江浙短尾蝮蛇纤溶酶原激活剂的原核表达、纯化及其鉴定
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摘要
目的
采用基因工程技术,构建江浙短尾蝮蛇纤溶酶原激活剂(plasminogen activator of Gloydius brevicaudus venom, GBV-PA )原核核表达载体(pET42a(+)-GBV-PA),并在大肠杆菌中的表达。为编码GBV-PA的基因克隆在大肠杆菌细胞中进行有效表达,批量生产GBV-PA;同时为研究GBV-PA的基因和蛋白质、结构和功能的关系及其作用机制创造条件。
方法
根据已知竹叶青蛇毒纤溶酶原激活剂(TSV-PA)基因的核苷酸序列设计上游引物,含EcoRⅠ酶切位点,和下游引物,含XhoⅠ酶切位点。以提取的江浙短尾蝮蛇毒腺总RNA为模板,RT-PCR方法合成扩增其中的纤溶酶原激活剂基因序列,将GBV-PA基因定向克隆到原核表达载体pET42a(+)中,经EcoRⅠ/XhoⅠ双酶切鉴定后,热激转化至宿主菌BL21(DE3),测序鉴定。IPTG诱导rGBV-PA融合蛋白表达,SDS-PAGE凝胶电泳鉴定表达产物;His亲和层析柱纯化融合蛋白rGBV-PA, Western blotting方法鉴定表达产物的抗原性。
结果
1.RT-PCR扩增得到的cDNA为800bp左右,与预期GBV-PA基因大小基本一致。
2.构建的表达质粒pET42a(+)-GBV-PA,经双酶切和测序鉴定证实GBV-PA基因已正确插入质粒载体的多克隆位点。得知GBV-PA的cDNA大小为777bp,推导出的氨基酸数目为259个,MW=27921。GBV-PA与TSV-PA核苷酸序列同源性为90.13%,氨基酸序列同源性为81.92%。
3.在大肠杆菌BL21中成功表达融合蛋白rGBV-PA,SDS-APGE凝胶电泳显示rGBV-PA主要以包涵体形式存在,分子量约61.7kDa。
4.纯化得到融合蛋白rGBV-PA,具有蝮蛇毒的抗原性。
结论
成功构建了GBV-PA基因的原核表达载体,并在大肠杆菌中表达,经His亲和层析柱纯化后,获得rGBV-PA融合蛋白具有蝮蛇毒的抗原性。这些结果为江浙短尾蝮蛇纤溶酶原激活剂作为溶栓药物的进一步研发提供实验依据。
Objective
The subject using gene techniques constructed the prokaryotic expression vector of pET42a(+)-GBV-PA (plasminogen activator of Gloydius brevicaudus venom,GBV-PA),and expressed recombinate GBV-PA(rGBV-PA) fusion protein in Escherichia coli(E. coli). These create the possibility for expression of GBV-PA cloning efficiently in E. coli and mass production of GBV-PA. These also provide the basis for study on the relationship of constitution between function and the pharmacological mechanism of GBV-PA.
Method
A couple of specific primers were designed on the basis of known nucleotide sequence of TSV-PA(Plasminogen Activator of Trimeresurus stejnegeri Venom) . The forward primer contained EcoRⅠrestriction site,and the reward prime contained XhoⅠrestriction site. GBV-PA gene fragment was amplified from total RNA of Gloydius brevicaudus venom gland with RT-PCR. The PCR products were cloned and inserted into prokaryotic expression plasmid pET42a(+).Recombinant plasmid (pET42a(+)-GBV-PA)were identified by restriction enzymes digestion,then transformed into E. coli strain BL21(DE3). pET42a(+)-GBV-PA was identified by sequencing. Expression of rGBV-PA fusion protein induced by IPTG,and the expressed product was analyzed by SDS- PAGE .The rGBV-PA fusion protein was purified with His-Bind resin protein purification procedure. The antigenicity of expressed product was identified by Western blotting.
Results
1.The cDNA in 800 bp was obtained by RT-PCR amplification,which was the same as GBV-PA gene.
2.The GBV-PA gene was synthesized sueeessuflly and inserted into the vector PET42a(+).The recombinant expression plasmid pET42a(+)-GBV-PA was constructed. The size of cDNA sequence of GBV-PA gene was 777 bp which encoding 259 amino acids. MW=27921. Homologies of nucleotide sequence and amino acid sequence of GBV-PA between TSV-PA were 90.13% and 81.92%, respectively.
