鸡CARP单克隆抗体制备及其在骨骼肌发育中的表达
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摘要
心肌锚定重复蛋白(CARP)是一种重要的核转录调控因子,是锚定重复序列家族成员之一,具有锚定重复区、两个PEST基序以及一个核定位信号。目前CARP已经被确定为一个新的心血管疾病的分子标志物,它在胚胎期、新生期和成年哺乳动物心脏中高水平表达。CARP具有多种功能,在血管发生,心脏发育,细胞凋亡,转录调控,纤维的装配和拉伸传感,核质通讯等方面具有重要作用。最近研究发现与哺乳动物相比,CARP在鸡骨骼肌组织中呈特异性表达,这暗示了CARP可能在鸡骨骼肌发育过程起重要作用,因此深入研究CARP在鸡骨骼肌发生中的调控机制具有重要意义。为了研究CARP在鸡骨骼肌发育过程中的表达,本试验克隆表达并纯化了鸡CARP重组蛋白,并制备了抗鸡CARP单克隆抗体,在蛋白质水平来检测其在骨骼肌发育过程中的表达。
利用RT-PCR的方法从鸡骨骼肌扩增获得CARP N端110个氨基酸的cDNA,将其克隆至原核表达载体pET28b中,然后转化至大肠杆菌BL21(DE3),提取质粒进行双酶切鉴定,并测序验证。重组菌经IPTG诱导表达并纯化重组蛋白。SDS-PAGE结果表明与未诱导菌株相比,诱导后的重组菌表达分子量约18000的重组蛋白,并主要以可溶性形式存在,该蛋白经灰度分析纯度能达到88.5%。
用纯化的CARP重组蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA法进行筛选阳性克隆,经多次克隆化培养,筛选高分泌滴度的阳性杂交瘤细胞株,制备腹水并纯化。采用McAb亚类鉴定试剂盒确定单克隆抗体亚型,对筛选得到的杂交瘤细胞染色体分析,检测细胞培养上清和腹水效价,并用Western blot方法对McAbs进行特异性鉴定。为研究CARP在肌肉不同发育时期的表达,本实验选取胚胎期,出生后幼雏,成年鸡为研究对象,从鸡肌骨骼肌提取总蛋白,采用Western blot检测肌肉发育过程中CARP在蛋白质水平的表达。
结果最终获得两株能稳定分泌抗鸡CARP杂交瘤细胞株,分命名为4A8、4E62,亚型都属于IgG1,轻链为κ链;融合细胞的染色体数目均大于89条,符合杂交瘤染色体数目。两株杂交瘤细胞免疫小鼠的腹水及其细胞培养液上清的效价分别为3.2×104、1.28×105和2.56×103、2.56×103。Western blot结果表明McAb能识别重组表达和内源性的CARP蛋白,并且与小鼠CARP蛋白无交叉反应。在蛋白质水平,CARP仅在出壳后的鸡骨骼肌中表达。
本研究成功获得2株能识别鸡CARP的杂交瘤及其分泌的特异性McAbs,为进一步研究CARP的功能奠定了物质基础;试验结果表明其CARP可能在鸡骨骼肌发育过程中发挥重要作用。
CARP, a protein containing an ankyrin repeat domain, two PEST sequence and a nucleus localization signal (NLS), is as a member of the Muscle Ankyrin Repeat Protein family (MARP). It has been reported to be a novel nuclear transcription cofactor. Recently, CARP has been identified as a novel marker of cardiovascular disease. There is a high expression of CARP in fetal, neonatal and adult heart. As a multifunctional factor, the information on the molecular functions of CARP suggests that the gene plays a critical role in angiogenesis and vasculogenesis, cardiac development, transcriptional regulation, myofibrillar assembly, stretch sensing, and in communication between the sarcoplasm and the nucleus. Later, it has been found that CARP is specifically expressed in skeletal muscle tissue of chicken, which is different from that in mammals, suggesting its potential functions in myogenesis. To investigate its expression during myogenesis of chicken, the recombinant chicken CARP protein and anti-chicken CARP McAbs were prepared and its expression at the levels of translation during skeletal muscle development was assayed in this study.
