓靶向肽RDG和PEG化,使该制剂将Ile-Glu-Gly-Arg序列连接在RGD肽和水蛭素的中间,可利于血栓部位的FXa特异质切割Ile-Glu-Gly-Arg序列的特点,让RGD肽和水蛭素在血栓部分定点释放激活,发挥各自的抗栓作用。同时,FXa对Ile-Glu-Gly-Arg序列的切割,也能对凝血酶原的激活起到竞争性的抑制作用。
本发明具有PAMAM/DNA纳米粒血栓靶向,是重组水蛭素融合蛋白血栓靶向和血栓部分定点激活的长效缓释双重靶向定位释放的纳米DNA制剂。
附图说明
图1 重组水蛭素基因结构示意图。
图2 显微镜观察图(A)PCR鉴定;(B)双酶切鉴定;(C)质粒抽提鉴定;(D)10X倍镜下转染的细胞。
图3 不同N/P比的质粒/纳米粒复合物电泳鉴定图。
图4 Westen Bloting结果:(A)无纳米粒包封的质粒转染(B)不同N/P比的质粒/纳米粒复合物转染。
图5 抗血小板聚集活性结果。
图6 (A)PT(B)APTT结果。
具体实施方式
以下具体实施例是对本发明提供的方法与技术方案的进一步说明,但不应理解成对本发明的限制。
(一)pSecTag2A-RGD-FXa-Hirudin-FXa-EGFP质粒构建
在水蛭素蛋白的编码基因的N端依次连接凝血因子活性形式Xa(FXa)识别序列和精氨酰-甘氨酰-天冬氨酸(RGD)序列,水蛭素蛋白的编码基因的C端依次连接凝血因子活性形式Xa(FXa)识别序列和增强绿色荧光蛋白(EGFP)的编码基因,得到重组水蛭素基因RGD-FXa-Hirudin-FXa-EGFP,参见图1。然后使用真核表达分泌型质粒载体pSecTag2A,在其多克隆位点上插入RGD-FXa-Hirudin-FXa-EGFP重组蛋白的基因。合成后水蛭素融合蛋白的核苷酸序列(SEQ ID NO.1)为:
GTGGAATTCTGATGAGAGGCGACATCGAGGGCAGGATCACCTACACCGACTGCACCGAGAGCGGCCAGAACCTGTGCCTGTGCGAGGGCAGCAACGTGTGCGGCAAGGGCAACAAGTGCATCCTGGGCAGCAACGGCAAGGGCAACCAGTGCGTGACCGGCGAGGGCACCCCCAACCCCGAGAGCCACAACAACGGCGACTTCGAGGAGATCCCCGAGGAGTACCTGCAGATCGAGGGCAGGACGGTACCGCGGGCCCGGGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGCTCGAGGA。
表达的融合蛋白实际序列为:
RDG三肽(3个氨基酸)+FXa识别序列(4个氨基酸)+水蛭素基因(不含终止子,为65个氨基酸)+FXa识别序列(4个氨基酸)+12个氨基酸(含KpnⅠ、SacⅡ、ApaⅠ、XmaⅠ、BamHⅠ、EagⅠ酶切位点)+EGFP(238个氨基酸)+5个氨基酸(含XholⅠ、DraⅡ、ApaⅠ酶切位点)+myc(10个氨基酸)+5个氨基酸+6个组氨酸标签。
随后设计引物并进行PCR鉴定,PCR反应体系:要保证操作中各溶液在冰上进行,并在无菌的Eppendorf管内按顺序加入相应反应物。PCR结束后琼脂糖凝胶电泳分析PCR结果。
取适量的质粒双酶切鉴定,在一个洁净的1.5mlEP管中按顺序依次加入相应试剂,小心混匀试剂,将EP管置于恒温水浴箱中37℃反应1.5小时。琼脂糖凝胶电泳分析双酶切结果。
取20uL质粒加入100uL感受态大肠杆菌,轻吹混匀,冰上孵育40min,42℃水浴,90s精确热休克后立即拿出置冰上3min,加入LB培养基700uL,轻轻混匀。将转化的大肠杆菌于37℃水浴培养45min,同时准备含博来霉素的LB液体培养基7mL,于37℃温箱预热。取适量大肠杆菌加入到培养基中,180转/分钟,37℃温箱中培养16h。按照质粒抽提试剂盒提取质粒并进行琼脂糖凝胶电泳分析。
将此质粒用脂质体转染法转入人脐静脉血管内皮细胞(HUVEC细胞),显微镜下观察表达情况。结果见图2,(A)-(C)结果显示重组质粒的成功合成。(D)10倍镜下细胞转染24h的情况,说明重组质粒在细胞内成功表达。
(二)pSecTag2A-RGD-FXa-Hirudin-FXa-EGFP的PAMAM/DNA纳米粒制备评价
选用非病毒基因传递载体材料5代PAMAM,并在PAMAM的表面用RGD肽和不同量的PEG修饰,然后包裹DNA质粒,具体制备方法如下:
称取4.65mg RGDyC,溶于2mL醋酸-醋酸钠缓冲液中(pH 6.0),向其中加入27.32mgMAL-PEG3500-NHS,涡旋30s,迅速加入至含有5.0mg PAMAM的硼砂-氢氧化钠缓冲液中(pH9.2),室温反应过夜。通过缓冲液调节体系pH至7.0,加入5μLβ-巯基乙醇反应1h以终止未反应完全的马来酰亚胺基团。超纯水超滤离心后,复溶于2mL磷酸盐缓冲液(PBS 8.0),向其加入适量mPEG3000-NHS,使PAMAM G5与PEG最终的反应摩尔比为1:64。室温反应48h,超滤离心,除去未反应的PEG,冷冻干燥。
取适量的DNA与PEG修饰的PEG-PAMAM、RGDyC-PEG-PAMAM分别溶解在去离子水中,配制成适当浓度,将各PAMAM复合物溶液加入到等体积DNA溶液中,涡旋30s,然后室温孵育20min,制成不同N/P比(0、0.5、1、2、5、8、10)的PAMAM/pDNA复合物,琼脂糖凝胶电泳分析其DNA压缩能力,Marvern measurement粒度/Zeta电位分析仪测定粒径分布和电位分布。其中氮磷比(N/P),即PAMAM上氨基与DNA的比例。比如:PAMAM G5分子量为28826,一个分子中有128个表面氨基N,因此,1μg PAMAM则平均含有4.44nmol氨基氮。1个碱基对DNA的平均分子量为647.5Da,1μgDNA平均含有3.08nmol个磷酸基团。
将不同量(以PAMAM:PEG=1:64为例)PEG修饰的RGD-PAMAM载体材料和pSecTag2A-RGD-FXa-Hirudin-FXa-EGFP按照不同N/P比(0.5、1、2、4、8、10)混合,迅速漩涡30s并室温孵育30min,制得质粒载体纳米粒,通过采用琼脂糖凝胶电泳法评价PAMAM聚合物包封效果(参见图3)、粒度/zeta电位测定仪测定纳米粒的光散射粒径。图3说明在不同N/P比下,纳米粒均能很好的包封质粒。
表1 不同N/P比的质粒/纳米粒复合物粒径
N/P | 0.5 | 1 | 2 | 4 | 8 | 10 | 无DNA |
粒径nm | 20.11 | 22.40 | 25.28 | 29.05 | 36.56 | 40.23 | 19.33 |
由表1可知,制备得到的PAMAM/DNA纳米粒溶液,颗粒粒径达到纳米级,且粒径分布小。
(三)Westen Bloting鉴定
将PAMAM/DNA纳米粒转染HUVEC细胞,培养48h后收集细胞上清,利用His标签蛋白纯化试剂盒纯化并用FXa酶切后进行Westen Bloting鉴定分析,鉴定结果参见图4所示。
(四)抗血小板聚集活性测定
兔耳缘静脉取血,置于含有1/10体积0.109M枸橼酸钠抗凝液(1份抗凝液+9份全血)的塑料管中,700rpm离心10min,收集上清得富血小板血浆(PRP),剩余部分4000rpm离心15min,收集上清得贫血小板血浆(PPP)。用PPP稀释PRP至血小板数量为25×104-30×104/μL,作为PRP试剂。取PRP试剂0.2mL,加入FXa酶切后与未切割后的融合蛋白,使其终浓度为0.5μg/mL、1.0μg/mL、5.0μg/mL、10.0μg/mL、25.0μg/mL、50.0μg/mL,室温静置15min。随后加入经37℃预热的200μmol/L的二磷酸腺苷(ADP)5μL,同时以生理盐水替代融合蛋白作为对照,用血小板聚集仪测定聚集率。
血小板聚集仪测定结果图5发现,重组水蛭素融合蛋白具有较好的抗血小板聚集活性。融合蛋白未经FXa切割时并不具有活性,从而降低了其从注射部位到到达血栓部位之前大量使用引起的出血风险。
(五)水蛭素融合蛋白抗凝血酶作用分析
采用活化部分凝血活酶时间(APTT)法检测聚合物纳米药物的抗凝血活性。兔耳缘静脉取血,置于含有1/10体积0.109M枸橼酸钠抗凝液(1份抗凝液+9份全血)的塑料管中,3000rpm离心15min,收集上清,得贫血小板血浆(PPP)。分别用PPP稀释融合蛋白至0.5μg/mL、1.0μg/mL、5.0μg/mL、10.0μg/mL、25.0μg/mL、50.0μg/mL,天然水蛭素替代融合蛋白作为对照,室温静置15min血液凝集仪测定PT、APPT,结果如图6所示。
以上实施例的说明只是用于帮助理解本发明方法及其核心思想。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,还可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利要求保护范围内。
序列表
<110> 浙江中医药大学
<120> 水蛭素靶向定位释放基因给药系统及其制备方法和用途
<130> 412
<141> 2018-02-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1004
<212> DNA
<213> 人工序列()
<400> 1