3. The rGBV-PA fusion protein was expressed in E. coli BL21(DE3). SDS - PAGE showed that the fusion protein was expressed as inclusion body formation in E. coli BL21(DE3). MW was 61.7kD.
4. The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Gloydius brevicaudus venom.
Conclusions
The prokaryotic expression recombinant plasmid pET42a(+)-GBV-PA were successfully constructed. The GBV-PA gene was expressed in E. coli BL21(DE3). The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Agkistrodon halys pallas venom. This can provide experimental basis for research and development of GBV-PA as thrombolytic drug.
采用基因工程技术,构建江浙短尾蝮蛇纤溶酶原激活剂(plasminogen activator of Gloydius brevicaudus venom, GBV-PA )原核核表达载体(pET42a(+)-GBV-PA),并在大肠杆菌中的表达。为编码GBV-PA的基因克隆在大肠杆菌细胞中进行有效表达,批量生产GBV-PA;同时为研究GBV-PA的基因和蛋白质、结构和功能的关系及其作用机制创造条件。
方法
根据已知竹叶青蛇毒纤溶酶原激活剂(TSV-PA)基因的核苷酸序列设计上游引物,含EcoRⅠ酶切位点,和下游引物,含XhoⅠ酶切位点。以提取的江浙短尾蝮蛇毒腺总RNA为模板,RT-PCR方法合成扩增其中的纤溶酶原激活剂基因序列,将GBV-PA基因定向克隆到原核表达载体pET42a(+)中,经EcoRⅠ/XhoⅠ双酶切鉴定后,热激转化至宿主菌BL21(DE3),测序鉴定。IPTG诱导rGBV-PA融合蛋白表达,SDS-PAGE凝胶电泳鉴定表达产物;His亲和层析柱纯化融合蛋白rGBV-PA, Western blotting方法鉴定表达产物的抗原性。
结果
1.RT-PCR扩增得到的cDNA为800bp左右,与预期GBV-PA基因大小基本一致。
2.构建的表达质粒pET42a(+)-GBV-PA,经双酶切和测序鉴定证实GBV-PA基因已正确插入质粒载体的多克隆位点。得知GBV-PA的cDNA大小为777bp,推导出的氨基酸数目为259个,MW=27921。GBV-PA与TSV-PA核苷酸序列同源性为90.13%,氨基酸序列同源性为81.92%。
3.在大肠杆菌BL21中成功表达融合蛋白rGBV-PA,SDS-APGE凝胶电泳显示rGBV-PA主要以包涵体形式存在,分子量约61.7kDa。
4.纯化得到融合蛋白rGBV-PA,具有蝮蛇毒的抗原性。
结论
成功构建了GBV-PA基因的原核表达载体,并在大肠杆菌中表达,经His亲和层析柱纯化后,获得rGBV-PA融合蛋白具有蝮蛇毒的抗原性。这些结果为江浙短尾蝮蛇纤溶酶原激活剂作为溶栓药物的进一步研发提供实验依据。
Objective
The subject using gene techniques constructed the prokaryotic expression vector of pET42a(+)-GBV-PA (plasminogen activator of Gloydius brevicaudus venom,GBV-PA),and expressed recombinate GBV-PA(rGBV-PA) fusion protein in Escherichia coli(E. coli). These create the possibility for expression of GBV-PA cloning efficiently in E. coli and mass production of GBV-PA. These also provide the basis for study on the relationship of constitution between function and the pharmacological mechanism of GBV-PA.
Method
A couple of specific primers were designed on the basis of known nucleotide sequence of TSV-PA(Plasminogen Activator of Trimeresurus stejnegeri Venom) . The forward primer contained EcoRⅠrestriction site,and the reward prime contained XhoⅠrestriction site. GBV-PA gene fragment was amplified from total RNA of Gloydius brevicaudus venom gland with RT-PCR. The PCR products were cloned and inserted into prokaryotic expression plasmid pET42a(+).Recombinant plasmid (pET42a(+)-GBV-PA)were identified by restriction enzymes digestion,then transformed into E. coli strain BL21(DE3). pET42a(+)-GBV-PA was identified by sequencing. Expression of rGBV-PA fusion protein induced by IPTG,and the expressed product was analyzed by SDS- PAGE .The rGBV-PA fusion protein was purified with His-Bind resin protein purification procedure. The antigenicity of expressed product was identified by Western blotting.
Results
1.The cDNA in 800 bp was obtained by RT-PCR amplification,which was the same as GBV-PA gene.
