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Construction,Expression of κ-bungarotoxin in ***

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Construction,Expression of-bungarotoxin in periplasm of ***

第十四届生物毒素毒理学术大会暨第一届生物毒素——从生存适应到转化医学专题学术会议

作者： Lin Zhao Bo Lin Yun Zhang Sheng-Zhou Zhang Wen-Hui Lee Key Laboratory of Animal Models and Human Disease Mechanisms of The Chinese Academy of Sciences/Key Laboratory of Bioactive peptides of Yunnan province Kunming Institute of Zoologythe Chinese Academy of Sciences College of Life Sciences Anhui Normal University

Bungarus multicinctus is one of the most deadly snakes in the *** lethality of biten by *** was ranked the first of snake-bite in *** lethal components of the *** venom are attributed to its neurotoxins,including α-bungarotoxin,β-bungarotoxin,γ-bungarotoxin and κ-***,litter is known on κ-bungarotoxin because of its lower quantity in crude venom and hard to *** better understand the characterics of κ-bungarotoxin,we inserted the κ-bungarotoxin cDNA into expression vector of pmal-p2 x and the MBp-κ-bungarotoxin recombinant protein was successfully generated in the periplasmic of Escherichia coli(TB1).To induce the recombinant protein expression,the IpTG was supplied to0.3 mM when the absorbance at 600 nm of the cultures reached about 0.6,and the culture media were grown for another 18 h in shaking flasks at 18℃.According to the meathod of periplasmic extract,the recombinant protein was acquired in a soluble form and at least accounts for 90% of total protein *** we can get a high purity recombinant protein MBp-κ-bungarotoxin by one step affinity *** method is simple and economical and might provide enough κ-bungarotoxin for further investigation.

关键词： B.multicinctus κ-bungarotoxin pmal-p2x prokaryotic Vector

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The recombinant expression of gamma-bungarotoxin

第十四届生物毒素毒理学术大会暨第一届生物毒素——从生存适应到转化医学专题学术会议

作者： Bo Lin Yun Zhang xiao-Bing Wu Wen-Hui Lee Key Laboratory of Animal Models and Human Disease Mechanisms of The Chinese Academy of Sciences/Key Laboratory of Bioactive peptides of Yunnan province Kunming Institute of Zoologythe Chinese Academy of Sciences College of Life Sciences Anhui Normal University

Bungarus multicinctus is one of the most deadly snakes in the *** lethality of biten by *** was ranked the first of snake-bite in *** lethal components of the *** venom are attributed to its neurotoxins,including a-bungarotoxin,β-bungarotoxin,ybungarotoxin and κ-*** researches were mainly focused on α-bungarotoxin andβ-bungarotoxin,the study on y-bungarotoxin was rare because of it’s little content in crude *** we inserted the y-bungarotoxin cDNA into expression vector of pmal-p2 x and the MBp-γ-bungarotoxin recombinant protein was generated in the periplasmic of Escherichia coli(TB1).To induce the recombinant protein expression,the IpTG was supplied to 0.3 mM when the absorbance at600 nm of the cultures reached 0.6,and the culture media were grown for another 18 h in shaking flasks at 18℃.According to the meathod of periplasmic extract,the recombinant protein was acquired in a soluble form and at least accounts for 90% of total periplasmic *** fusion protein MBp-γ-bungarotoxin could be purified at high purity by one step affinity chromatography and used as antigen to produce specific anti-y-bungarotoxin antisera for further *** method is simple and economical,it might provide enough y-bungarotoxin for further research.

关键词： B.multicinctus γ-bungarotoxin pmal-p2x prokaryotic Vector

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The recombinant expression of gamma-bungarotoxin

凋亡抑制因子Survivin在大肠杆菌TB1中的表达纯化及鉴定

中国生物化学与分子生物学会2019年全国学术会议暨学会成立四十周年

作者： Bo Lin Lin Zhao xue-Shong Wei xiao-Bing Wu Wen-Hui Lee Key Laboratory of Animal Models and Human Disease Mechanisms of The Chinese Academy of Sciences/Key Laboratory of Bioactive peptides of Yunnan province Kunming Institute of Zoology the Chinese Academy of Sciences College of Life Sciences Anhui Normal University

Bungarus multicinctus is one of the most deadly snakes in the world. The lethality of biten by B. multicinctus was ranked first in China. The main components responsible for the death of the victims are attributed to neurotoxins contained in B. multicinctus venoms, including alpha-bungarotoxin, γ-bungarotoxin,beta-bungarotoxin and kappa-bungarotoxin. previous researches were mainly focused on α-bungarotoxin and γ-bungarotoxin, while the study on γ-bungarotoxin was rare because of it’s lower content in crude venom. To obtain enough γ-bungarotoxin for investigation, we inserted the γ-bungarotoxin cDNA into expression vector of pmal-p2 x, and the MBp-γ-bungarotoxin recombinant protein was successively generated in the periplasm of Escherichia coli(TB1) after IpTG inducing and 18 h culture at 18°C. According to the method of periplasmic extract provided by NEB, the recombinant protein was collected in soluble form and at least accounts for 90% of total periplasmic proteins. The recombinant protein MBp-γ-bungarotoxin was isolated by one step affinity chromatography on an MBp-affinity column. The recombinant protein collected from affinity chromatography can be further fractionated by using a source 15 S cationic ion-exchange chromatography column. Finally, an electrophoretic purity recombinant MBp-γ-bungarotoxin was prepared for the next step of factor xa enzyme digestion to release γ-bungarotoxin. This method is simple and economical, it might provide enough γ-bungarotoxin to investigate the biological activities of γ-bungarotoxin.

