原核表达小鼠嗅觉受体MOR28及其多克隆抗体的制备
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摘要
哺乳动物每个嗅觉感受神经(OSN, olfactory sense neurones)从超过数百个甚至上千个嗅觉受体(OR, olfactory receptor)基因中,只选择一个进行表达,这种表达调控方式是产生嗅觉的遗传学基础。经过相关研究实验推测,OR的这种表达方式的调控机制包括OR的负调控作用:当每个OSN成功表达一个有功能的OR后,此OR将抑制其它OR的表达,使OSN只表达这个OR。但在此过程中,OR具体是如何反馈抑制其他OR的表达,现在仍不清楚。
OR蛋白进行反馈调控的具体途径有以下几种可能性:1.OR结合到其它转录因子或上游激活因子上,使其失活或活性大大降低,以进行反馈调控;2.OR直接或与其它转录因子共同作用,结合到相关的顺式调控元件上,使此顺式调控元件的活性受到抑制,以进行负调控。
本实验旨在原核表达小鼠嗅觉受体28(MOR28),并制备其多克隆抗体。此抗体将用于对上述OR蛋白反馈调控具体途径的推理进行研究。
以小鼠基因组DNA为模板扩增出小鼠嗅觉受体28基因的部分序列smor28(shortmice olfactory receptor),将其连接到pET28a(+)骨架载体上,构建原核表达载体pMOR28。pMOR28表达载体诱导表达,SDS-PAGE检测目的蛋白的表达情况。利用Ni-NTA琼脂糖提纯表达的目的蛋白SMOR28,SMOR28经透析除盐,冻干浓缩后,免疫兔子制备其抗兔多克隆抗体。经westen blotting检测,SMOR28的抗兔多克隆抗体特异性及滴度较好。
Each olfactory sense neuron (OSN) choose just one olfactory receptor (OR) fromthe more than 1000 possibilities encoded in the genome and transcribes it from just oneallele. This process generates great neuronal diversity and forms the basis for thedevelopment and logic of the olfactory circuit between the nose and brain.
The mechanism behind this monoallelic regulation has been the subject of intensespeculation. Recent genetic experiments have brought the outline of the process intosharper relief, identifying a feedback mechanism in which the first olfactory receptorexpressed, generates a signal that stabilizes its choice,thus maintaining singularselection.
There are two possible feedback ways: (1) acting as a feedback factor, OR binds toother transcriptional factor (2) acting as a feedback factor, OR binds to regulationregion of other OR genes.
To study the two possible feedback ways just mentioned, we express and purifypart amino acid of mice olfactory receptor28(SMOR28) in E.coli and prepare rabbitanti-SMOR28 antibody. The anti-serum will be used to study OR feedback mechanismby immunoprecipitation and other methods.
Part sequence of mice olfactory receptor28 gene (smor28) has been amplifiedfrom the mice genome by utilizing PCR. The products have been ligated into thepET28a ( + ) vector plasmid. We obtain SMOR28 expression vectors pMOR28successfully. Expression vectors pMOR28 were induced, SDS-PAGE assay detectexpressed products. We purify SMOR28 by utilizing Ni-NTA agarose. Purified proteinwas dialysed and freezed-dry to inject rabbit preparing rabbit anti-SMOR28 antibody.Western blotting assay shows that the potency of this rabbit anti-SMOR28 antibody isgood.
