东亚钳蝎氯离子通道毒素的生物活性研究
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摘要
蝎(scorpion)是一种古老的物种,蝎尾部毒腺内储存的蝎毒素不仅是其捕食、御敌的重要器官,也是治疗人类某些疾病的良药。蝎毒素的主要成分是20～90个氨基酸构成的小分子多肽,一般含3～4对二硫键,可以选择性的与动物可兴奋细胞膜上的钠、钾、钙、氯离子通道结合,引起通道电流的阻断或门控动力学的改变。
本课题组的前期研究根据大肠杆菌偏爱密码子,优化了东亚钳蝎(Buthusmartrnsii Karsch,BmK)氯离子通道毒素BmK CT基因序列(Accession Number:AF135821),并将此人工合成的基因命名为BmK CTa。该序列编码的氯离子通道毒素由35个氨基酸组成,含4对二硫键,与从以色列蝎(Leiurus quinquestriatus)中分离得到的氯离子通道毒素chlorotoxin有68%的同源性,推测它们可能具有近似的生物学功能。Chlorotoxin可以选择性的结合并抑制人脑神经胶质瘤细胞上的基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2),从而抑制神经胶质瘤细胞的转移扩散。前期的体外研究表明,rBmK CTa对神经胶质瘤细胞有较强的毒性作用,对神经胶质瘤具有潜在的治疗价值。所以,对BmK CT生物学功能的进一步研究具有一定的理论意义和应用价值。
本研究将重组东亚钳蝎氯离子通道毒素rBmK CTa进行原核可溶性表达,并获得较高纯度的rBmK CTa,分析了该毒素对神经胶质瘤细胞的迁移、侵袭能力的影响,并研究了它与细胞表面可能的受体蛋白MMP-2的相互作用关系。通过人神经胶质瘤的原位荷瘤裸鼠动物模型观察了rBmK CT在小鼠体内的代谢动力学分布。本研究的主要结果如下:
1.建立了rBmK CTa(recombinant BmK CT artifact)的可溶性表达与纯化方法。通过pRSETc表达系统将按照大肠杆菌偏爱密码子优化后的BmK CT基因在大肠杆菌BL21(DE3)中获得表达。Western blot表明,目的蛋白主要以单体形式存在。利用Ni-NTA亲和层析一步纯化得到了纯度较高的单体重组BmK CTa,最终每升培养液中可获得纯蛋白1.5～2.1 mg,为下一步研究其生物学功能奠定了基础。
2.分析了rBmK CTa对神经胶质瘤细胞SHG-44的迁移、侵袭能力的影响,并通过细胞免疫组化、明胶酶谱与Western blot方法,分析rBmK CTa作用前后SHG-44细胞中MMP-2的表达及活性变化。结果表明,rBmK CTa可减慢SHG-44细胞的生长与迁移速度,其抑制细胞迁移IC_(50)为0.28μM。同时,rBmK CTa能够抑制在侵袭转移中起重要作用的MMP-2的表达,使MMP-2分泌减少,这种减少呈剂量依赖性趋势。上述结果表明,rBmKCTa通过抑制MMP-2的表达,从而抑制了肿瘤细胞与细胞外基质(ECM)的黏附及ECM的降解,降低了肿瘤细胞迁移能力。流式细胞术分析表明,0.4μM～3.2μM重组BmK CTa作用后,SHG-44细胞G0/G1期所占比例增加,S期所占比例减少,使细胞生长阻滞于G0/G1期。在3.2μM的rBmK CTa作用24 h时,细胞的凋亡率接近50%。结果提示,rBmK CTa对于神经胶质瘤具有潜在的治疗价值,有望成为新型的靶向性药物。
3.利用RT-PCR法从SHG-44细胞中克隆得到基质金属蛋白酶2(MMP-2)基因,将其C端的胞外区部分构建入表达载体pExSecI中,并在大肠杆菌中获得表达。MMP-2C主要以包涵体的形式存在,通过变复性和亲和层析,从而获得可溶的MMP-2C蛋白。利用pull down方法确定MMP-2C与rBmK CTa可以在体外相互作用。据此推测,rBmK CTa抑制MMP-2的表达,可能是通过二者的直接相互作用而实现。
4.将rBmK CTa的C末端引入2个Tyr残基,并在pExSecI分泌型表达系统中表达ZZ-rBmK CTa-Tyr,通过IgG Sepharose纯化该蛋白。用标准Iodogen法进行ZZ-rBmK CTa的~(131)I标记,纸层析测定放化纯为90%。建立了含人神经胶质瘤的荷瘤鼠模型,经尾静脉和颅内注射分别将17.9μCi和2μCi标记蛋白注入小鼠体内。在注射给药后的10个时间段,通过SPECT观察小鼠体内标记蛋白的动力学分布。结果显示,~(131)I-ZZ-rBmK CTa通过两种给药方式均可快速通过体液循环穿过小鼠的血脑屏障。在给药后24 h,检测标记蛋白在小鼠左脑、右脑、胃、肾脏、脾脏、肝脏、后肢肌肉、肺、心脏等12个器官的分布情况。分析表明,~(131)I-ZZ-rBmK CTa在大脑中的比放射性量,在右脑(肿瘤)处蛋白含量明显高于左脑正常组织,T/NT值分别为1.25和4.13,说明ZZ-rBmK CTa与神经胶质瘤细胞可以特异性结合。以上结果提示,脑神经胶质细胞和神经细胞膜上的某些受体分子对rBmK CTa有较强的亲和性。