2.The GBV-PA gene was synthesized sueeessuflly and inserted into the vector PET42a(+).The recombinant expression plasmid pET42a(+)-GBV-PA was constructed. The size of cDNA sequence of GBV-PA gene was 777 bp which encoding 259 amino acids. MW=27921. Homologies of nucleotide sequence and amino acid sequence of GBV-PA between TSV-PA were 90.13% and 81.92%, respectively.
3. The rGBV-PA fusion protein was expressed in E. coli BL21(DE3). SDS - PAGE showed that the fusion protein was expressed as inclusion body formation in E. coli BL21(DE3). MW was 61.7kD.
4. The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Gloydius brevicaudus venom.
Conclusions
The prokaryotic expression recombinant plasmid pET42a(+)-GBV-PA were successfully constructed. The GBV-PA gene was expressed in E. coli BL21(DE3). The rGBV-PA fusion protein was obtained by His-bind Purification system,which has the antigenicity of Agkistrodon halys pallas venom. This can provide experimental basis for research and development of GBV-PA as thrombolytic drug.
引文
[1] 汪钟,郑植荃. 现代血栓病学[M].北京:北京医科大学中国协和医科大学联合出版社,1997,523-529.
[2] 王振义,阮长耿等. 血栓与止血--基础理论与临床( 第三版)[M] .南京:上海科技技术出版社,2004,809-810.
[3] 段智变等.纳豆激酶粗制液对家兔溶血栓作用及其机制研究[J].营养学报 ,2003, 25(1):46-51.
[4] Markland F S. Snake venoms and the hemostatic system [J]. Toxicon, 1998,36(12): 1749-1800.
[5] Zhang Y,Wisner A,Xiong Y et al. A novel plasminogen activator from snake venom [J].Biol Chem,1995,270(12):10246-10255.
[6] Zhang Y,Wisner A,Maroun R C et al .Trimeresurus stejnegeri snake venom plasminogen activator[J].Biol Chem,1997,272(33):20531-20537.
[7] 张志强,张进华等.短尾蝮蛇毒纤溶酶原激活剂的分离纯化及活性测定[J].中国药理学通报,2007,23(12):1639-1644.
[8]Sambrook J and Russell DW.Molecular Cloning: A Laboarroy Manual,3rd[M].Cold Spring Habror Loratory Press,2001.
[9] Studier F W,Rosenberg A H,Dunn J J et al.Use of T7 RNA polymerase to direct expression of cloned genes[J].Methods Enzymol,1990,185:60-89.
[10] Zhang,-X,Studier,-F-W.Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme[J].J-Mol-Biol,1997 May 30,269(1):10-27.
[11] Lyakhov,-D-L,He,-B,Zhang,-X.Pausing and termination by bacteriophage T7 RNA polymerase[J].J-Mol-Biol,1998 Jul 10,280(2):201-13.
[12] Zhang,-X,Studier,-F-W.Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection[J].J-Mol-Biol,2004 Jul 16,340(4):707-30.
[13] MECK.pET系统操作手册,第十版.上海,2002:31.
[14] Anton P.J.Middelberg.Preparative protein refolding[J].Trend in Biotechnology,2002,20(10):437-443.
[15] 李校坤,袁辉.药物蛋白质分离纯化技术,第一版[M].北京:化学工业出版社,2005:239.
[16] 卢圣栋主编.现代分子生物学实验技术,第二版[M].北京:中国协和医科大学出版社,1999:377.
[17] 袁勤生,赵健主编.酶与酶工程,第一版[M].上海:华东理工大学出版社,2005:192.
[18] Zhao Qin-yi. Irreversible Thermodynamic Theory for Protein Floding and Protein thermodynamic Structures.Prog[J].Biochem Biophys,2001,28(3):429-435.
[19] Lilie H,Schwarz E, Rudolph R.Advances in refolding of proteins produced in E.coli.Curr.Opin[J].Biotechnol.,1998,9(5):497-501.
[20] Rudolph R,Lillie H.In vitro folding of inclusion body proteins[J]..FASEB J.,1996,10(1):29-56.
[1] 段 清 海 , 张 兆 山 , 李 淑 琴 等 .α- 银 环 蛇 毒 素 的 克 隆 和 表 达 [J]. 生 物 技 术 通讯,1998,9(4):247-252.
[2] Lathan LO, Staggers SL. Ancrod: the use of snake venom in the treatment of patients with heparin-induced thrombocytopenia and thrombosis undergoing coronary hypass grafting: Nursing management[J]. Heart Lung,1996,25(6):451.
