v-Src的基因重组、表达及活性分析
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摘要
蛋白酪氨酸激酶(PTK)是具有酪氨酸激酶活性的一类蛋白质,能催化ATP上的β-磷酸基团转移到其它重要蛋白质的酪氨酸残基上,使其发生磷酸化,并介导细胞生长、分裂和信号传导等一系列生理生化反应。Src家族激酶(SFKs)包括Src及其他八种蛋白,隶属于蛋白酪氨酸激酶。Src蛋白主要以两种形式存在：病毒癌基因表达蛋白v-Src和细胞原癌基因表达蛋白c-Src。c-Src蛋白在细胞内参与信号传导,与细胞生长、分化、死亡等过程密切相关。目前,国内外对Src家族的研究大都集中在c-Src蛋白上,然而c-Src蛋白在体外活性低,导致对它的结构和活性无法深入研究。与c-Src蛋白同源的病毒v-Src蛋白,两者的结构与功能非常相似,且后者在体外活性基本不变。因此,本论文拟以大肠杆菌为宿主,大量表达重组v-Src蛋白,并以此建立体外抑制Src活性药物筛选体系。
首先,利用实验室保存携带v-src基因的pFastbac Htb质粒PCR扩增目的基因,经克隆载体pUCm-T连接至表达载体pGEX-KT构建表达质粒pGEX-KT/v-src,经PCR、双酶切、测序鉴定其序列正确,转化大肠杆菌BL21(DE3).其次,构建好的工程菌株经正交实验优化诱导条件,确定最佳的表达条件为37℃、1 mM IPTG和5h诱导时间。在该诱导条件下v-Src蛋白以包涵体形式大量表达,包涵体经洗涤、溶解、GST亲和层析并经凝血酶酶切后,再经过尿素连续梯度透析复性后,获得有一定活性的v-Src蛋白。再次,用重组的v-Src蛋白来筛选中药半枝莲提取物(ESB),发现乙醇提取物对重组v-Src活性具有抑制作用,且该抑制作用具有剂量依赖性。细胞实验表明该成分对HepG2肝癌细胞活性同样具有抑制作用。
本研究利用大肠杆菌表达系统获得了具有活性的v-Src蛋白,并利用其筛选出中药半枝莲的抑制v-Src活性、抗肿瘤成分,这为进一步研究Src蛋白的结构和功能以及构建体外抗肿瘤药物筛选系统奠定基础。
Protein tyrosine kinase (PTK) which has tyrosine kinase activity, can transfer theβ-phosphate group of ATP to other important protein tyrosine residues, make them phosphorylated,and also mediate cell growth, cell division, signaling and a series of physiological and biochemical reactions and so on. Src family kinases (SFKs) include eight proteins besides Src, belonging to PTK. Src protein mainly exists in two forms:protein expressed by oncogene of Rous sarcoma virus and protein expressed by proto-oncogene of eukaryocytes. c-Src protein involves in intracellular signal transduction, closely relevant to cell growth, differentiation, and apoptosis and so on. At present, the domestic and foreign research of Src family are focused on the c-Src protein, whose structure, physical and chemical properties cannot be further studied. c-Src protein's homologue, the v-Src protein, both the structure and function of which are quite similar to the former, seldom shows any change in the activity of in vitro in contrast to in vivo, while c-Src protein in vitro shows little activity. Therefore, the physical and chemical properties of v-Src protein can be easily studied through constructing restructured v-src gene and expressing the v-Src protein in E. coli in great quantities. Therefore, the physical and chemical properties of v-Src protein can be well studied by expressing the v-Src protein in E.coli in great quantities, and a model system for screening of Src tyrosine kinase inhibitors is estabilished.
First, through gene engineering technology,v-src gene was cloned into the middle cloning vector pUCm-T and then into the expression vector pGEX-KT, then the recombined expression plasmid pGEX-KT/v-src was completed. PCR, double digests and sequencing identify the successfully constructed expression plasmid. Secondly, by orthogonal experiment optimization, the optical condition for expressing was determined:37℃,1 mM IPTG and 5 hours induced expressing. The v-Src protein was bacterially expressed and collected in the form of inclusion-body under that condition, and then, was washed、dissolved、affinity chromatographied、digested by thrombin and renatured by continuous gradient of urea. Again, screening the extract of Scutellaria barba(ESB), the ethanol extract is found to have inhibitory effect on v-Src protein, and the inhibition is relevant to dose. Cell experiments show that the composent also has inhibition over mitochondria activity.
This research has obtained active v-Src protein by E.coli expression system, and selected the anti-tumor composent of ESB. It can provide a basis for further study on the structure and function of Src protein, and lay the foundation of the anti-tumor drug screening system in vitro.
