hGH，HSA-双精氨酸C肽人胰岛素原的表达、加工及重组胰岛素的降糖活性分析
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摘要
胰岛素是1型和中重度2型糖尿病患者临床治疗中必不可少的药物,随着世界糖尿病患者的急剧增加,市场对胰岛素的需求量也迅速攀升,由此开发成本低、活性高和副作用小的人重组胰岛素原料药已成为国际医药业研究和竞争热点。
由于胰岛素分子量较小,其表达在转录、翻译水平不稳定,易被降解,表达量和复性率均较低,导致重组胰岛素原料药成本较高。由于胰岛素药物临床使用多为终生用药,给糖尿病患者带来大的经济负担。目前国内外重组胰岛素原料药生产厂家主要通过优化发酵工艺来提高表达量,降低重组人胰岛素成本。
本研究通过基因工程手段对人胰岛素原基因进行改造,并与前导序列重组,构建一个新的hGH/HSA-双精氨酸C肽人胰岛素原核表达载体,并在大肠杆菌中获得稳定和较高的表达,表达产物经加工纯化后获得有降糖活性的重组胰岛素,初步建立起一整套新的生产重组人胰岛素的方法,除为该重组胰岛素的产业化生产提供技术支撑外,还为进一步对该重组胰岛素表达载体的前导序列的功能分析,提高表达量打下基础,具有重要的实际意义,结果概括如下：
人胰岛素原N端分别融合上人生长素N端的一段序列(hGH)和人血白蛋白N端的一段序列(HSA)来充当前导肽,将C肽设计为两个精氨酸。分10段合成长链寡核苷酸链,利用重叠延伸PCR技术(SOE PCR)扩增hGH-双精氨酸C肽人胰岛素原基因片段；同时以实验室保存的HSA-PUC19质粒为模板扩增HSA-双精氨酸C肽人胰岛素原基因。将上述两个基因分别与表达载体PET-30a连接,转化Ecoli BL21(DE3)菌株。
表达菌株用IPTG来诱导表达,hGH-双精氨酸C肽人胰岛素原蛋白在大肠杆菌BL21(DE3)中得到了表达,表达产物以不溶性包涵体形式纯在,约占大肠杆菌总蛋白的30%；而对照HSA-双精氨酸C肽人胰岛素原蛋白则没有观察到特异性表达条带。表达的融合蛋白采用Ni-NTA亲和层析纯化,纯化后的蛋白经复性,冻干等步骤后用胰蛋白酶,羧肽酶B双酶切再经DEAE Sepharose Fast Flow离子交换层析得到了单组分胰岛素。制备所得的胰岛素经SDS-PAGE; Western blot进行性质鉴定。建立降血糖动物模型,皮下注射小鼠表明制备的人重组胰岛素和标准品具有相当的降血糖活性。
结论：本研究通过对人胰岛素原基因进行改造,设计和组建了hGH/HSA-双精氨酸C肽人胰岛素新组合,该组合在大肠杆菌中获得稳定和较高的表达,并且初步建立了一套相应的人重组胰岛素加工纯化方法,为重组胰岛素的产业化生产提供基础,同时也为胰岛素前导序列和胰岛素突变体的研究提供技术平台。
Insulin is a kind of essential medicine in the clinical treatment of patients with severe type 1 and type 2 diabetes.As the dramatic increase in diabetes, the insulin market demand is increasing rapidly. Thus,the development of low cost, high activity and low side effect recombinant human insulin raw materials has become a hot point in the world.
At present, the way to enhance the expression of recombinant human insulin is mainly by optimizing the fermentation technology. However, it existed neight expression limitation nor the renaturation level is relatively low. As the molecular weight of insulin is relatively small, individual expression at the transcription、translation level is unstable which is vulnerable to degradate.
In this study,we used genetic engineering technique to achieve human proinsulin (containing only two amino acids for the C peptide) fusion protein with the leader sequence at N-terminal side, improving the expression of genetic recombinant insulin in E. coli by optimization the conditions at all aspects,Improving the stability and expression level of inclusion body protein which lay the basis for the development of low cost and high activity of genetically engineered human insulin raw materials.The results are summarized as follows:
Two precursor sequences choosed as a precursor peptide in this experiment were available:hGH,N-terminal section of human growth hormone and HSA,N-terminal section of human serum albumin, meanwhile,the C peptide is designed for two arginines,synthesizing 10-long oligonucleotide chains to amplify hGH-double Arg C peptide human proinsulin gene by overlap extension PCR technique (SOE PCR); at the same time,taking HSA-PUC19 plasmid kept in our laboratory as template to get HSA-double Arg C peptide human proinsulin gene. Then the two genes were inserted into PET-30a expression vector before transformed into Ecoli BL21 (DE3) strain.
The high efficiency expression vector expressed in E.coli BL21(DE3) was successfully induced by IPTG in the form of insoluble inclusion bodies, accounting for about 30% of total proteins in E.coli.However,we do not observe the specific band expressed by HSA-double Arg C peptide human proinsulin gene.Then the expressed fusion protein was purified by Ni-NTA Affinity chromatography,After Renaturation, Freeze-dried procedures.The purified protein was digested by trypsinase and carboxypeptidase B,at last the digestion products were under DEAE Sepharose Fast Flow purification procedure in order to get the purified human insulin. After these processes we got a single band protein detected by SDS-PAGE analysis. Western Blot
Analysius showed that the recombiant protein possessed Insulin antigenicity, the subcutaneous injection of mice showed the insulin have a significant ability to lower blood glucose.
