新型转录因子Mdfic的原核表达、抗体制备以及组织分布研究
|
摘要
Mdfic(MyoD family inhibitor domain containing)基因，是一个近年新发现的基因，其编码I-mfa结构域，但其功能研究尚属空白。本实验室的前期研究初步证实Mdfic蛋白与Rhox5蛋白间的相互作用，暗示Mdfic蛋白有可能是与Rhox5蛋白组成转录调控复合体，共同参与胚胎发育过程中的分化调控。为了进一步研究Mdfic蛋白的功能，利用原核表达系统表达并纯化Mdfic重组蛋白，制备其特异性多克隆抗血清，为Mdfic的功能研究奠定基础。
目的：构建pGEX-4T-3-Mdfic重组表达质粒，在大肠杆菌中诱导表达GST-Mdfic蛋白，纯化GST-Mdfic蛋白，制备其特异性多克隆抗血清，定位Mdfic的亚细胞分布研究，定量小鼠各主要组织器官中Mdfic的表达水平。
方法：生物信息学分析Mdfic基因序列；设计引物，PCR扩增Mdfic全长cDNA序列，并将其克隆至原核表达载体pGEX-4T-3，构建成pGEX-4T-3-Mdfic重组表达质粒；转化大肠杆菌表达菌株BL21，IPTG诱导表达GST-Mdfic融合蛋白，亲和纯化GST-Mdfic蛋白；以纯化后的GST-Mdfic蛋白为抗原，皮内注射免疫雄性新西兰兔制备Mdfic蛋白的特异性多克隆抗血清，ELISA测定其效价，Western Blotting检测其特异性；免疫荧光检测Mdfic的亚细胞分布；荧光定量PCR方法检测小鼠各组织器官中Mdfic mRNA的表达水平。
结果：Mdfic的生物信息学分析表明：Mdfic基因可能是HIC(Human I-mfa domain containing protein)的鼠基因类似物，同属于I-mfa结构域家族，推测I-mfa结构域在Mdfic蛋白的功能发挥中发挥重要作用；成功构建pGEX-4T-3-Mdfic重组表达质粒，IPTG诱导获得GST-Mdfic的高效表达；亲和纯化GST-Mdfic蛋白并对其超滤除盐；获得Mdfic蛋白的特异多克隆抗血清，ELISA测定抗血清效价约为1∶640000，Western Blotting表明该多克隆抗血清能特异识别结合内源性及外源性Mdfic蛋白；间接免疫荧光实验表明Mdfic在细胞中遍在分布，其中以细胞质为主；荧光定量PCR显示Mdfic在成鼠肝、脾、肾、睾丸中高表达。
结论：成功制备获得高效价、高特异性的Mdfic多克隆抗血清；定位Mdfic蛋白的亚细胞分布并指出成鼠不同组织器官组织中Mdfic RNA的表达量存在差异，为Mdfic的功能研究奠定基础。
Mdfic(MyoD family inhibitor domain containing) gene, was a newly discovered gene in recent years. It encode a protein containing the I-mfa domain with unknown function. Mdfic was shown to interact with Rhox5 in our previous study. It suggested that Mdfic might be involved in the transcription that was regulated by Rhox5. Thus, in turn, participateds in the embryonic development process and the differentiation regulation together with Rhox5 protein. In order to further investigate the function of the Mdfic, the Mdfic recombinant protein was expressed and purified using the prokaryotic expression system. Polyclonal anti-Mdfic antiserum was subsequently generated for future studies.
Object: Overexpression of GST-Mdfic protein in E. coli; getting specific polyclonal antiserum of Mdfic protein; characterize the subcellular distribution of Mdfic; quantitate the Mdfic expression leveling different mouse tissue.
Method: Sequence analysis showed that Mdfic, a mouse orthologue of HIC(Human I-mfa domain containing protein), belonged to I-mfa superfamily. The Mdfic ORF was cloned into E. coli expression vector pEGX-4T-3 and the GST fusion protein was expressed in BL21. GST-Mdfic fusion protein was purified by affinity chromatography and was then used to immunize male New Zealand white rabbit by intradermal injection. The titer and the specificity of the rabbit antiserum were shown by ELISA and Western blotting, respectively. The antiserum was then used in localizing Mdfic by immunofluorescence. The expressions of Mdfic in different mouse tissues were studied by real-time PCR.