Scorpion is an animal which preys on mollusk insect.Its venom is rich sources of toxic poly peptides that affect ion-channel function of excitable cells,containing 20-90 amino acids linked by three or four disulfide bridges. Four different families of scorpion toxins have been described,which interact with ion channels:Na~+,K~+,Cl~- and Ca~(2+).They are proved to be important tools for clarifying the physical effects and functional mapping of ion-channels.Scorpion venoms are contain rich source of small,mainly neurotoxic proteins which interacting with ionic channels in excitable cell membranes.
In our previous work,a choride channel peptide BmK CT(Accession Number:AF 135821) was synthesized with a sequence optimized for codon usage in E.coli encoding.This 35 amino acids peptide linked by four disulfide bridges,and shares 68%of identity with the sequence of chlorotoxin,an inhibitor of Cl~- channels from the Leiurus quinquestriatus which was purified and characterized by DeBin et al.The results determined by MTT assay showed that rBmK CTa could inhibited the survival of glioma cellsand had less toxicity on cortical astrocytes.
In this study,we expressed rBmK CTa in E.coli BL21(DE3).The protein was purified by Ni-NTA resin to analyze the influence of rBmK CTa on the migration and invasion of glioma cells.Gelatin zymography,western blot, and pull down assay were used to determine the interaction between rBmK CTa and its putative receptors of glima cells—MMP-2(matrix metalloproteinase-2).The ~(131)I labeled ZZ-rBmK CTa was injected into the athymic nude mice whose right brain were implanted with astrocytoma,to investigate the biodistribution and pharmacokinetics of BmK CT.The results were obtained as followed:
1.The recombinant expression plasmid pRSETc-rBmK CTa was constructed and the fusion protein rBmK CTa(Recombinant BmK CT artifact) was expressed in E.coli BL21(DE3).rBmK CTa was eluted after purificated on the Ni-NTA resin.However,it was found that most of rBmK CTa was existed as monomer Western blot.Finally,purified protein was obtained by centrifugation.The final product by this procedure was about 1.5-2.1 mg/L cultured cells.This step suggested the pharmaceutical function of rBmK CTa.