OR蛋白进行反馈调控的具体途径有以下几种可能性:1.OR结合到其它转录因子或上游激活因子上,使其失活或活性大大降低,以进行反馈调控;2.OR直接或与其它转录因子共同作用,结合到相关的顺式调控元件上,使此顺式调控元件的活性受到抑制,以进行负调控。
本实验旨在原核表达小鼠嗅觉受体28(MOR28),并制备其多克隆抗体。此抗体将用于对上述OR蛋白反馈调控具体途径的推理进行研究。
以小鼠基因组DNA为模板扩增出小鼠嗅觉受体28基因的部分序列smor28(shortmice olfactory receptor),将其连接到pET28a(+)骨架载体上,构建原核表达载体pMOR28。pMOR28表达载体诱导表达,SDS-PAGE检测目的蛋白的表达情况。利用Ni-NTA琼脂糖提纯表达的目的蛋白SMOR28,SMOR28经透析除盐,冻干浓缩后,免疫兔子制备其抗兔多克隆抗体。经westen blotting检测,SMOR28的抗兔多克隆抗体特异性及滴度较好。
Each olfactory sense neuron (OSN) choose just one olfactory receptor (OR) fromthe more than 1000 possibilities encoded in the genome and transcribes it from just oneallele. This process generates great neuronal diversity and forms the basis for thedevelopment and logic of the olfactory circuit between the nose and brain.
The mechanism behind this monoallelic regulation has been the subject of intensespeculation. Recent genetic experiments have brought the outline of the process intosharper relief, identifying a feedback mechanism in which the first olfactory receptorexpressed, generates a signal that stabilizes its choice,thus maintaining singularselection.
There are two possible feedback ways: (1) acting as a feedback factor, OR binds toother transcriptional factor (2) acting as a feedback factor, OR binds to regulationregion of other OR genes.
To study the two possible feedback ways just mentioned, we express and purifypart amino acid of mice olfactory receptor28(SMOR28) in E.coli and prepare rabbitanti-SMOR28 antibody. The anti-serum will be used to study OR feedback mechanismby immunoprecipitation and other methods.
Part sequence of mice olfactory receptor28 gene (smor28) has been amplifiedfrom the mice genome by utilizing PCR. The products have been ligated into thepET28a ( + ) vector plasmid. We obtain SMOR28 expression vectors pMOR28successfully. Expression vectors pMOR28 were induced, SDS-PAGE assay detectexpressed products. We purify SMOR28 by utilizing Ni-NTA agarose. Purified proteinwas dialysed and freezed-dry to inject rabbit preparing rabbit anti-SMOR28 antibody.Western blotting assay shows that the potency of this rabbit anti-SMOR28 antibody isgood.
引文
[1] Buck,L. and Axel,R. (1991) A novel multigene family may encode odorant receptor:a molecular basis for odor recognition[J]. Cell 65,175-187
[2] Buck,L.B. (2000) The molecular architecture of odor and pheromone sensing in mammals[J]. Cell 100,611-618
[3] Chess,A. et al. (1994) Allelic inactivation regulates olfactory receptor gene expression[J]. Cell 78,823-834
[4] Feinstein,P. and Mombaerts,P. (2004) A contextual model for axonal sorting into glomeruli in the mouse olfactory system[J]. Cell 117,817-831
[5] Feinstein,P. et al. (2004) Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 advenergic receptor[J]. Cell 117,833-846
[6] Glusman,G. et al. (2001) The complete human olfactory subgenome[J]. Genome Res. 11,685-702
[7] Hirota,J. and Mombaerts,P. (2004) The LIM-homeodomain protein Lhx2 is required for complete development of mouse olfactory sensory neurons[J]. Proc. Natl. Acad. Sci. U.S.A. 101,8751-8755
[8] Ishii,T. et al. (2001) Monoallelic expression of the odourant receptor gene and axonal projection of olfactory sensory neurons[J]. Genes Cell 6,71-78
[9] Kratz,E. et al. (2002) Odorants receptor gene regulation: implications from genomic organization[J]. Trends in Genetics 18,29-34
[10] Lancet,D. and Arie,N.B.(1993) Olfactory receptor[J]. Current Biology 3,668-674
[11] Lapidot,M. et al. (2000) Mouse-Human orthology relationships in an olfactory receptor gene cluster[J]. Genomics 71,296-306