Result: Sequence analyze of Mdfic indicated that the Mdfic gene was mouse gene orthologue of HIC (Human I-mfa domain containing protein), belonging to the I-mfa domain family. The pGEX-4T-3-Mdfic E. coli expression plasmid was constructed and expressed successfully; the GST-Mdfic was purified with affinity chromatography and the purified product was ultrafiltered to desalt. The polyclonal antiserum was collected from the immunized rabbit. Its titer was 1:640, 000 and could specifically recognize endogenous and exogenous Mdfic protein, according to the result of ELISA and Western blotting, respectively. The indirect immunofluorescence using this antiserum indicated that Mdfic was distributed in both cytoplasm and nuclear. Real Time PCR demonstrated Mdfic was highly expressed in the livers, spleen, kidney and testicle in adult mice.
Conclusion: Mdfic specific polyclonal antiserum was collected and used successfully. In localizing subceUular Mdfic by immunofluorescence; different levels of expression of Mdfic were observed in different mouse tissues.
目的：构建pGEX-4T-3-Mdfic重组表达质粒，在大肠杆菌中诱导表达GST-Mdfic蛋白，纯化GST-Mdfic蛋白，制备其特异性多克隆抗血清，定位Mdfic的亚细胞分布研究，定量小鼠各主要组织器官中Mdfic的表达水平。
方法：生物信息学分析Mdfic基因序列；设计引物，PCR扩增Mdfic全长cDNA序列，并将其克隆至原核表达载体pGEX-4T-3，构建成pGEX-4T-3-Mdfic重组表达质粒；转化大肠杆菌表达菌株BL21，IPTG诱导表达GST-Mdfic融合蛋白，亲和纯化GST-Mdfic蛋白；以纯化后的GST-Mdfic蛋白为抗原，皮内注射免疫雄性新西兰兔制备Mdfic蛋白的特异性多克隆抗血清，ELISA测定其效价，Western Blotting检测其特异性；免疫荧光检测Mdfic的亚细胞分布；荧光定量PCR方法检测小鼠各组织器官中Mdfic mRNA的表达水平。
结果：Mdfic的生物信息学分析表明：Mdfic基因可能是HIC(Human I-mfa domain containing protein)的鼠基因类似物，同属于I-mfa结构域家族，推测I-mfa结构域在Mdfic蛋白的功能发挥中发挥重要作用；成功构建pGEX-4T-3-Mdfic重组表达质粒，IPTG诱导获得GST-Mdfic的高效表达；亲和纯化GST-Mdfic蛋白并对其超滤除盐；获得Mdfic蛋白的特异多克隆抗血清，ELISA测定抗血清效价约为1∶640000，Western Blotting表明该多克隆抗血清能特异识别结合内源性及外源性Mdfic蛋白；间接免疫荧光实验表明Mdfic在细胞中遍在分布，其中以细胞质为主；荧光定量PCR显示Mdfic在成鼠肝、脾、肾、睾丸中高表达。
结论：成功制备获得高效价、高特异性的Mdfic多克隆抗血清；定位Mdfic蛋白的亚细胞分布并指出成鼠不同组织器官组织中Mdfic RNA的表达量存在差异，为Mdfic的功能研究奠定基础。
Mdfic(MyoD family inhibitor domain containing) gene, was a newly discovered gene in recent years. It encode a protein containing the I-mfa domain with unknown function. Mdfic was shown to interact with Rhox5 in our previous study. It suggested that Mdfic might be involved in the transcription that was regulated by Rhox5. Thus, in turn, participateds in the embryonic development process and the differentiation regulation together with Rhox5 protein. In order to further investigate the function of the Mdfic, the Mdfic recombinant protein was expressed and purified using the prokaryotic expression system. Polyclonal anti-Mdfic antiserum was subsequently generated for future studies.