2.The wound healing assay and matrigel invasion assay observation were showed that rBmK CTa decreased human glioma cell SHG-44 migration and cell invasion(IC_(50) is 0.28μM).Immunochemistry,zymography and western blot results demonstrated that rBmK CTa could inhibit expression of MMP-2. Otherwise,the results of flow cytometry showed that the cell cycle of glioma was arrested in G0/G1 phase after treated.And the rate of S phase was decreased.The apoptosis rate of glioma cell was approximately 50%when treated with 3.2μM rBmK CTa.These result suggested that neurotoxin BmK CT may have a therapeutic role on the medical application of glioma.
3.The gene sequence MMP-2C was encoding from the total RNA of human glioma cell SHG-44 by RT-PCR and was cloned into prokaryotic expression vector pRSETc for expression in E.coli.The product was found in inclusion bodies.After denaturation and renaureation procedure,soluble MMP-2C was obtained.The results indicated MMP-2C and rBmK CTa could interact in vitro by using in vitro pull down assay.
4.The fusion protein,ZZ-rBmK CTa-Tyr,was expressed in E.coli BL21 (DE3) using a pExSecI expression system and was purified using IgG sepharose column.The ZZ-rBmK CTa-Tyr was then radiolabeled with ~(131)I using standard Iodogen Bead method.After purified with Sepharose G-25,the radio-chemical purity of the sample was reach to 90%.~(131)I-ZZ-rBmK CTa was injected into the athymic nude mice whose right brain were implanted with astrocytoma SHG-44 at the dose of 2μCi and 17.9μCi.SPECT analysis were performed on the mice received ~(131)I-ZZ-rBmk CTa at 10 period of timeinjection.The results showed that ZZ-rBmk CTa can cross blood and tissue barriers immediately.Mice were sacrificed 24 h later,the blood,heart, liver,spleen,lung,kidney,stomach,small intestine,skeleton,muscle and tumors were weighed and counted to determine ~(131)I-ZZ-rBmk CTa concentration.The binding amount of ~(131)I- ZZ-rBmk CTa in the right brain was much higher than the left brain,and T/NT is 1.25 and 4.13 respectively. Suggesting that ZZ-rBmk CTa can binds to brain glioma cells by the high affinity action power with glioma cells,reflecting non-specific uptake of rBmk CTa in normal tissues in the brain.