[12] Lewcock,J.W. and Reed,R.R. (2004) A feedback mechanism regulates monoallelic odorant receptor expression[J]. Proc. Natl. Acad. Sci. U.S.A. 101,1069-1074
[13] Malnic,B. et al. (2004) The human olfactory receptor gene family[J]. Proc. Natl. Acad. Sci. U.S.A. 101,2584-2589
[14] Mombaerts,p. et al. (1996) Visualizing and olfactory sensory map[J]. Cell 87,675-685
[15] Mombaerts,p. (2001) How smell develops[J]. Nat. Neurosci. 4,1192-1198
[16] Mombaerts,p. (2004) Odorant receptor gene choice in olfactory sensory neurons: the one receptor-one neuron hypothesis revisited[J]. Curr. Opin. Neurobiol. 14,31-36
[17] Nagawa,F. et al. (2002) Genomic analysis of the murine odorant receptor MOR28 cluster: a possible role of gene conversion in maintaining the olfactory map[J]. Gene 292,73-80
[18] Niimura,Y. and Nei,M. (2005) Comparative evolutionary analysis of olfactory receptor gene cluster between humans and mice[J]. Gene 346,13-21
[19] Qasba,P. and Reed,R.R. (1998) Tissue and zonal-specific expression of an olfactory receptor transgene[J]. J. Neurosci. 18,227-236
[20] Raming,K. et al. (1993) Cloning and expression of odorant receptor[J]. Nature 361,353-356
[21] Reed,R.R. (2004) After the holy grail: establishing a molecular basis for mammalian olfaction[J]. Cell 116,329-336
[22] Ressler,K.J. et al. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium[J]. Cell 73,597-609
[23] Ressler,K.J. et al. (1994) Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb[J]. Cell 97,1245-1255
[24] Serizawa,S. et al. (2000) Mutually exclusive expression of odorant receptor transgenes[J]. Nat. Neurosci. 3,687-693
[25] Serizawa,S. et al. (2003) Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse[J]. Science 302,2088-2094
[26] Shykind,B.M. et al. (2004) Gene switching and stability of odorant receptor gene choice[J]. Cell 117,801-815
[27] Shykind,B.M. (2005) Regulation of odorant receptor: one allele at a time[J]. Hum. Mol. Genet. 14,33-39
[28] Tsuboi,A. et al. (1999) Olfactory neurons expression closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb[J]. J. Neurosci. 19,8409-8418
[29] Vassar,R. et al. (1994) Topographic organization of sensory projection to the olfactory bulb[J]. Cell 79,981-991
[30] Vasslli,A. et al. (2000) Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb[J]. Neuron 35,681-696
[31] Wang,F. et al. (1998) Odorant receptors govern the formation of a precise topographic map[J]. Cell 93,47-60
[32] Young,J.M. et al. (2002) The sense of smell: genomics of vertebrate odorant receptor[J]. Hum. Mol. Genet. 11,1153-1160
[33] Zhang,X. and Firestein,S. (2002) The olfactory receptor gene superfamily of mouse[J]. Nat. Neurosci. 5,124-133
[34] Deleage,G. et al.(2001) ANTHEPROT: an integrated protein sequence analysis software with client/server capabilities[J]. Comput. Biol. Med. 4,259-267
[2] Buck,L.B. (2000) The molecular architecture of odor and pheromone sensing in mammals[J]. Cell 100,611-618
[3] Chess,A. et al. (1994) Allelic inactivation regulates olfactory receptor gene expression[J]. Cell 78,823-834
[4] Feinstein,P. and Mombaerts,P. (2004) A contextual model for axonal sorting into glomeruli in the mouse olfactory system[J]. Cell 117,817-831
[5] Feinstein,P. et al. (2004) Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 advenergic receptor[J]. Cell 117,833-846
[6] Glusman,G. et al. (2001) The complete human olfactory subgenome[J]. Genome Res. 11,685-702