Object: Overexpression of GST-Mdfic protein in E. coli; getting specific polyclonal antiserum of Mdfic protein; characterize the subcellular distribution of Mdfic; quantitate the Mdfic expression leveling different mouse tissue.
Method: Sequence analysis showed that Mdfic, a mouse orthologue of HIC(Human I-mfa domain containing protein), belonged to I-mfa superfamily. The Mdfic ORF was cloned into E. coli expression vector pEGX-4T-3 and the GST fusion protein was expressed in BL21. GST-Mdfic fusion protein was purified by affinity chromatography and was then used to immunize male New Zealand white rabbit by intradermal injection. The titer and the specificity of the rabbit antiserum were shown by ELISA and Western blotting, respectively. The antiserum was then used in localizing Mdfic by immunofluorescence. The expressions of Mdfic in different mouse tissues were studied by real-time PCR.
Result: Sequence analyze of Mdfic indicated that the Mdfic gene was mouse gene orthologue of HIC (Human I-mfa domain containing protein), belonging to the I-mfa domain family. The pGEX-4T-3-Mdfic E. coli expression plasmid was constructed and expressed successfully; the GST-Mdfic was purified with affinity chromatography and the purified product was ultrafiltered to desalt. The polyclonal antiserum was collected from the immunized rabbit. Its titer was 1:640, 000 and could specifically recognize endogenous and exogenous Mdfic protein, according to the result of ELISA and Western blotting, respectively. The indirect immunofluorescence using this antiserum indicated that Mdfic was distributed in both cytoplasm and nuclear. Real Time PCR demonstrated Mdfic was highly expressed in the livers, spleen, kidney and testicle in adult mice.
Conclusion: Mdfic specific polyclonal antiserum was collected and used successfully. In localizing subceUular Mdfic by immunofluorescence; different levels of expression of Mdfic were observed in different mouse tissues.
引文
[1] 田璐，李俊雅等，动物生肌决定因子的研究进展，黄牛杂志，2005，31(1)：43-46．
[2] Berkes CA, Tapscott SJ. MyoD and the transcriptional control of myogenesis, Semin Cell Dev Biol. 2005, 16(4-5):585-595.
[3] 王先梅，MyoD家族与分子心肌成形术，国外医学心血管疾病分册，2001，28(1)：6-9．
[4] Davis RL, Cheng PF, Lassar AB, et al. Cell, 1990, 60(5): 733-746.
[5] Weintraub H. The MyoD family and myogenesis: redundancy, networks and theresholds, Cell,1993, 75: 1241-1244.
[6] 刘丑生，赵兴波等，动物肌肉生长发育调控的功能基因研究进展，中国畜牧杂志，2003，39(5)：48-49．
[7] 党喜同，贾弘禔，张乃蘅，生肌蛋白的功能与调节，生理科学进展，1996，27(2)：146-148．
[8] Weintraub, H., Tapscott, S. J., Davis, R. L.,et al. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc. Natl. Acad. Sci. USA, 1989, 86: 5434-5438.
[9] Thayer, M. J., Tapscott, S. J., Davis, R. L.,et al. Positive autoregulation of the myogenic determination gene MyoD1. Cell, 1989, 58: 241-248.
[10] Pownall, M. E. and Emerson, C. E., Jr. Sequential activation of three myogenic regulatory genes during somite morphogenesis in quails. Dev. Biol. 1992, 151: 67-79.
[11] Pownall, M. E. and Emerson, C. E., Jr. Molecular and embryological studies of avian myogenesis. Semin. Dev. Biol. 1992, 3: 229-241.
[12] Lyons, G. E. and Buckingham, M. E. Developmental regulation ofmyogenesis in the mouse. Semin. Dev. Biol. 1992, 3: 243-253.
[13] Braun, T. and Arnold, H.-H. Myf-5 and myoD genes are activated in distinct mesenchymal stem cells and determine different skeletal muscle lineages. EMBO J. 1996, 15: 310-318.
[14] Cossu, G., Kelly, R., Tajbakhsh, S., et al. Activation of different myogenic pathways: myf-5 is induced by the neural tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm. Development, 1996, 122: 429-437.