关键词： Bungarus multicinctus γ-bungarotoxin pmal-p2x prokaryotic vector

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中国生物化学与分子生物学报 2007年第3期23卷 200-204页

作者：刘晔张青云山西医科大学基础医学院微生物与免疫教研室太原030001 北京大学临床肿瘤学院北京肿瘤医院检验科北京100036

本文旨在克隆凋亡抑制因子Survivin基因,并在大肠杆菌中进行可溶性表达与初步纯化.采用RT-pCR法,扩增人凋亡抑制因子survivincDNA,并克隆入原核表达载体pmal-p2x中,转化TB1大肠杆菌感受态细胞.经0.3mmol/LIpTG诱导2h后,收集菌体蛋白,进... 详细信息

本文旨在克隆凋亡抑制因子Survivin基因,并在大肠杆菌中进行可溶性表达与初步纯化.采用RT-pCR法,扩增人凋亡抑制因子survivincDNA,并克隆入原核表达载体pmal-p2x中,转化TB1大肠杆菌感受态细胞.经0.3mmol/LIpTG诱导2h后,收集菌体蛋白,进行SDS-pAGE、ELISA及Western印迹鉴定.实验获得凋亡抑制因子survivin编码区cDNA,以构建的原核表达载体pmal-p2x-survivin转化菌株后,可表达凋亡抑制因子survivin和麦芽糖结合蛋白(MBp)的融合蛋白,相对分子质量(Mr)为58000.并成功利用Factorxa将融合蛋白裂解开.ELISA和Western印迹表明,融合蛋白能与抗凋亡抑制因子survivin单克隆抗体特异性结合.获得的凋亡抑制因子survivin全长cDNA可在大肠杆菌TB1中以MBp-survivin融合蛋白的形式表达,成功地将survivin目的蛋白和MBp蛋白分离,为深入研究survivin的结构和功能奠定了基础.

关键词：凋亡抑制因子 survivin 麦芽糖结合蛋白 pmal-p2x 可溶性表达纯化

HBV preS2-MBp融合蛋白在大肠杆菌不同部位的表达

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生物技术 2006年第3期16卷 17-19页

作者：刘照惠邵丽军金立杰李利长春生物制品研究所疫苗一室吉林长春130062

目的:分析比较HBV preS2蛋白在大肠杆菌细胞周质和细胞质中的融合表达情况。方法:构建大肠杆菌细胞周质中融合表达HBV preS2蛋白的载体pmal-p2x/S2和细胞质中融合表达载体pmal-C2x/S2,IpTG诱导表达,产物经SDS-pAGE和Western Blot检测,分... 详细信息

目的:分析比较HBV preS2蛋白在大肠杆菌细胞周质和细胞质中的融合表达情况。方法:构建大肠杆菌细胞周质中融合表达HBV preS2蛋白的载体pmal-p2x/S2和细胞质中融合表达载体pmal-C2x/S2,IpTG诱导表达,产物经SDS-pAGE和Western Blot检测,分析HBV preS2-MBp融合蛋白在菌体不同部位的表达情况及表达产物的存在形式。结果:细胞周质中表达的融合蛋白有一部分被降解了,而细胞质中表达出了完整的融合蛋白。细胞质中表达的融合蛋白以包涵体和可溶性蛋白两种形式存在。结论:HBV preS2-MBp融合蛋白适合在大肠杆菌细胞质中表达。

关键词： HBV preS2-MBp融合蛋白原核表达 pmal-p2x pmal-C2x

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MBp-hmcm6融合蛋白的构建与表达

实用肿瘤学杂志 2005年第5期19卷 321-324页

作者：王蕾余波光吴文言广州中山大学有害生物控制与资源利用国家重点实验室广州510278 广州中山大学生命科学学院

目的构建含微小染色体维持蛋白MBp-hmcm6的原核表达重组质粒,并进行可溶性表达与初步纯化。方法以常规克隆方法将目的片段连接至pmal-p2x载体,获得的阳性克隆转化大肠杆菌Rosetta(DE3)进行表达。结果经Western blot鉴定,获得了分子量大... 详细信息

目的构建含微小染色体维持蛋白MBp-hmcm6的原核表达重组质粒,并进行可溶性表达与初步纯化。方法以常规克隆方法将目的片段连接至pmal-p2x载体,获得的阳性克隆转化大肠杆菌Rosetta(DE3)进行表达。结果经Western blot鉴定,获得了分子量大小为139 KD的MBp-hMCM6p蛋白。结论成功构建了pmal-p2x-hmcm6重组质粒和工程菌,实现了重组蛋白可溶性表达,有助于进一步研究MCM蛋白的结构与功能。

关键词：微小染色体维持蛋白 pmal-p2x 可溶性表达融合蛋白 Western 可溶性表达重组质粒克隆转化原核表达初步纯化大肠杆菌 blot