The chicken CARP cDNA encoding the N-terminal 110aa was obtained by RT-PCR and cloned into prokaryotic expression vector pET28b, and then recombinant plasmid was transformed into E. coli strain BL21 (DE3). The recombinant bacterias were induced with IPTG, and the expressed CARP recombinant protein was purified with MagneHis? Protein Purification and analyzed by SDS-PAGE. A soluble protein Mr about 18000 was detected, and its purity can reach 88.5% by analysis with the Bandscan software.
The BALB/c mice were immunized with the purified recombinant chicken CARP, the spleen cells from the immuned mice were obtained and fused with myeloma cells. The positive clones were screened by indirect ELISA, and cloned by limiting dilutions.The hybridoma cells secreting McAB at high titers were selected, the ascitic fluid from the imice inoculated with hybridomas in abdominal cavity was prepared and the McAbs were purified. Then the total proteins of skeletal muscle from chicken embryogenesis to adult were extracted, the expression level of CARP was detected by Western blot.
The subtype of the monoclonal antibody was detected by isotype analysis kit, and the chromosomal amounts of hybridoma cells were acounted. The specificity of the monoclonal antibody was tested by Western blot. Finally, two hybridoma strains were proved to have the capability of stably secreting monoclonal antibody against chicken CARP, designated as 4A8 and 4E6. The subtype of monoclonal antibodies was IgG1 and the light chain wasκ. The chromosomal amounts of them were more than 89. The McAb titers of ascites were 3.2×104, 1.28×105 and that of the culture supernatant of hybridomas were 2.56×103, 2.56×103 respectively. The McAbs could recognize a protein with approximate molecular weight 36 000 in chicken skeletal muscle by Western blot and they had no cross reaction with CARP of mouse. At the level of translation, CARP was expressed only in skeletal muscle after birth in chicken.
Two mouse anti-chicken hybridomas and their secreted monoclonal antibodies have been successfully obtained. They provide useful reagent for further study on the biological function of CARP chicken myogenesis, and the results suggest that CARP play an important role during in skeletal muscle development of chicken.
利用RT-PCR的方法从鸡骨骼肌扩增获得CARP N端110个氨基酸的cDNA,将其克隆至原核表达载体pET28b中,然后转化至大肠杆菌BL21(DE3),提取质粒进行双酶切鉴定,并测序验证。重组菌经IPTG诱导表达并纯化重组蛋白。SDS-PAGE结果表明与未诱导菌株相比,诱导后的重组菌表达分子量约18000的重组蛋白,并主要以可溶性形式存在,该蛋白经灰度分析纯度能达到88.5%。
用纯化的CARP重组蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA法进行筛选阳性克隆,经多次克隆化培养,筛选高分泌滴度的阳性杂交瘤细胞株,制备腹水并纯化。采用McAb亚类鉴定试剂盒确定单克隆抗体亚型,对筛选得到的杂交瘤细胞染色体分析,检测细胞培养上清和腹水效价,并用Western blot方法对McAbs进行特异性鉴定。为研究CARP在肌肉不同发育时期的表达,本实验选取胚胎期,出生后幼雏,成年鸡为研究对象,从鸡肌骨骼肌提取总蛋白,采用Western blot检测肌肉发育过程中CARP在蛋白质水平的表达。
结果最终获得两株能稳定分泌抗鸡CARP杂交瘤细胞株,分命名为4A8、4E62,亚型都属于IgG1,轻链为κ链;融合细胞的染色体数目均大于89条,符合杂交瘤染色体数目。两株杂交瘤细胞免疫小鼠的腹水及其细胞培养液上清的效价分别为3.2×104、1.28×105和2.56×103、2.56×103。Western blot结果表明McAb能识别重组表达和内源性的CARP蛋白,并且与小鼠CARP蛋白无交叉反应。在蛋白质水平,CARP仅在出壳后的鸡骨骼肌中表达。
本研究成功获得2株能识别鸡CARP的杂交瘤及其分泌的特异性McAbs,为进一步研究CARP的功能奠定了物质基础;试验结果表明其CARP可能在鸡骨骼肌发育过程中发挥重要作用。
CARP, a protein containing an ankyrin repeat domain, two PEST sequence and a nucleus localization signal (NLS), is as a member of the Muscle Ankyrin Repeat Protein family (MARP). It has been reported to be a novel nuclear transcription cofactor. Recently, CARP has been identified as a novel marker of cardiovascular disease. There is a high expression of CARP in fetal, neonatal and adult heart. As a multifunctional factor, the information on the molecular functions of CARP suggests that the gene plays a critical role in angiogenesis and vasculogenesis, cardiac development, transcriptional regulation, myofibrillar assembly, stretch sensing, and in communication between the sarcoplasm and the nucleus. Later, it has been found that CARP is specifically expressed in skeletal muscle tissue of chicken, which is different from that in mammals, suggesting its potential functions in myogenesis. To investigate its expression during myogenesis of chicken, the recombinant chicken CARP protein and anti-chicken CARP McAbs were prepared and its expression at the levels of translation during skeletal muscle development was assayed in this study.
