人表皮生长因子（hEGF）的原核表达
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摘要
1960年Cohen 首次从小鼠颌下腺中发现了表皮生长因子,之后的40几年又陆续在人体和哺乳动物中发现了与表皮生长因子结构与性质相近的生长因子,转化生长因子α（transforming growth factorα，TGFα）、牛痘病毒生长因子（vaccinia growth factor，VGF）、休普氏纤维瘤生长因子（shope fibroma growth factor,SFGF）、粘液瘤生长因子（myxoma growth factor，MGF）、两性调节蛋白（ampiregulin）、双向调节素(arnphireguli，AR)、betacellulin(BTC) 、表皮调节素（epiregulin，EPl) 、结合肝素的EGF样生长因子(heparin-binging EGF-like growth factor，HB-EGF)、痘苗病毒生长因子(vaccinia virus growth factor, VVGF)等，大小都在50～80个氨基酸之间，在一级结构上有20%～90%的同源性,它们都含有六个半胱氨酸,形成3对二硫键和表皮细胞生长因子受体结合且具有相似的生物活性,构成了表皮生长因子家族。
人表皮生长因子是由53个氨基酸组成的小分子量单链多肽类细胞因子，它对酸和热稳定。它的显著生物学效应是能够促进内外表皮细胞的生长，在临床上可用于治疗烧伤、创伤、角膜的移植、胃酸、十二指肠溃疡等；还可用于化妆品。由于其具有特殊的生物学效应使其有很大的潜在经济效益。
本实验运用RT-PCR技术从人胎盘组织中获得了人表皮生长因子的cDNA，将其分别克隆至PGEX-4T-1和PGEX-6P-1融合表达载体中，转花入BL21-CodonPlus?–RIL; BL21-CodonPlus?–RP表达宿主菌，该宿主菌带有稀有tRNA额外基因拷贝,解决了大肠杆菌密码子偏爱性问题,不需要附加程序就可以编码稀有密码子的重组基因。表达产物进行光密度扫描，目的蛋白占总菌体蛋白的30%左右。表达产物主要以包涵体的形式表达。收集并溶解包涵体后,在还原型谷胱甘肽存在下进行复性，之后过Glutathione SepharoseTM 4B柱，分离纯化得到有生物活性的重组人表皮生长因子。以兔抗人EGF单克隆抗体为一抗，进行Western blotting鉴定，证明分离纯化得到的融合蛋白含有目的蛋白。表皮生长因子为无血清细胞培养培养基的成分，采用添加试验证明分离纯化得到的融合蛋白具有良好的hEGF生物学活性。
Since Cohen firstly discovered epidermal growth factor(EGF) from a mouse in 1960, many growth factors have identified one after another mainly from human and mammilla after 40 years such as transforming growth factorα(TGFα)、vaccinia growth factor(VGF)、Shope fibroma growth factor(SFGF)、myxoma growth factor(MGF)、ampiregulin、arnphireguli(AR)、betacellulin(BTC) 、epiregulin(EPl) 、heparin-binging EGF-like growth factor(HB-EGF)、vaccinia virus growth factor(VVGF) ect. They have 50-80 amino acids and homology about 20%~90% in primary structure. In their molecules there have conservatively six cysteines, which form three disulfide bonds, make their bind to EGF receptor inducing similar biological activity. These molecules are defined as an EGF family.
Human epidermal growth factor is a small single-chain polypeptide of 53 amino acids. It has heat-stable and acid-stable. It's main biology effects are to stimulate the growth and proliferation of epidermal and epithelial cell both in vivo and in vitro. It is often applied to treat burns, scald, cornea transplant, gastric acid, duodenal ulcered and also use as cosmetic. It has big latency economic value because it has special and potential biological effects.