本课题组的前期研究根据大肠杆菌偏爱密码子,优化了东亚钳蝎(Buthusmartrnsii Karsch,BmK)氯离子通道毒素BmK CT基因序列(Accession Number:AF135821),并将此人工合成的基因命名为BmK CTa。该序列编码的氯离子通道毒素由35个氨基酸组成,含4对二硫键,与从以色列蝎(Leiurus quinquestriatus)中分离得到的氯离子通道毒素chlorotoxin有68%的同源性,推测它们可能具有近似的生物学功能。Chlorotoxin可以选择性的结合并抑制人脑神经胶质瘤细胞上的基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2),从而抑制神经胶质瘤细胞的转移扩散。前期的体外研究表明,rBmK CTa对神经胶质瘤细胞有较强的毒性作用,对神经胶质瘤具有潜在的治疗价值。所以,对BmK CT生物学功能的进一步研究具有一定的理论意义和应用价值。
本研究将重组东亚钳蝎氯离子通道毒素rBmK CTa进行原核可溶性表达,并获得较高纯度的rBmK CTa,分析了该毒素对神经胶质瘤细胞的迁移、侵袭能力的影响,并研究了它与细胞表面可能的受体蛋白MMP-2的相互作用关系。通过人神经胶质瘤的原位荷瘤裸鼠动物模型观察了rBmK CT在小鼠体内的代谢动力学分布。本研究的主要结果如下:
1.建立了rBmK CTa(recombinant BmK CT artifact)的可溶性表达与纯化方法。通过pRSETc表达系统将按照大肠杆菌偏爱密码子优化后的BmK CT基因在大肠杆菌BL21(DE3)中获得表达。Western blot表明,目的蛋白主要以单体形式存在。利用Ni-NTA亲和层析一步纯化得到了纯度较高的单体重组BmK CTa,最终每升培养液中可获得纯蛋白1.5～2.1 mg,为下一步研究其生物学功能奠定了基础。
2.分析了rBmK CTa对神经胶质瘤细胞SHG-44的迁移、侵袭能力的影响,并通过细胞免疫组化、明胶酶谱与Western blot方法,分析rBmK CTa作用前后SHG-44细胞中MMP-2的表达及活性变化。结果表明,rBmK CTa可减慢SHG-44细胞的生长与迁移速度,其抑制细胞迁移IC_(50)为0.28μM。同时,rBmK CTa能够抑制在侵袭转移中起重要作用的MMP-2的表达,使MMP-2分泌减少,这种减少呈剂量依赖性趋势。上述结果表明,rBmKCTa通过抑制MMP-2的表达,从而抑制了肿瘤细胞与细胞外基质(ECM)的黏附及ECM的降解,降低了肿瘤细胞迁移能力。流式细胞术分析表明,0.4μM～3.2μM重组BmK CTa作用后,SHG-44细胞G0/G1期所占比例增加,S期所占比例减少,使细胞生长阻滞于G0/G1期。在3.2μM的rBmK CTa作用24 h时,细胞的凋亡率接近50%。结果提示,rBmK CTa对于神经胶质瘤具有潜在的治疗价值,有望成为新型的靶向性药物。
3.利用RT-PCR法从SHG-44细胞中克隆得到基质金属蛋白酶2(MMP-2)基因,将其C端的胞外区部分构建入表达载体pExSecI中,并在大肠杆菌中获得表达。MMP-2C主要以包涵体的形式存在,通过变复性和亲和层析,从而获得可溶的MMP-2C蛋白。利用pull down方法确定MMP-2C与rBmK CTa可以在体外相互作用。据此推测,rBmK CTa抑制MMP-2的表达,可能是通过二者的直接相互作用而实现。
4.将rBmK CTa的C末端引入2个Tyr残基,并在pExSecI分泌型表达系统中表达ZZ-rBmK CTa-Tyr,通过IgG Sepharose纯化该蛋白。用标准Iodogen法进行ZZ-rBmK CTa的~(131)I标记,纸层析测定放化纯为90%。建立了含人神经胶质瘤的荷瘤鼠模型,经尾静脉和颅内注射分别将17.9μCi和2μCi标记蛋白注入小鼠体内。在注射给药后的10个时间段,通过SPECT观察小鼠体内标记蛋白的动力学分布。结果显示,~(131)I-ZZ-rBmK CTa通过两种给药方式均可快速通过体液循环穿过小鼠的血脑屏障。在给药后24 h,检测标记蛋白在小鼠左脑、右脑、胃、肾脏、脾脏、肝脏、后肢肌肉、肺、心脏等12个器官的分布情况。分析表明,~(131)I-ZZ-rBmK CTa在大脑中的比放射性量,在右脑(肿瘤)处蛋白含量明显高于左脑正常组织,T/NT值分别为1.25和4.13,说明ZZ-rBmK CTa与神经胶质瘤细胞可以特异性结合。以上结果提示,脑神经胶质细胞和神经细胞膜上的某些受体分子对rBmK CTa有较强的亲和性。
Scorpion is an animal which preys on mollusk insect.Its venom is rich sources of toxic poly peptides that affect ion-channel function of excitable cells,containing 20-90 amino acids linked by three or four disulfide bridges. Four different families of scorpion toxins have been described,which interact with ion channels:Na~+,K~+,Cl~- and Ca~(2+).They are proved to be important tools for clarifying the physical effects and functional mapping of ion-channels.Scorpion venoms are contain rich source of small,mainly neurotoxic proteins which interacting with ionic channels in excitable cell membranes.