gtggaattct gatgagaggc gacatcgagg gcaggatcac ctacaccgac tgcaccgaga 60
gcggccagaa cctgtgcctg tgcgagggca gcaacgtgtg cggcaagggc aacaagtgca 120
tcctgggcag caacggcaag ggcaaccagt gcgtgaccgg cgagggcacc cccaaccccg 180
agagccacaa caacggcgac ttcgaggaga tccccgagga gtacctgcag atcgagggca 240
ggacggtacc gcgggcccgg gatccaccgg tcgccaccat ggtgagcaag ggcgaggagc 300
tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 360
tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca 420
tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg 480
gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 540
ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 600
agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 660
gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 720
gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga 780
tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc 840
ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc 900
tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg 960
ccgggatcac tctcggcatg gacgagctgt acaaggctcg agga 1004
Claims (8)
1.一种水蛭素靶向定位释放基因给药系统,其特征在于,该给药系统为纳米基因给药系统,包括给药载体以及分泌表达质粒,所述分泌表达质粒由真核表达分泌型质粒载体pSecTag2A和重组水蛭素基因组成,所述重组水蛭素基因是在水蛭素蛋白的编码基因的N端依次连接凝血因子活性形式Xa(FXa)识别序列和精氨酰-甘氨酰-天冬氨酸(RGD)序列,水蛭素蛋白的编码基因的C端依次连接凝血因子活性形式Xa(FXa)识别序列和增强绿色荧光蛋白(EGFP)的编码基因而得到。
2.根据权利要求1所述的水蛭素靶向定位释放基因给药系统,其特征在于,所述给药载体为聚酰胺-胺型树枝状高分子(PAMAM) 。
3.根据权利要求2所述的水蛭素靶向定位释放基因给药系统,其特征在于,聚酰胺-胺型树枝状高分子为5代PAMAM。
4.根据权利要求1所述的水蛭素靶向定位释放基因给药系统,其特征在于,所述分泌表达质粒为在真核表达分泌型质粒载体pSecTag2A的多克隆位点上插入重组水蛭素。
5.根据权利要求1所述的水蛭素靶向定位释放基因给药系统,其特征在于,所述重组水蛭素基因序列为SEQ 1D NO.1。
6.根据权利要求1所述的水蛭素靶向定位释放基因给药系统,其特征在于,所述给药载体包裹分泌表达质粒,并且给药载体表面通过RGD肽和PEG进行修饰。
7.根据权利要求1-6中任一项所述的一种水蛭素靶向定位释放基因给药系统的制备方法,包括如下步骤:
(1)合成重组水蛭素基因片段,在水蛭素蛋白的编码基因的N端依次连接凝血因子活性形式Xa(FXa)识别序列和精氨酰-甘氨酰-天冬氨酸(RGD)序列,水蛭素蛋白的编码基因的C端依次连接凝血因子活性形式Xa(FXa)识别序列和增强绿色荧光蛋白(EGFP)的编码基因;
(2)构建分泌表达质粒,在真核表达分泌型质粒载体pSecTag2A的多克隆位点上插入重组水蛭素;
(3)在PAMAM表面用RGD肽和PEG进行修饰,然后包裹步骤(2)得到的分泌表达质粒,最后得到水蛭素靶向定位释放基因给药系统。
8.根据权利要求1-7中任一项所述的水蛭素靶向定位释放基因给药系统的应用,包括在抗凝、抗血栓药物中的应用。
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