抗CDK4人源单链抗体的原核表达、纯化及活性分析
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摘要
细胞周期调节失控是造成肿瘤细胞恶性增殖的主要原因之一。周期蛋白依赖蛋白激酶(cyclin-dependent kinase, CDK)的时相性激活是细胞周期调控机制的核心。CDK4作为细胞进入增殖周期第一个被激活的周期蛋白依赖蛋白激酶,是细胞周期进程中G1/S期转换的限速因子。研究发现在许多肿瘤细胞和组织中都存在着CDK4的过度表达和过度活化,与肿瘤的发生和发展有着密切的关系。
作为研究和治疗肿瘤的重要靶点,如何下调过度表达和活化的CDK4已经成为研究热点。单链抗体(single chain antibody, scFv)以其分子量小,免疫原性低等优点,在肿瘤诊断和治疗中具有独特的优势,目前已经有多种基于单链抗体的抗肿瘤药物进入临床试验。
本研究构建了pET28a-CDK4表达载体,获得有活性的重组人CDK4蛋白后,以其为抗原,从噬菌体库中筛选得到抗CDK4人源单链抗体基因,将其转化进原核细胞中进行了诱导、表达和纯化,并通过ELISA、Western blot、竞争性抑制、亲和力测定、免疫荧光和免疫沉淀等实验方法对单链抗体进行了活性表征。结果表明纯化的单链抗体不仅能与重组人CDK4蛋白特异性结合并具有较高亲和力,而且也能与细胞内源性的CDK4蛋白特异性结合。
本实验得到的结果对后续开展抗CDK4胞内抗体肿瘤基因治疗的研究奠定了基础。
Cell cycle regulation is a very complex and sophisticated regulative process, which is closely related to the division, growth and death of cells. Many key factors involves in the process. Once the abnormal regulation happens, it will lead to unlimited cell proliferation, and thus to form tumors.
The activation of CDK4 in different phases of cell cycle is the core of cell cycle regulation. As the first activated cyclin-dependent kinase since cells enter the cell cycle, CDK4 is the rate limiting factor during G1 / S phase conversion. Studies found that there were over-expression and excessive activation of CDK4 in many tumor cells and tissues, which was closely related to tumor’s happening, development and prognosis. More and more studies have shown CDK4 was an important target to research and treat cancers.
Facing the difficulty of cancer treatment, people have been in the search for finding effective methods and strategies. Antibody-based bio-immunotherapy shows an attractive prospect. With the development of antibody engineering technology, now people have developed a number of new antibodies. Because human single-chain antibody (ScFv) has many advantages such as low immunogenicity, small molecular weight and strong penetration of tissues, it has been receiving more and more attention. In recent years it is reported that intracellular antibody (intrabody) can target antigen specifically locating at definite position of cell. It can obtain the effect of targeted therapy and widen the application of single-chain antibody. That provides a new idea for biological treatment of tumor. So far, there has not any report about using intracellular single-chain antibody to inactivate CDK4.
According to those ideas above, the present study constructed the pET28a-CDK4 expression vector. After we obtained recombinant human CDK protein, we used it as antigen to screen anti-CDK4 scFv gene from phage display library. Then the pDAN5-scFv plasmid was transformed into E.coli HB2151 and expression of scFv was induced by IPTG. The expressed products were purified. The binding activity of purified anti-CDK4 scFv to CDK4 was identified by ELISA, Western blot, competitive inhibition, affinity analysis, immunofluorescence and immunoprecipitation. The follows are the results:
1. pET28a-CDK4 expression vector was successfully constructed and then transformed into E.coli BL21(DE3). CDK4 expression was induced by IPTG. CDK4 inclusion was obtained. After degeneration, pufication and renaturation of CDK4 inclusion, soluble CDK4 protein was obtained with purity up to 98%. Western blot showed it had biological activity.
2. The pDAN5-scFv plasmid screened from the phage display library was transformed into E.coli HB2151 and induced by IPTG. ELISA result showed that the expressed products existed in supernatant and had specific ability to bind to recombinant human CDK4 protein. The supernatant was concentrated with sulfate ammonium and then purified with His Trap HP column. Western blot results showed that the purified anti-CDK4 scFv could bind to recombinant human CDK4 and its molecular weight was about 30 kD.
3. The competitive inhibition of anti-CDK4 polyclonal antibody to scFv for binding recombinant human CDK4 was analyzed by competitive ELISA. The results revealed that the rabbit anti-CDK4 antibody could inhibit scFv to bind to recombinant human CDK4. The index of inhibition was 31.9%.
