基因工程小肽——[Gly～（14）]-Humanin及人胰岛素突变体原核表达克隆的构建、表达及初步纯化
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摘要
目的
1．基因工程[Gly~(11)]-Humanin原核表达体系的构建、表达及初步纯化
2．基因工程人胰岛素突变体原核表达体系的构建、表达、初步纯化及测活
方法
采用基因工程手段，基因合成法分别获得[Gly~(11)]-Humanin(HNG)及人胰岛素突变体(M-insulin)的cDNA序列，构建了pBV220-HNG、pTXB1-HNG及pTXB1—M-insulin原核表达质粒，基因测序确定质粒的正确性。
三个质粒转化大肠杆菌工程菌株，诱导表达，Western-Blot鉴定目的蛋白。
采用超声加洗涤液破碎菌体；离心加冻融分离纯化融合蛋白，研究不同的超声次数和冻融对包涵体洗涤效果的影响。
放免法测定人胰岛素突变体融合蛋白的活性。
结果
1．克隆构建
成功构建了pBV220—HNG、pTXB1—HNG及pTXB1—M-insulin三个质粒，基因测序显示重组质粒的大小、方向、长度均正确。pBV220—HNG在大肠杆菌中未检测到表达，后两个克隆在大肠杆菌BL21(DE3)中获得高效表达，HNG及M-insulin融合蛋白表达量分别占全菌蛋白的40％及50％左右；经Western-Blot鉴定M-insulin融合蛋白可以与小鼠抗人胰岛素单克隆抗体(IgG)发生抗原抗体结合反应。
2．工程菌表达条件的研究
山西医科大学
2003届硕士学位论文
表达凿l株BLZI(DE3)一pTXBI一HNG及BL21(DE3)一PTXBI一
介insulin均在BL培养基，37oC、ZOOrpm摇床培养4小时45分时进入
对数生民中后期，此时加入终浓度0.Olmlnol/L的I尸TG开始诱导，再持
续培养5小时可达到最人表达量。
3.表达产物初步纯化
仁Gly”〕一Flumanin融合蛋白和人胰岛素突变体融合蛋白包涵体的洗涤
方案为:lj]体沉淀溶于含Zmol，/L尿索洗涤液，超声(200W、巧O次、3
秒/次、间隔3秒)，4℃、I000Orpm离心15min后沉淀用洗涤液充分重
悬，一2()℃冻存过夜，次日融化后4℃、10000rPm离心15min，取上清，
即得到初步纯化的融合蛋白，可去除土要的杂蛋白，融合蛋白溶解于上
清液中，纯度达到80%以_1:。
4.活性测定
采用放射免疫法，对人胰岛素突变体融合蛋白初步纯化产物，进行
放免计数，结果:每升诱导培养后的菌液表达产物中，具有的放射免疫
活性为0.5单位。
今士考个
二目卜乙
成功构建了pTXBI一HNG及pTXBI一M一insulin原核表达克隆，并获得
了高效表达，经过纯化得到纯度人于80%的融合蛋白，并对人胰岛素突
变体融合蛋白进行了初步活性测定。为用融合表达方式表达小分子量多
肤及规模化制备小分子蛋自奠定了初步的实验和理论基础。
Objective
1 . Construction of prokaryotic high expression vector of [Gly14]-Humanin (HNG) and its expression and purification.
2. Construction of prokaryotic high expression vector of human insulin mutant (M-insulin) and its expression, purification and bioassay.
Methods
By gene synthesis, we get the cDNAs of [Gly14]-Humanin and human insulin mutant, and then we construct three prokaryotic expression vectors: pBV220-HNG, pTXB1-HNG and pTXB1 - M-insulin by DNA polymerase chain reaction(PCR) and DMA recombinant technics. The expression plasmids are identified with PCR and DNA sequencing.
pBV220-HNG is transformed into E. coli DH5 a , BL21(DE3) and induced by increasing the temperature to 42℃. pTXB1-HNG and pTXB1-M-insulin are transformed into BL21(DE3) and induced by adding IPTG. We identified the expressed proteins by SDS-PAGE and Western-blot. After the expression form analysis, the insoluble recombinant proteins was purified by destraction and abstersion of inclusion bodies. To study the abstersion condition of the inclusion bodies, we adopted ultrasound crushing and freezing-melting methods. Radioimmunoassay was used to test the immune activity of M-insulin fusion protein.