首先,利用实验室保存携带v-src基因的pFastbac Htb质粒PCR扩增目的基因,经克隆载体pUCm-T连接至表达载体pGEX-KT构建表达质粒pGEX-KT/v-src,经PCR、双酶切、测序鉴定其序列正确,转化大肠杆菌BL21(DE3).其次,构建好的工程菌株经正交实验优化诱导条件,确定最佳的表达条件为37℃、1 mM IPTG和5h诱导时间。在该诱导条件下v-Src蛋白以包涵体形式大量表达,包涵体经洗涤、溶解、GST亲和层析并经凝血酶酶切后,再经过尿素连续梯度透析复性后,获得有一定活性的v-Src蛋白。再次,用重组的v-Src蛋白来筛选中药半枝莲提取物(ESB),发现乙醇提取物对重组v-Src活性具有抑制作用,且该抑制作用具有剂量依赖性。细胞实验表明该成分对HepG2肝癌细胞活性同样具有抑制作用。
本研究利用大肠杆菌表达系统获得了具有活性的v-Src蛋白,并利用其筛选出中药半枝莲的抑制v-Src活性、抗肿瘤成分,这为进一步研究Src蛋白的结构和功能以及构建体外抗肿瘤药物筛选系统奠定基础。
Protein tyrosine kinase (PTK) which has tyrosine kinase activity, can transfer theβ-phosphate group of ATP to other important protein tyrosine residues, make them phosphorylated,and also mediate cell growth, cell division, signaling and a series of physiological and biochemical reactions and so on. Src family kinases (SFKs) include eight proteins besides Src, belonging to PTK. Src protein mainly exists in two forms:protein expressed by oncogene of Rous sarcoma virus and protein expressed by proto-oncogene of eukaryocytes. c-Src protein involves in intracellular signal transduction, closely relevant to cell growth, differentiation, and apoptosis and so on. At present, the domestic and foreign research of Src family are focused on the c-Src protein, whose structure, physical and chemical properties cannot be further studied. c-Src protein's homologue, the v-Src protein, both the structure and function of which are quite similar to the former, seldom shows any change in the activity of in vitro in contrast to in vivo, while c-Src protein in vitro shows little activity. Therefore, the physical and chemical properties of v-Src protein can be easily studied through constructing restructured v-src gene and expressing the v-Src protein in E. coli in great quantities. Therefore, the physical and chemical properties of v-Src protein can be well studied by expressing the v-Src protein in E.coli in great quantities, and a model system for screening of Src tyrosine kinase inhibitors is estabilished.
First, through gene engineering technology,v-src gene was cloned into the middle cloning vector pUCm-T and then into the expression vector pGEX-KT, then the recombined expression plasmid pGEX-KT/v-src was completed. PCR, double digests and sequencing identify the successfully constructed expression plasmid. Secondly, by orthogonal experiment optimization, the optical condition for expressing was determined:37℃,1 mM IPTG and 5 hours induced expressing. The v-Src protein was bacterially expressed and collected in the form of inclusion-body under that condition, and then, was washed、dissolved、affinity chromatographied、digested by thrombin and renatured by continuous gradient of urea. Again, screening the extract of Scutellaria barba(ESB), the ethanol extract is found to have inhibitory effect on v-Src protein, and the inhibition is relevant to dose. Cell experiments show that the composent also has inhibition over mitochondria activity.
This research has obtained active v-Src protein by E.coli expression system, and selected the anti-tumor composent of ESB. It can provide a basis for further study on the structure and function of Src protein, and lay the foundation of the anti-tumor drug screening system in vitro.
引文
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[1]张燕,李洪燕,张翼,苏富琴,冯志强,陈晓光哈尔滨商业大学学报.2009.25(2)：129-134
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[2]Jacobs C., Riibsamen H., Expression of pp60c-Src protein kinase in adult and fetal human tissue:high activities in some sarcomas and mammary carcinomas. Cancer Res,1983 Apr. 43(4):p.1696-702.
[3]Irby R.B., Yeatman T.J., Increased Src activity disrupts cadherin/catenin-mediated homotypic adhesion in human colon cancer and transformed rodent cells. Cancer Research,2002.62(9):p. 2669-2674.
[4]Frame M.C., Src in cancer:deregulation and consequences for cell behaviour. Biochim Biophys Acta.2002.1602:p.114-130.
[5]Jankowski J., Coghill G., Hopwood D., et al., Oncogens and oncosuppressor genes in adenocarcinoma of esophagus. Gut,1992.33(8):p.1033
[6]Takekura N., Yasul W., Yoshida K., et al., pp60c-Src protein kinase activity in human gastric carcinoma. Int J Cancer,1990.45:p.847
[7]Cartwright C.A., Kamps M.P., Meisler A.I., et al., pp60c-Src activation in human colon carcinoma. JClin Invest,1989.83:p.2025
[8]Fanning P., Bulovas K., Saini K.S., et al., Elevated expression of pp60c-Src in low grade human bladder carcinoma. Cancer Res,1992.32(6):p.1457
[9]Salni K., Fanning P., Bulovas K., et al., Possible role for pp60c-Src in urothelial cell differentiation. Proc Amer Associ Cancer Res,1991.32:p.283
[10]Fizazi K., The role of Src in prostate cancer. Annals of Oncology,2007.18:p.1765-1773
[11]Hunter T., Sefton B.M., Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc. Nat. Acad. Sci. USA,1980.77:p.1311-1315.
[12]Sawyer T., Boyce, B., et al., Src inhibitors:genomics to therapeutics. Expert Opin Investig Drugs,2001.10(7):p.1327-1344.
[13]Sudol M., et al., A novel Yes-related kinase, Yrk, is expressed at elevated levels in neural and hematopoietic tissues. Oncogene,1993 Apr.8(4):p.823-31.
[14]Talmor-Cohen A., Tomashov-Matar R., Eliyahu E,Shapiro R., Shalgi R., Are Src Family kinases involved in cell cycle resumption in rat eggs? Reproduction.2004.127:p.455-463.
[15]Martin G.S., The hunting of the Src. Nat. Rev. Mol. Cell Biol.,2001.2(6):p.467-475.
[16]Stehelin D., Varmus H.E., et al., DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature,1976.260(5547):p.170-173.
[17]Sato K., Iwasaki T., Hirahara S., et al., Molecilar dissection of eggfertilization signaling with the aid of tyrosine kinase specific inhibitor and activator strategies. Biochim Biophys Acta, 2004.1697:p.103-121.
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