Conclusion:The precursor sequence combined with c peptide was highly expressed in E.coli succefully,establishing an efficient method for recombinant human insulin production,which also lay the foundation for precursor sequence mutation in order to promote the folding efficiency of recombinant insulin based on the precursor peptide.
由于胰岛素分子量较小,其表达在转录、翻译水平不稳定,易被降解,表达量和复性率均较低,导致重组胰岛素原料药成本较高。由于胰岛素药物临床使用多为终生用药,给糖尿病患者带来大的经济负担。目前国内外重组胰岛素原料药生产厂家主要通过优化发酵工艺来提高表达量,降低重组人胰岛素成本。
本研究通过基因工程手段对人胰岛素原基因进行改造,并与前导序列重组,构建一个新的hGH/HSA-双精氨酸C肽人胰岛素原核表达载体,并在大肠杆菌中获得稳定和较高的表达,表达产物经加工纯化后获得有降糖活性的重组胰岛素,初步建立起一整套新的生产重组人胰岛素的方法,除为该重组胰岛素的产业化生产提供技术支撑外,还为进一步对该重组胰岛素表达载体的前导序列的功能分析,提高表达量打下基础,具有重要的实际意义,结果概括如下：
人胰岛素原N端分别融合上人生长素N端的一段序列(hGH)和人血白蛋白N端的一段序列(HSA)来充当前导肽,将C肽设计为两个精氨酸。分10段合成长链寡核苷酸链,利用重叠延伸PCR技术(SOE PCR)扩增hGH-双精氨酸C肽人胰岛素原基因片段；同时以实验室保存的HSA-PUC19质粒为模板扩增HSA-双精氨酸C肽人胰岛素原基因。将上述两个基因分别与表达载体PET-30a连接,转化Ecoli BL21(DE3)菌株。
表达菌株用IPTG来诱导表达,hGH-双精氨酸C肽人胰岛素原蛋白在大肠杆菌BL21(DE3)中得到了表达,表达产物以不溶性包涵体形式纯在,约占大肠杆菌总蛋白的30%；而对照HSA-双精氨酸C肽人胰岛素原蛋白则没有观察到特异性表达条带。表达的融合蛋白采用Ni-NTA亲和层析纯化,纯化后的蛋白经复性,冻干等步骤后用胰蛋白酶,羧肽酶B双酶切再经DEAE Sepharose Fast Flow离子交换层析得到了单组分胰岛素。制备所得的胰岛素经SDS-PAGE; Western blot进行性质鉴定。建立降血糖动物模型,皮下注射小鼠表明制备的人重组胰岛素和标准品具有相当的降血糖活性。
结论：本研究通过对人胰岛素原基因进行改造,设计和组建了hGH/HSA-双精氨酸C肽人胰岛素新组合,该组合在大肠杆菌中获得稳定和较高的表达,并且初步建立了一套相应的人重组胰岛素加工纯化方法,为重组胰岛素的产业化生产提供基础,同时也为胰岛素前导序列和胰岛素突变体的研究提供技术平台。
Insulin is a kind of essential medicine in the clinical treatment of patients with severe type 1 and type 2 diabetes.As the dramatic increase in diabetes, the insulin market demand is increasing rapidly. Thus,the development of low cost, high activity and low side effect recombinant human insulin raw materials has become a hot point in the world.
At present, the way to enhance the expression of recombinant human insulin is mainly by optimizing the fermentation technology. However, it existed neight expression limitation nor the renaturation level is relatively low. As the molecular weight of insulin is relatively small, individual expression at the transcription、translation level is unstable which is vulnerable to degradate.
In this study,we used genetic engineering technique to achieve human proinsulin (containing only two amino acids for the C peptide) fusion protein with the leader sequence at N-terminal side, improving the expression of genetic recombinant insulin in E. coli by optimization the conditions at all aspects,Improving the stability and expression level of inclusion body protein which lay the basis for the development of low cost and high activity of genetically engineered human insulin raw materials.The results are summarized as follows:
Two precursor sequences choosed as a precursor peptide in this experiment were available:hGH,N-terminal section of human growth hormone and HSA,N-terminal section of human serum albumin, meanwhile,the C peptide is designed for two arginines,synthesizing 10-long oligonucleotide chains to amplify hGH-double Arg C peptide human proinsulin gene by overlap extension PCR technique (SOE PCR); at the same time,taking HSA-PUC19 plasmid kept in our laboratory as template to get HSA-double Arg C peptide human proinsulin gene. Then the two genes were inserted into PET-30a expression vector before transformed into Ecoli BL21 (DE3) strain.
The high efficiency expression vector expressed in E.coli BL21(DE3) was successfully induced by IPTG in the form of insoluble inclusion bodies, accounting for about 30% of total proteins in E.coli.However,we do not observe the specific band expressed by HSA-double Arg C peptide human proinsulin gene.Then the expressed fusion protein was purified by Ni-NTA Affinity chromatography,After Renaturation, Freeze-dried procedures.The purified protein was digested by trypsinase and carboxypeptidase B,at last the digestion products were under DEAE Sepharose Fast Flow purification procedure in order to get the purified human insulin. After these processes we got a single band protein detected by SDS-PAGE analysis. Western Blot
Analysius showed that the recombiant protein possessed Insulin antigenicity, the subcutaneous injection of mice showed the insulin have a significant ability to lower blood glucose.
Conclusion:The precursor sequence combined with c peptide was highly expressed in E.coli succefully,establishing an efficient method for recombinant human insulin production,which also lay the foundation for precursor sequence mutation in order to promote the folding efficiency of recombinant insulin based on the precursor peptide.
引文
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