[7] Hirota,J. and Mombaerts,P. (2004) The LIM-homeodomain protein Lhx2 is required for complete development of mouse olfactory sensory neurons[J]. Proc. Natl. Acad. Sci. U.S.A. 101,8751-8755
[8] Ishii,T. et al. (2001) Monoallelic expression of the odourant receptor gene and axonal projection of olfactory sensory neurons[J]. Genes Cell 6,71-78
[9] Kratz,E. et al. (2002) Odorants receptor gene regulation: implications from genomic organization[J]. Trends in Genetics 18,29-34
[10] Lancet,D. and Arie,N.B.(1993) Olfactory receptor[J]. Current Biology 3,668-674
[11] Lapidot,M. et al. (2000) Mouse-Human orthology relationships in an olfactory receptor gene cluster[J]. Genomics 71,296-306
[12] Lewcock,J.W. and Reed,R.R. (2004) A feedback mechanism regulates monoallelic odorant receptor expression[J]. Proc. Natl. Acad. Sci. U.S.A. 101,1069-1074
[13] Malnic,B. et al. (2004) The human olfactory receptor gene family[J]. Proc. Natl. Acad. Sci. U.S.A. 101,2584-2589
[14] Mombaerts,p. et al. (1996) Visualizing and olfactory sensory map[J]. Cell 87,675-685
[15] Mombaerts,p. (2001) How smell develops[J]. Nat. Neurosci. 4,1192-1198
[16] Mombaerts,p. (2004) Odorant receptor gene choice in olfactory sensory neurons: the one receptor-one neuron hypothesis revisited[J]. Curr. Opin. Neurobiol. 14,31-36
[17] Nagawa,F. et al. (2002) Genomic analysis of the murine odorant receptor MOR28 cluster: a possible role of gene conversion in maintaining the olfactory map[J]. Gene 292,73-80
[18] Niimura,Y. and Nei,M. (2005) Comparative evolutionary analysis of olfactory receptor gene cluster between humans and mice[J]. Gene 346,13-21
[19] Qasba,P. and Reed,R.R. (1998) Tissue and zonal-specific expression of an olfactory receptor transgene[J]. J. Neurosci. 18,227-236
[20] Raming,K. et al. (1993) Cloning and expression of odorant receptor[J]. Nature 361,353-356
[21] Reed,R.R. (2004) After the holy grail: establishing a molecular basis for mammalian olfaction[J]. Cell 116,329-336
[22] Ressler,K.J. et al. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium[J]. Cell 73,597-609
[23] Ressler,K.J. et al. (1994) Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb[J]. Cell 97,1245-1255
[24] Serizawa,S. et al. (2000) Mutually exclusive expression of odorant receptor transgenes[J]. Nat. Neurosci. 3,687-693
[25] Serizawa,S. et al. (2003) Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse[J]. Science 302,2088-2094
[26] Shykind,B.M. et al. (2004) Gene switching and stability of odorant receptor gene choice[J]. Cell 117,801-815
[27] Shykind,B.M. (2005) Regulation of odorant receptor: one allele at a time[J]. Hum. Mol. Genet. 14,33-39
[28] Tsuboi,A. et al. (1999) Olfactory neurons expression closely linked and homologous odorant receptor genes tend to project their axons to neighboring glomeruli on the olfactory bulb[J]. J. Neurosci. 19,8409-8418
[29] Vassar,R. et al. (1994) Topographic organization of sensory projection to the olfactory bulb[J]. Cell 79,981-991
[30] Vasslli,A. et al. (2000) Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb[J]. Neuron 35,681-696
[31] Wang,F. et al. (1998) Odorant receptors govern the formation of a precise topographic map[J]. Cell 93,47-60
[32] Young,J.M. et al. (2002) The sense of smell: genomics of vertebrate odorant receptor[J]. Hum. Mol. Genet. 11,1153-1160
[33] Zhang,X. and Firestein,S. (2002) The olfactory receptor gene superfamily of mouse[J]. Nat. Neurosci. 5,124-133
[34] Deleage,G. et al.(2001) ANTHEPROT: an integrated protein sequence analysis software with client/server capabilities[J]. Comput. Biol. Med. 4,259-267