Results
1. DNA sequencing demonstrated that we have constructed the expression plasmids pBV220-HNG, pTXB1-HNG and pTXB1-M-insulin successfully. Expression of pBV220-HNG can not be detected in E.coli. pTXB1-HNG and pTXB1-M-insulin are expressed in E.coli successfully. After SDS-PAGE and densitometric scan analysis, the results show that the expression level of HNG fusion protein is above 40% and M-insulin fusion protein above 50% of total bacterial proteins. Western-blot result demonstrated M-insulin fusion protein had specific reaction with mouse anti human insulin antibody(IgG).
2. The results show that the optimal conditions of the expression of BL21(DE3)- pTXBl -HNG and BL21(DE3)-pTXB1-M-insuIin are: BL Medium, 37℃, 200rpm for 4.75h, then 1PTG was added to the medium to 0.01mmol/L , induce for another 5h.
3. HNG fusion protein and M-insulin fusion protein are expressed as inclusion bodies in E.coli. After washing with reagent (50mmol/L pH8.0 Tris-HCl, 100mmol/L NaCl, 0.5mmol/L EDTA, 2mol/L carbamide, 0.2% Triton X-100, 0.2% DOC ) , ultrasound crushing (200 W, 150 times, 3 seconds per time, spacing
3 seconds) and freezing-melting methods, we got HNG fusion protein and M-insulin fusion protein with purity of above 80%.
4. Radioimmunoassay result shows that the radioimmune activity of of M-insulin fusion protein is 0.5 unit per litre bacterial liquid.
Conclusion
We have constructed the expression plasmids pTXBl-HNG and pTXBl -M-insulin successfully. After SDS-PAGE and densitometric scan analysis, the expression level of HNG fusion protein is above 40% and M-insulin fusion protein above 50%. Western-blot result demonstrated M-insulin fusion protein had specific reaction with mouse anti human insulin antibody, we got HNG fusion protein and M-insulin fusion protein with purity of above 80%. Radioimmunoassay
gets the radioimmune activity of of M-insulin fusion protein is 0.5 unit per litre bacterial liquid. Our study establish a stable base for further reseach of HNG and human insulin mutant and provide a theoretics gist for expression of low molecular weight proteins in prokaryotic cells.
1．基因工程[Gly~(11)]-Humanin原核表达体系的构建、表达及初步纯化
2．基因工程人胰岛素突变体原核表达体系的构建、表达、初步纯化及测活
方法
采用基因工程手段，基因合成法分别获得[Gly~(11)]-Humanin(HNG)及人胰岛素突变体(M-insulin)的cDNA序列，构建了pBV220-HNG、pTXB1-HNG及pTXB1—M-insulin原核表达质粒，基因测序确定质粒的正确性。
三个质粒转化大肠杆菌工程菌株，诱导表达，Western-Blot鉴定目的蛋白。
采用超声加洗涤液破碎菌体；离心加冻融分离纯化融合蛋白，研究不同的超声次数和冻融对包涵体洗涤效果的影响。
放免法测定人胰岛素突变体融合蛋白的活性。
结果
1．克隆构建
成功构建了pBV220—HNG、pTXB1—HNG及pTXB1—M-insulin三个质粒，基因测序显示重组质粒的大小、方向、长度均正确。pBV220—HNG在大肠杆菌中未检测到表达，后两个克隆在大肠杆菌BL21(DE3)中获得高效表达，HNG及M-insulin融合蛋白表达量分别占全菌蛋白的40％及50％左右；经Western-Blot鉴定M-insulin融合蛋白可以与小鼠抗人胰岛素单克隆抗体(IgG)发生抗原抗体结合反应。
2．工程菌表达条件的研究
山西医科大学
2003届硕士学位论文
表达凿l株BLZI(DE3)一pTXBI一HNG及BL21(DE3)一PTXBI一
介insulin均在BL培养基，37oC、ZOOrpm摇床培养4小时45分时进入
对数生民中后期，此时加入终浓度0.Olmlnol/L的I尸TG开始诱导，再持
续培养5小时可达到最人表达量。
3.表达产物初步纯化
仁Gly”〕一Flumanin融合蛋白和人胰岛素突变体融合蛋白包涵体的洗涤
方案为:lj]体沉淀溶于含Zmol，/L尿索洗涤液，超声(200W、巧O次、3
秒/次、间隔3秒)，4℃、I000Orpm离心15min后沉淀用洗涤液充分重
悬，一2()℃冻存过夜，次日融化后4℃、10000rPm离心15min，取上清，
即得到初步纯化的融合蛋白，可去除土要的杂蛋白，融合蛋白溶解于上
清液中，纯度达到80%以_1:。
4.活性测定
采用放射免疫法，对人胰岛素突变体融合蛋白初步纯化产物，进行
放免计数，结果:每升诱导培养后的菌液表达产物中，具有的放射免疫
活性为0.5单位。
今士考个
二目卜乙
成功构建了pTXBI一HNG及pTXBI一M一insulin原核表达克隆，并获得
了高效表达，经过纯化得到纯度人于80%的融合蛋白，并对人胰岛素突
变体融合蛋白进行了初步活性测定。为用融合表达方式表达小分子量多
肤及规模化制备小分子蛋自奠定了初步的实验和理论基础。
Objective
1 . Construction of prokaryotic high expression vector of [Gly14]-Humanin (HNG) and its expression and purification.