The chicken CARP cDNA encoding the N-terminal 110aa was obtained by RT-PCR and cloned into prokaryotic expression vector pET28b, and then recombinant plasmid was transformed into E. coli strain BL21 (DE3). The recombinant bacterias were induced with IPTG, and the expressed CARP recombinant protein was purified with MagneHis? Protein Purification and analyzed by SDS-PAGE. A soluble protein Mr about 18000 was detected, and its purity can reach 88.5% by analysis with the Bandscan software.
The BALB/c mice were immunized with the purified recombinant chicken CARP, the spleen cells from the immuned mice were obtained and fused with myeloma cells. The positive clones were screened by indirect ELISA, and cloned by limiting dilutions.The hybridoma cells secreting McAB at high titers were selected, the ascitic fluid from the imice inoculated with hybridomas in abdominal cavity was prepared and the McAbs were purified. Then the total proteins of skeletal muscle from chicken embryogenesis to adult were extracted, the expression level of CARP was detected by Western blot.
The subtype of the monoclonal antibody was detected by isotype analysis kit, and the chromosomal amounts of hybridoma cells were acounted. The specificity of the monoclonal antibody was tested by Western blot. Finally, two hybridoma strains were proved to have the capability of stably secreting monoclonal antibody against chicken CARP, designated as 4A8 and 4E6. The subtype of monoclonal antibodies was IgG1 and the light chain wasκ. The chromosomal amounts of them were more than 89. The McAb titers of ascites were 3.2×104, 1.28×105 and that of the culture supernatant of hybridomas were 2.56×103, 2.56×103 respectively. The McAbs could recognize a protein with approximate molecular weight 36 000 in chicken skeletal muscle by Western blot and they had no cross reaction with CARP of mouse. At the level of translation, CARP was expressed only in skeletal muscle after birth in chicken.
Two mouse anti-chicken hybridomas and their secreted monoclonal antibodies have been successfully obtained. They provide useful reagent for further study on the biological function of CARP chicken myogenesis, and the results suggest that CARP play an important role during in skeletal muscle development of chicken.
引文
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[2] Ikeda K, Emoto N, Matsuo M, et al. Molecular identification and characterization of a novel nuclear protein whose expression is up-regulated in insulin-resistant animals [J]. J Biol Chem, 2003, 278(6): 3514-20.
[3] Samaras SE, Shi Y, Davidson JM. CARP: Fishing for Novel Mechanisms of Neovascularization [J]. J Investig Dermatol Symp Proc, 2006, 11(1): 124-31.
[4] Chu W, Burns DK, Swerlick RA, et al. Identification and characterization of a novelcytokineinducible nuclear protein from human endothelial cells [J]. J Biol Chem, 1995, 270(17): 10236-45.
[5] Jeyaseelan R, Poizat C, Baker RK, et al. A novel cardiac-restricted target for doxorubicin. CARP, a nuclear modulator of gene expression in cardiac progenitor cells and cardiomyocytes [J]. J Biol Chem, 1997, 272(36): 22800-8.
[6] Baumeister A, Arber S, Caroni P. Accumulation of muscle ankyrin repeat protein transcript reveals local activation of primary myotube endcompartments during muscle morphogenesis [J]. J Cell Biol, 1997, 139(5): 1231-1242.
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