[3] Leonard A S , Mark A O , Pierre J L , et al. Cloning and expression of mamba toxins[J]. Toxicon,1995,33(4):439-474.
[4] 万榕一,宋军,林旭等.中华眼镜蛇毒腺cDNA表达文库的建立和分析[J]. 第四军医大学学报,2004,25(13):1210-1211.
[5] Zhang S P,Zubay G,Goldman E.Low usage codons in Escheriehiaooli,yeast,fruit fly and primates[J].Gene,1991,105(1):61-72.
[6] DuanHQ,ZhangZS,LiSQ,et al.Cloning and expression of α-bungarotoxin gene[J].Letters in Biotechnology ,1998 ,9(4):247-252.
[7] 汪 芳 , 王 义 权 .a- 银 环 蛇 毒 素 同 工 毒 素 基 因 的 克 隆 和 表 达 [J]. 遗 传 学报.2005,32(7):684-688.
[8] Seo J H ,Bailev J E. Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli[J].Biotechnol Bioeng, 1985,27:1668-1674.
[9] 黄星,杨青,闫明等.在大肠杆菌中表达蝮蛇毒类凝血酶基因gloshedobin[J].过程工程学报,2004,4(1):23-26.
[10] 刘小龙,钟晓燕,吴祥甫等.尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征[J].生物化学与生物物理进展,2000,27(3):271.
[11] 查向东,张华远,肖亚中等.尖吻蝮蛇未知C 类凝集素蛋白cDNA 扩增、克隆与表达[J].生物化学与生物物理进展,2001,28(5):754.
[12] Qing Yang, Jianqiang Xu, Min L.High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions[J].Biotechnology Letters,2003,25: 607–610.
[13] 林鲁萍,汪芳,章晓波等.重组α-银环蛇毒素P22-A31的原核表达及功能分析[J].动物学报,2005,51(6):1102-1108.
[14] N G Qing,HU Xue-J un,XU Xiao-Ming.Expression , Purification and Partial Characterizationof Recombinant Gloshedobin , a Thrombin-like Enzymefrom The Venom of Gloydius shedaoensis[J].Prog. Biochem. Biophys,2002,29(3)390-393.
[15] 杨旭宇,杨青,包永明等.大连蛇岛蝮蛇类凝血酶在大肠杆菌的表达与纯化[J].中国工程生物杂志,2004,24(9):45-46.
[16] 向开军,余红秀,邹春森等.皖南尖吻蝮蛇毒I 型金属蛋白酶Acutolysin A 的表达、重折叠及其生物学活性[J].生物化学与生物物理学报,2002,34(5):675-679.
[17] 钱友纯,沈雁,范春阳等.蛇神经毒素的表达和鉴定[J].生物工程学报,2000,16(3): 312-313.
[18] 白霞,傅建新,苏健等.绿色荧光蛋白-去整合素融合蛋白的表达及其结合功能的研究[J].中国免疫学杂志,2005,21(10):739-743.
[2] 王振义,阮长耿等. 血栓与止血--基础理论与临床( 第三版)[M] .南京:上海科技技术出版社,2004,809-810.
[3] 段智变等.纳豆激酶粗制液对家兔溶血栓作用及其机制研究[J].营养学报 ,2003, 25(1):46-51.
[4] Markland F S. Snake venoms and the hemostatic system [J]. Toxicon, 1998,36(12): 1749-1800.
[5] Zhang Y,Wisner A,Xiong Y et al. A novel plasminogen activator from snake venom [J].Biol Chem,1995,270(12):10246-10255.
[6] Zhang Y,Wisner A,Maroun R C et al .Trimeresurus stejnegeri snake venom plasminogen activator[J].Biol Chem,1997,272(33):20531-20537.
[7] 张志强,张进华等.短尾蝮蛇毒纤溶酶原激活剂的分离纯化及活性测定[J].中国药理学通报,2007,23(12):1639-1644.
[8]Sambrook J and Russell DW.Molecular Cloning: A Laboarroy Manual,3rd[M].Cold Spring Habror Loratory Press,2001.
[9] Studier F W,Rosenberg A H,Dunn J J et al.Use of T7 RNA polymerase to direct expression of cloned genes[J].Methods Enzymol,1990,185:60-89.
[10] Zhang,-X,Studier,-F-W.Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme[J].J-Mol-Biol,1997 May 30,269(1):10-27.
[11] Lyakhov,-D-L,He,-B,Zhang,-X.Pausing and termination by bacteriophage T7 RNA polymerase[J].J-Mol-Biol,1998 Jul 10,280(2):201-13.