4. The affinity of anti-CDK4 scFv was determined by noncompetitive ELISA, and the affinity constant binding to recombinant human CDK4 was (2.78±0.78)×10~(-8) mol/L. It suggested that the anti-CDK4 scFv had affinity of binding to recombinant human CDK4.
5. To identify the interaction between anti-CDK4 scFv and CDK4 in HeLa and MCF-7 cells under fluorescence microscope, the fixed cells were incubated with the anti-CDK4 scFv, and then with anti-V5 antibody, followed by the incubation with FITC-conjugation goat anti-mouse IgG antibody and Hoechst 33342 staining nucleus. The results showed that anti-CDK4 scFv can interact with CDK4 in tumor cells.
6. To identify the interaction between anti-CDK4 scFv and CDK4 in MCF-7 cell lysate, the cell lysate were subjected to SDS-PAGE and transferred to NC membrane. The NC memebrane was incubated with the anti-CDK4 scFv, and then with anti-V5 antibody, followed by the incubation with HRP-conjugation goat anti-mouse IgG antibody. The results showed that anti-CDK4 scFv can interact with CDK4 in MCF-7 cell lysate.
7. CDK4 was immunoprecipitated from the MCF-7 cell extracts using anti-CDK4 scFv and V5 tag antibody. After separating the immunoprecipitated complex by SDS-PAGE, they were identified by western blot and probed with anti-CDK4 rabbit polyclonal antibody and HRP-conjugation goat anti-rabbit IgG antibody. The results showed that there was a band in the region of 34 kD. This result reveals that the purified anti-CDK4 scFv can bind to CDK4 in tumor cells.
The above results revealed that the purified anti-CDK4 scFv could not only bind to recombinant CDK4 protein but also bind to CDK4 in tumor cells. And its binding affinity was high. This study not only provided a new idea of tumor biological treatment, but also laid the foundation for treating tumor using anti-CDK4 intracellular scFv.
作为研究和治疗肿瘤的重要靶点,如何下调过度表达和活化的CDK4已经成为研究热点。单链抗体(single chain antibody, scFv)以其分子量小,免疫原性低等优点,在肿瘤诊断和治疗中具有独特的优势,目前已经有多种基于单链抗体的抗肿瘤药物进入临床试验。
本研究构建了pET28a-CDK4表达载体,获得有活性的重组人CDK4蛋白后,以其为抗原,从噬菌体库中筛选得到抗CDK4人源单链抗体基因,将其转化进原核细胞中进行了诱导、表达和纯化,并通过ELISA、Western blot、竞争性抑制、亲和力测定、免疫荧光和免疫沉淀等实验方法对单链抗体进行了活性表征。结果表明纯化的单链抗体不仅能与重组人CDK4蛋白特异性结合并具有较高亲和力,而且也能与细胞内源性的CDK4蛋白特异性结合。
本实验得到的结果对后续开展抗CDK4胞内抗体肿瘤基因治疗的研究奠定了基础。
Cell cycle regulation is a very complex and sophisticated regulative process, which is closely related to the division, growth and death of cells. Many key factors involves in the process. Once the abnormal regulation happens, it will lead to unlimited cell proliferation, and thus to form tumors.
The activation of CDK4 in different phases of cell cycle is the core of cell cycle regulation. As the first activated cyclin-dependent kinase since cells enter the cell cycle, CDK4 is the rate limiting factor during G1 / S phase conversion. Studies found that there were over-expression and excessive activation of CDK4 in many tumor cells and tissues, which was closely related to tumor’s happening, development and prognosis. More and more studies have shown CDK4 was an important target to research and treat cancers.
Facing the difficulty of cancer treatment, people have been in the search for finding effective methods and strategies. Antibody-based bio-immunotherapy shows an attractive prospect. With the development of antibody engineering technology, now people have developed a number of new antibodies. Because human single-chain antibody (ScFv) has many advantages such as low immunogenicity, small molecular weight and strong penetration of tissues, it has been receiving more and more attention. In recent years it is reported that intracellular antibody (intrabody) can target antigen specifically locating at definite position of cell. It can obtain the effect of targeted therapy and widen the application of single-chain antibody. That provides a new idea for biological treatment of tumor. So far, there has not any report about using intracellular single-chain antibody to inactivate CDK4.