In our previous work,a choride channel peptide BmK CT(Accession Number:AF 135821) was synthesized with a sequence optimized for codon usage in E.coli encoding.This 35 amino acids peptide linked by four disulfide bridges,and shares 68%of identity with the sequence of chlorotoxin,an inhibitor of Cl~- channels from the Leiurus quinquestriatus which was purified and characterized by DeBin et al.The results determined by MTT assay showed that rBmK CTa could inhibited the survival of glioma cellsand had less toxicity on cortical astrocytes.
In this study,we expressed rBmK CTa in E.coli BL21(DE3).The protein was purified by Ni-NTA resin to analyze the influence of rBmK CTa on the migration and invasion of glioma cells.Gelatin zymography,western blot, and pull down assay were used to determine the interaction between rBmK CTa and its putative receptors of glima cells—MMP-2(matrix metalloproteinase-2).The ~(131)I labeled ZZ-rBmK CTa was injected into the athymic nude mice whose right brain were implanted with astrocytoma,to investigate the biodistribution and pharmacokinetics of BmK CT.The results were obtained as followed:
1.The recombinant expression plasmid pRSETc-rBmK CTa was constructed and the fusion protein rBmK CTa(Recombinant BmK CT artifact) was expressed in E.coli BL21(DE3).rBmK CTa was eluted after purificated on the Ni-NTA resin.However,it was found that most of rBmK CTa was existed as monomer Western blot.Finally,purified protein was obtained by centrifugation.The final product by this procedure was about 1.5-2.1 mg/L cultured cells.This step suggested the pharmaceutical function of rBmK CTa.
2.The wound healing assay and matrigel invasion assay observation were showed that rBmK CTa decreased human glioma cell SHG-44 migration and cell invasion(IC_(50) is 0.28μM).Immunochemistry,zymography and western blot results demonstrated that rBmK CTa could inhibit expression of MMP-2. Otherwise,the results of flow cytometry showed that the cell cycle of glioma was arrested in G0/G1 phase after treated.And the rate of S phase was decreased.The apoptosis rate of glioma cell was approximately 50%when treated with 3.2μM rBmK CTa.These result suggested that neurotoxin BmK CT may have a therapeutic role on the medical application of glioma.
3.The gene sequence MMP-2C was encoding from the total RNA of human glioma cell SHG-44 by RT-PCR and was cloned into prokaryotic expression vector pRSETc for expression in E.coli.The product was found in inclusion bodies.After denaturation and renaureation procedure,soluble MMP-2C was obtained.The results indicated MMP-2C and rBmK CTa could interact in vitro by using in vitro pull down assay.
4.The fusion protein,ZZ-rBmK CTa-Tyr,was expressed in E.coli BL21 (DE3) using a pExSecI expression system and was purified using IgG sepharose column.The ZZ-rBmK CTa-Tyr was then radiolabeled with ~(131)I using standard Iodogen Bead method.After purified with Sepharose G-25,the radio-chemical purity of the sample was reach to 90%.~(131)I-ZZ-rBmK CTa was injected into the athymic nude mice whose right brain were implanted with astrocytoma SHG-44 at the dose of 2μCi and 17.9μCi.SPECT analysis were performed on the mice received ~(131)I-ZZ-rBmk CTa at 10 period of timeinjection.The results showed that ZZ-rBmk CTa can cross blood and tissue barriers immediately.Mice were sacrificed 24 h later,the blood,heart, liver,spleen,lung,kidney,stomach,small intestine,skeleton,muscle and tumors were weighed and counted to determine ~(131)I-ZZ-rBmk CTa concentration.The binding amount of ~(131)I- ZZ-rBmk CTa in the right brain was much higher than the left brain,and T/NT is 1.25 and 4.13 respectively. Suggesting that ZZ-rBmk CTa can binds to brain glioma cells by the high affinity action power with glioma cells,reflecting non-specific uptake of rBmk CTa in normal tissues in the brain.
引文
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