[15] Rudnicki, M. A., Braun, T., Hinuma, S. and Jaenisch, R. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Call 1992, 71: 383-390.
[16] Olson, E. N. Interplay between proliferation and differentiation within myogenic lineage. Dev. Biol. 1992, 154: 261-272.
[17] Li, L., Zhou, J., Guy, J., et al. FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell 1992, 71:1181-1194.
[18] Kopan, R., Nye, J. S. and Weintraub, H. The intracellular domain of mouse Notch: a constutively actived repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Developmen, 1994, 120:2421-2430.
[19] Jarriault, S., Brou, C., Logeat, C., et al. Signalling downstream of activated mammalian Notch. Nature 1995, 377: 355-358.
[20] 王蕾，赵玉莲，安利国等，脊椎动物骨骼肌细胞发生的分子机制，海洋科学，2004，28(12)：54-58．
[21] Kunikazu T, Norbert K, Mark G, et al. Vitamin D_3 enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts. Biochimica et Biophysica Acta, 2001, 1539: 122-130.
[22] Chen CM, Kraut N, Groudine M, Weintraub H. I-mf, a movel myogenic repressor, interacts with members of the MyoD family. Cell, 1996, 86(5): 731-741.
[23] kiyomi M, Minoru H, Takahide T, et al. Myogenic repressor I-mfa interferes with the function of Zic Family proteins. Biochemical and Biophysical Research Communication, 2004, 320: 233-240.
[24] Pan W, Jia Y, Wang J, et al. {beta}-Catenin regulates myogenesis by relieving I-mfa-mediated suppression of myogenic regulatory factory factors in P 19 cells. Pro Natl Aead Sei USA, 2005, 102(48): 17378-383
[25] Kaut N, Snider L, Chen CM, et al. Requirement of the mouse I-mfa gene for placental development and skeletal patterning. The EMBO Journal, 1998, 17:6276-6288
[26] Sabine T, Jihane B, Bernard G, et al. Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40). European Journal of Cell Biology, 2000, 79: 834-838.
[27] Snider, L., H. Thidwell, J. R. Miller, et al. Inhibition of Tcf3 binding by I-mfa domain proteins. Mol. Cell. Biology. 2001, 21:1866-1873.
[28] The RIKEN Genome Exploration Research Group Phase Ⅱ Team and the FANTOM Consortium Functional annotation of a full-length mouse eDNA collection. Nature, 2001,409 (6821): 685-690
[29] 罗志文，郭芬，李月琴等．mPem蛋白相作用分子的筛选与鉴定，生物工程学报，2006，22(1)：125-130．
[30] Yuan Xin HU, Ju Yuan GUO, Lu SHEN, Yan CHEN, Zu Chuan ZHANG, Yong Lian ZHANG. Get effective polyclonal antisera in one month. Cell Research.2002, 12(2):157-160
[31] Saluta M, Bell P A. Troubleshooting GST fusion protein expression in E.coli. Life Sci News, 1997, 1:201-209
[2] Berkes CA, Tapscott SJ. MyoD and the transcriptional control of myogenesis, Semin Cell Dev Biol. 2005, 16(4-5):585-595.
[3] 王先梅，MyoD家族与分子心肌成形术，国外医学心血管疾病分册，2001，28(1)：6-9．
[4] Davis RL, Cheng PF, Lassar AB, et al. Cell, 1990, 60(5): 733-746.
[5] Weintraub H. The MyoD family and myogenesis: redundancy, networks and theresholds, Cell,1993, 75: 1241-1244.
[6] 刘丑生，赵兴波等，动物肌肉生长发育调控的功能基因研究进展，中国畜牧杂志，2003，39(5)：48-49．
[7] 党喜同，贾弘禔，张乃蘅，生肌蛋白的功能与调节，生理科学进展，1996，27(2)：146-148．
[8] Weintraub, H., Tapscott, S. J., Davis, R. L.,et al. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc. Natl. Acad. Sci. USA, 1989, 86: 5434-5438.
[9] Thayer, M. J., Tapscott, S. J., Davis, R. L.,et al. Positive autoregulation of the myogenic determination gene MyoD1. Cell, 1989, 58: 241-248.