According to those ideas above, the present study constructed the pET28a-CDK4 expression vector. After we obtained recombinant human CDK protein, we used it as antigen to screen anti-CDK4 scFv gene from phage display library. Then the pDAN5-scFv plasmid was transformed into E.coli HB2151 and expression of scFv was induced by IPTG. The expressed products were purified. The binding activity of purified anti-CDK4 scFv to CDK4 was identified by ELISA, Western blot, competitive inhibition, affinity analysis, immunofluorescence and immunoprecipitation. The follows are the results:
1. pET28a-CDK4 expression vector was successfully constructed and then transformed into E.coli BL21(DE3). CDK4 expression was induced by IPTG. CDK4 inclusion was obtained. After degeneration, pufication and renaturation of CDK4 inclusion, soluble CDK4 protein was obtained with purity up to 98%. Western blot showed it had biological activity.
2. The pDAN5-scFv plasmid screened from the phage display library was transformed into E.coli HB2151 and induced by IPTG. ELISA result showed that the expressed products existed in supernatant and had specific ability to bind to recombinant human CDK4 protein. The supernatant was concentrated with sulfate ammonium and then purified with His Trap HP column. Western blot results showed that the purified anti-CDK4 scFv could bind to recombinant human CDK4 and its molecular weight was about 30 kD.
3. The competitive inhibition of anti-CDK4 polyclonal antibody to scFv for binding recombinant human CDK4 was analyzed by competitive ELISA. The results revealed that the rabbit anti-CDK4 antibody could inhibit scFv to bind to recombinant human CDK4. The index of inhibition was 31.9%.
4. The affinity of anti-CDK4 scFv was determined by noncompetitive ELISA, and the affinity constant binding to recombinant human CDK4 was (2.78±0.78)×10~(-8) mol/L. It suggested that the anti-CDK4 scFv had affinity of binding to recombinant human CDK4.
5. To identify the interaction between anti-CDK4 scFv and CDK4 in HeLa and MCF-7 cells under fluorescence microscope, the fixed cells were incubated with the anti-CDK4 scFv, and then with anti-V5 antibody, followed by the incubation with FITC-conjugation goat anti-mouse IgG antibody and Hoechst 33342 staining nucleus. The results showed that anti-CDK4 scFv can interact with CDK4 in tumor cells.
6. To identify the interaction between anti-CDK4 scFv and CDK4 in MCF-7 cell lysate, the cell lysate were subjected to SDS-PAGE and transferred to NC membrane. The NC memebrane was incubated with the anti-CDK4 scFv, and then with anti-V5 antibody, followed by the incubation with HRP-conjugation goat anti-mouse IgG antibody. The results showed that anti-CDK4 scFv can interact with CDK4 in MCF-7 cell lysate.
7. CDK4 was immunoprecipitated from the MCF-7 cell extracts using anti-CDK4 scFv and V5 tag antibody. After separating the immunoprecipitated complex by SDS-PAGE, they were identified by western blot and probed with anti-CDK4 rabbit polyclonal antibody and HRP-conjugation goat anti-rabbit IgG antibody. The results showed that there was a band in the region of 34 kD. This result reveals that the purified anti-CDK4 scFv can bind to CDK4 in tumor cells.
The above results revealed that the purified anti-CDK4 scFv could not only bind to recombinant CDK4 protein but also bind to CDK4 in tumor cells. And its binding affinity was high. This study not only provided a new idea of tumor biological treatment, but also laid the foundation for treating tumor using anti-CDK4 intracellular scFv.
引文
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[2] Ito Y, Matsuura N, Sakon M, et al. Expression and prognostic roles of the G1 modulators in hepato cellular carcinoma: p27 independently predicts the recurrence [J]. Hepatology, 1999, 30:90-99.
[3] Hunter T, Pines J. Cyclin D and CDK inhibitors come of age [J].Cell, 1994, 79(4): 547-550.
[4] Coats S, Flanagan W M, Nourse J, et al. Requirement of p27 Kip1 for restriction point control of the fibroblast cell cycle [J]. Scicence, 1996, 27(563): 8771-8776.
[5] Marcos Malumbers, Mariano Barbacid. Mammalian cyclin-dependent kinase [J]. TRENDS in Biochemical Sciences, 2005, 30(11):630-641.
[6]高燕,林莉萍,丁健.细胞周期调控的研究进展.生命科学, 2005,17(4): 318-322.
[7] King R W.How proteolysis drives the cell cycle [J].Science, 1996, 274:1652-1659.
[8] Amit D, Peter S, Philip W H. Cyclins and cdks in development and cancer: a perspective [J]. Oncogene, 2005, 24: 2909–2915.
[9]王瑞.人CDK4基因克隆与其在大肠杆菌中的表达[D].吉林:吉林大学医学院,2006.
[10]李娟,吴平平,姜叙诚. CDK4调控机制在肿瘤发生中的作用和意义[J].临床与实验病理学杂志,2006,22(4): 485 - 487.
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