Human EGF gene was successfully amplified from human placenta with RT-PCR method, then cloned it into expression vector: pGEX-4T-1 and pGEX-6P-1 and transformed them into BL21-CodonPlus?–RIL and BL21-CodonPlus?–RP, which don't need added program to solve the coding of rare codons. The fusion hEGF was about 30% of total protein measured with SDS-PAGE. The expression product is mainly inclusion bodies. Collecting and dissolving of bodies was renaturation with reduced Glu. Separating and purifing the inclusion bodies and obtained fusion hEGF has biological activity then passing through Glutathione SepharoseTM 4B. Western blotting test identified that the separated and purificated protein is objective protein hEGF. Because hEGF is a kind of content of cell culture medium without blood serum, using addition the GST-hEGF fusion protein to cell culture medium of EP, it has been shown the fusion proteins have good biological activity.
人表皮生长因子是由53个氨基酸组成的小分子量单链多肽类细胞因子，它对酸和热稳定。它的显著生物学效应是能够促进内外表皮细胞的生长，在临床上可用于治疗烧伤、创伤、角膜的移植、胃酸、十二指肠溃疡等；还可用于化妆品。由于其具有特殊的生物学效应使其有很大的潜在经济效益。
本实验运用RT-PCR技术从人胎盘组织中获得了人表皮生长因子的cDNA，将其分别克隆至PGEX-4T-1和PGEX-6P-1融合表达载体中，转花入BL21-CodonPlus?–RIL; BL21-CodonPlus?–RP表达宿主菌，该宿主菌带有稀有tRNA额外基因拷贝,解决了大肠杆菌密码子偏爱性问题,不需要附加程序就可以编码稀有密码子的重组基因。表达产物进行光密度扫描，目的蛋白占总菌体蛋白的30%左右。表达产物主要以包涵体的形式表达。收集并溶解包涵体后,在还原型谷胱甘肽存在下进行复性，之后过Glutathione SepharoseTM 4B柱，分离纯化得到有生物活性的重组人表皮生长因子。以兔抗人EGF单克隆抗体为一抗，进行Western blotting鉴定，证明分离纯化得到的融合蛋白含有目的蛋白。表皮生长因子为无血清细胞培养培养基的成分，采用添加试验证明分离纯化得到的融合蛋白具有良好的hEGF生物学活性。
Since Cohen firstly discovered epidermal growth factor(EGF) from a mouse in 1960, many growth factors have identified one after another mainly from human and mammilla after 40 years such as transforming growth factorα(TGFα)、vaccinia growth factor(VGF)、Shope fibroma growth factor(SFGF)、myxoma growth factor(MGF)、ampiregulin、arnphireguli(AR)、betacellulin(BTC) 、epiregulin(EPl) 、heparin-binging EGF-like growth factor(HB-EGF)、vaccinia virus growth factor(VVGF) ect. They have 50-80 amino acids and homology about 20%~90% in primary structure. In their molecules there have conservatively six cysteines, which form three disulfide bonds, make their bind to EGF receptor inducing similar biological activity. These molecules are defined as an EGF family.
Human epidermal growth factor is a small single-chain polypeptide of 53 amino acids. It has heat-stable and acid-stable. It's main biology effects are to stimulate the growth and proliferation of epidermal and epithelial cell both in vivo and in vitro. It is often applied to treat burns, scald, cornea transplant, gastric acid, duodenal ulcered and also use as cosmetic. It has big latency economic value because it has special and potential biological effects.
Human EGF gene was successfully amplified from human placenta with RT-PCR method, then cloned it into expression vector: pGEX-4T-1 and pGEX-6P-1 and transformed them into BL21-CodonPlus?–RIL and BL21-CodonPlus?–RP, which don't need added program to solve the coding of rare codons. The fusion hEGF was about 30% of total protein measured with SDS-PAGE. The expression product is mainly inclusion bodies. Collecting and dissolving of bodies was renaturation with reduced Glu. Separating and purifing the inclusion bodies and obtained fusion hEGF has biological activity then passing through Glutathione SepharoseTM 4B. Western blotting test identified that the separated and purificated protein is objective protein hEGF. Because hEGF is a kind of content of cell culture medium without blood serum, using addition the GST-hEGF fusion protein to cell culture medium of EP, it has been shown the fusion proteins have good biological activity.
引文
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