[10] Pownall, M. E. and Emerson, C. E., Jr. Sequential activation of three myogenic regulatory genes during somite morphogenesis in quails. Dev. Biol. 1992, 151: 67-79.
[11] Pownall, M. E. and Emerson, C. E., Jr. Molecular and embryological studies of avian myogenesis. Semin. Dev. Biol. 1992, 3: 229-241.
[12] Lyons, G. E. and Buckingham, M. E. Developmental regulation ofmyogenesis in the mouse. Semin. Dev. Biol. 1992, 3: 243-253.
[13] Braun, T. and Arnold, H.-H. Myf-5 and myoD genes are activated in distinct mesenchymal stem cells and determine different skeletal muscle lineages. EMBO J. 1996, 15: 310-318.
[14] Cossu, G., Kelly, R., Tajbakhsh, S., et al. Activation of different myogenic pathways: myf-5 is induced by the neural tube and MyoD by the dorsal ectoderm in mouse paraxial mesoderm. Development, 1996, 122: 429-437.
[15] Rudnicki, M. A., Braun, T., Hinuma, S. and Jaenisch, R. Inactivation of MyoD in mice leads to up-regulation of the myogenic HLH gene Myf-5 and results in apparently normal muscle development. Call 1992, 71: 383-390.
[16] Olson, E. N. Interplay between proliferation and differentiation within myogenic lineage. Dev. Biol. 1992, 154: 261-272.
[17] Li, L., Zhou, J., Guy, J., et al. FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell 1992, 71:1181-1194.
[18] Kopan, R., Nye, J. S. and Weintraub, H. The intracellular domain of mouse Notch: a constutively actived repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Developmen, 1994, 120:2421-2430.
[19] Jarriault, S., Brou, C., Logeat, C., et al. Signalling downstream of activated mammalian Notch. Nature 1995, 377: 355-358.
[20] 王蕾，赵玉莲，安利国等，脊椎动物骨骼肌细胞发生的分子机制，海洋科学，2004，28(12)：54-58．
[21] Kunikazu T, Norbert K, Mark G, et al. Vitamin D_3 enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts. Biochimica et Biophysica Acta, 2001, 1539: 122-130.
[22] Chen CM, Kraut N, Groudine M, Weintraub H. I-mf, a movel myogenic repressor, interacts with members of the MyoD family. Cell, 1996, 86(5): 731-741.
[23] kiyomi M, Minoru H, Takahide T, et al. Myogenic repressor I-mfa interferes with the function of Zic Family proteins. Biochemical and Biophysical Research Communication, 2004, 320: 233-240.
[24] Pan W, Jia Y, Wang J, et al. {beta}-Catenin regulates myogenesis by relieving I-mfa-mediated suppression of myogenic regulatory factory factors in P 19 cells. Pro Natl Aead Sei USA, 2005, 102(48): 17378-383
[25] Kaut N, Snider L, Chen CM, et al. Requirement of the mouse I-mfa gene for placental development and skeletal patterning. The EMBO Journal, 1998, 17:6276-6288
[26] Sabine T, Jihane B, Bernard G, et al. Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40). European Journal of Cell Biology, 2000, 79: 834-838.
[27] Snider, L., H. Thidwell, J. R. Miller, et al. Inhibition of Tcf3 binding by I-mfa domain proteins. Mol. Cell. Biology. 2001, 21:1866-1873.
[28] The RIKEN Genome Exploration Research Group Phase Ⅱ Team and the FANTOM Consortium Functional annotation of a full-length mouse eDNA collection. Nature, 2001,409 (6821): 685-690
[29] 罗志文，郭芬，李月琴等．mPem蛋白相作用分子的筛选与鉴定，生物工程学报，2006，22(1)：125-130．
[30] Yuan Xin HU, Ju Yuan GUO, Lu SHEN, Yan CHEN, Zu Chuan ZHANG, Yong Lian ZHANG. Get effective polyclonal antisera in one month. Cell Research.2002, 12(2):157-160
[31] Saluta M, Bell P A. Troubleshooting GST fusion protein expression in E.coli. Life Sci News, 1997, 1:201-209