2. Construction of prokaryotic high expression vector of human insulin mutant (M-insulin) and its expression, purification and bioassay.
Methods
By gene synthesis, we get the cDNAs of [Gly14]-Humanin and human insulin mutant, and then we construct three prokaryotic expression vectors: pBV220-HNG, pTXB1-HNG and pTXB1 - M-insulin by DNA polymerase chain reaction(PCR) and DMA recombinant technics. The expression plasmids are identified with PCR and DNA sequencing.
pBV220-HNG is transformed into E. coli DH5 a , BL21(DE3) and induced by increasing the temperature to 42℃. pTXB1-HNG and pTXB1-M-insulin are transformed into BL21(DE3) and induced by adding IPTG. We identified the expressed proteins by SDS-PAGE and Western-blot. After the expression form analysis, the insoluble recombinant proteins was purified by destraction and abstersion of inclusion bodies. To study the abstersion condition of the inclusion bodies, we adopted ultrasound crushing and freezing-melting methods. Radioimmunoassay was used to test the immune activity of M-insulin fusion protein.
Results
1. DNA sequencing demonstrated that we have constructed the expression plasmids pBV220-HNG, pTXB1-HNG and pTXB1-M-insulin successfully. Expression of pBV220-HNG can not be detected in E.coli. pTXB1-HNG and pTXB1-M-insulin are expressed in E.coli successfully. After SDS-PAGE and densitometric scan analysis, the results show that the expression level of HNG fusion protein is above 40% and M-insulin fusion protein above 50% of total bacterial proteins. Western-blot result demonstrated M-insulin fusion protein had specific reaction with mouse anti human insulin antibody(IgG).
2. The results show that the optimal conditions of the expression of BL21(DE3)- pTXBl -HNG and BL21(DE3)-pTXB1-M-insuIin are: BL Medium, 37℃, 200rpm for 4.75h, then 1PTG was added to the medium to 0.01mmol/L , induce for another 5h.
3. HNG fusion protein and M-insulin fusion protein are expressed as inclusion bodies in E.coli. After washing with reagent (50mmol/L pH8.0 Tris-HCl, 100mmol/L NaCl, 0.5mmol/L EDTA, 2mol/L carbamide, 0.2% Triton X-100, 0.2% DOC ) , ultrasound crushing (200 W, 150 times, 3 seconds per time, spacing
3 seconds) and freezing-melting methods, we got HNG fusion protein and M-insulin fusion protein with purity of above 80%.
4. Radioimmunoassay result shows that the radioimmune activity of of M-insulin fusion protein is 0.5 unit per litre bacterial liquid.
Conclusion
We have constructed the expression plasmids pTXBl-HNG and pTXBl -M-insulin successfully. After SDS-PAGE and densitometric scan analysis, the expression level of HNG fusion protein is above 40% and M-insulin fusion protein above 50%. Western-blot result demonstrated M-insulin fusion protein had specific reaction with mouse anti human insulin antibody, we got HNG fusion protein and M-insulin fusion protein with purity of above 80%. Radioimmunoassay
gets the radioimmune activity of of M-insulin fusion protein is 0.5 unit per litre bacterial liquid. Our study establish a stable base for further reseach of HNG and human insulin mutant and provide a theoretics gist for expression of low molecular weight proteins in prokaryotic cells.
引文
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