[12] Zhang,-X,Studier,-F-W.Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection[J].J-Mol-Biol,2004 Jul 16,340(4):707-30.
[13] MECK.pET系统操作手册,第十版.上海,2002:31.
[14] Anton P.J.Middelberg.Preparative protein refolding[J].Trend in Biotechnology,2002,20(10):437-443.
[15] 李校坤,袁辉.药物蛋白质分离纯化技术,第一版[M].北京:化学工业出版社,2005:239.
[16] 卢圣栋主编.现代分子生物学实验技术,第二版[M].北京:中国协和医科大学出版社,1999:377.
[17] 袁勤生,赵健主编.酶与酶工程,第一版[M].上海:华东理工大学出版社,2005:192.
[18] Zhao Qin-yi. Irreversible Thermodynamic Theory for Protein Floding and Protein thermodynamic Structures.Prog[J].Biochem Biophys,2001,28(3):429-435.
[19] Lilie H,Schwarz E, Rudolph R.Advances in refolding of proteins produced in E.coli.Curr.Opin[J].Biotechnol.,1998,9(5):497-501.
[20] Rudolph R,Lillie H.In vitro folding of inclusion body proteins[J]..FASEB J.,1996,10(1):29-56.
[1] 段 清 海 , 张 兆 山 , 李 淑 琴 等 .α- 银 环 蛇 毒 素 的 克 隆 和 表 达 [J]. 生 物 技 术 通讯,1998,9(4):247-252.
[2] Lathan LO, Staggers SL. Ancrod: the use of snake venom in the treatment of patients with heparin-induced thrombocytopenia and thrombosis undergoing coronary hypass grafting: Nursing management[J]. Heart Lung,1996,25(6):451.
[3] Leonard A S , Mark A O , Pierre J L , et al. Cloning and expression of mamba toxins[J]. Toxicon,1995,33(4):439-474.
[4] 万榕一,宋军,林旭等.中华眼镜蛇毒腺cDNA表达文库的建立和分析[J]. 第四军医大学学报,2004,25(13):1210-1211.
[5] Zhang S P,Zubay G,Goldman E.Low usage codons in Escheriehiaooli,yeast,fruit fly and primates[J].Gene,1991,105(1):61-72.
[6] DuanHQ,ZhangZS,LiSQ,et al.Cloning and expression of α-bungarotoxin gene[J].Letters in Biotechnology ,1998 ,9(4):247-252.
[7] 汪 芳 , 王 义 权 .a- 银 环 蛇 毒 素 同 工 毒 素 基 因 的 克 隆 和 表 达 [J]. 遗 传 学报.2005,32(7):684-688.
[8] Seo J H ,Bailev J E. Effects of recombinant plasmid content on growth properties and cloned gene product formation in Escherichia coli[J].Biotechnol Bioeng, 1985,27:1668-1674.
[9] 黄星,杨青,闫明等.在大肠杆菌中表达蝮蛇毒类凝血酶基因gloshedobin[J].过程工程学报,2004,4(1):23-26.
[10] 刘小龙,钟晓燕,吴祥甫等.尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征[J].生物化学与生物物理进展,2000,27(3):271.
[11] 查向东,张华远,肖亚中等.尖吻蝮蛇未知C 类凝集素蛋白cDNA 扩增、克隆与表达[J].生物化学与生物物理进展,2001,28(5):754.
[12] Qing Yang, Jianqiang Xu, Min L.High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions[J].Biotechnology Letters,2003,25: 607–610.
[13] 林鲁萍,汪芳,章晓波等.重组α-银环蛇毒素P22-A31的原核表达及功能分析[J].动物学报,2005,51(6):1102-1108.
[14] N G Qing,HU Xue-J un,XU Xiao-Ming.Expression , Purification and Partial Characterizationof Recombinant Gloshedobin , a Thrombin-like Enzymefrom The Venom of Gloydius shedaoensis[J].Prog. Biochem. Biophys,2002,29(3)390-393.
[15] 杨旭宇,杨青,包永明等.大连蛇岛蝮蛇类凝血酶在大肠杆菌的表达与纯化[J].中国工程生物杂志,2004,24(9):45-46.
[16] 向开军,余红秀,邹春森等.皖南尖吻蝮蛇毒I 型金属蛋白酶Acutolysin A 的表达、重折叠及其生物学活性[J].生物化学与生物物理学报,2002,34(5):675-679.
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