猪CCL17基因的克隆与原核和真核表达
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摘要
趋化因子( chemokine)由多种细胞产生,机体在防御和清除入侵病原体等异物时,趋化因子具有激活和趋化白细胞的功能,细胞沿着趋化因子浓度的增加向趋化因子源处迁徙,从而吸引免疫细胞到免疫应答局部,参与免疫调节和免疫病理反应。趋化因子超家族在机体的生理和病理调节中均显示出了重要意义,特别是其在自身免疫、炎症等疾病中发挥重要作用。
趋化因子配体17(chemokine ligand 17,CCL17)又称为胸腺活化调节趋化因子(thymusand activation-regulated chemokine, TARC),是C-C亚族趋化因子家族中首个被认为具有高度选择性趋化Th2细胞从外周血募集至炎症部位的作用。该研究进行了猪趋化因子CCL17基因的克隆与原核和真核表达,得到的结果如下:
(1)本研究根据NCBI猪的EST数据库中得到登录号为DB794536的EST序列,通过NCBI的Blast工具及ORF Finder软件发现该序列含有完整的CCL17基因的ORF,依据该序列设计引物,以RT-PCR克隆了猪CCL17基因,验证了该基因在猪体内的存在,其ORF共309个核苷酸,编码102个氨基酸。
(2)扩增CCL17基因,并将其克隆至表达载体pET-32a(+)上,转化大肠埃希菌BL21,进行IPTG诱导表达。SDS-PAGE分析融合蛋白大小约为36ku。通过不断反复摸索表达条件,表明在28℃, IPTG为0.5mol/L,诱导4-6h,有大量的融合蛋白表达。
(3)将CCL17基因与高效的带有绿色荧光标记的真核表达载体pEGFP-C1相连,构建了重组质粒pEGFP-CCL17。利用Lipofectamine 2000 Regent将pEGFP-CCL17和pEGFP-C1质粒转染于SUVEC细胞中,转染48h显微镜下观察到典型的带有绿色荧光标记的细胞形态,进一步用Western-blot方法验证CCL17基因得到了成功表达。以上结果初步掌握了猪体内CCL17基因在体外表达的条件和特点,为进一步对猪体内CCL17蛋白的免疫学特性、功能的研究奠定了基础。
Chemokines are generated by a wide variety of cells and function as activators and chemoattractants for leukocytes while the organism is defending and eliminating invading pathogen as well as other non-self molecules. Chemokines also act as a chemoattractant to guide the migration of cells. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine, thereby immune cells are attracted to the site of immune response and participate in immunomodulation and immunopathological reactions. Finally, chemokine superfamily play significant roles in both the physiological and pathological modulation especially in the autoimmune and inflammatory diseases.
Chemokine ligand 17, aka( thymus and activation-regulated chemokine ,TARC) is the first recognized member in CC subfamiliy with super selective chemotactic capability to recruit Th2 cells from peripheral blood to the site of inflammation. Current perspectives focused on the role of CCL17 and its receptor in autoimmune diseases. However, the pathogenesis and immune mechanism of CC17 remains to be explained thoroughly. This study has cloned the porcine CCL17 gene and performed both prokaryotic and eukaryotic expression. The results are as follows.
(1)We obtained an EST sequence with accession number of DB794536 from porcine EST database in NCBI. Using blast tools and ORF Finder on NCBI, an ORF of this sequence containing the intact CCL17 gene was found. Designing primers based on this sequence, we cloned porcine CCL17 gene using RT-PCR method and validated the existence of this gene. Results suggested that the ORF contained 309 nucleotides, coding 102 amino acids.
(2)CCL17 gene was amplified and cloned to its prokaryotic vector pET-32a(+), and then the recombinant plasmids were transformed to E.Coli BL21 (DE3). By the challenge of variant concentrations of IPTG and other conditions, SDS-PAGE analysis showed that the target protein was overexpressed in E. coli BL21 (DE3) with the optimal condition of 28℃, 0.5mol/L IPTG and 4-6 induction hours
(3)CCL17 fragment was ligated into eukaryotic expression vector pEGFP-C1 which contained an enhanced green fluorescence reporter gene, thus recombinant plasmid pEGFP- CCL17 was constructed. The recombinant plasmid and the control plasmid pEGFP-C1 were respectively transfected to SUVEC cells by Lipofectamine 2000 Regent. Positively transfected clones were observed with inversion fluorescence microscope, and CCL17 gene expression were further verified by western-blot. From the results above, conditions and profiles of CCL17 gene expression in vitro were acquired preliminarily. And the findings may offer some references to the further revealing of its immunological properties, detection kit preparation and functional exploration.
趋化因子配体17(chemokine ligand 17,CCL17)又称为胸腺活化调节趋化因子(thymusand activation-regulated chemokine, TARC),是C-C亚族趋化因子家族中首个被认为具有高度选择性趋化Th2细胞从外周血募集至炎症部位的作用。该研究进行了猪趋化因子CCL17基因的克隆与原核和真核表达,得到的结果如下:
(1)本研究根据NCBI猪的EST数据库中得到登录号为DB794536的EST序列,通过NCBI的Blast工具及ORF Finder软件发现该序列含有完整的CCL17基因的ORF,依据该序列设计引物,以RT-PCR克隆了猪CCL17基因,验证了该基因在猪体内的存在,其ORF共309个核苷酸,编码102个氨基酸。
(2)扩增CCL17基因,并将其克隆至表达载体pET-32a(+)上,转化大肠埃希菌BL21,进行IPTG诱导表达。SDS-PAGE分析融合蛋白大小约为36ku。通过不断反复摸索表达条件,表明在28℃, IPTG为0.5mol/L,诱导4-6h,有大量的融合蛋白表达。
(3)将CCL17基因与高效的带有绿色荧光标记的真核表达载体pEGFP-C1相连,构建了重组质粒pEGFP-CCL17。利用Lipofectamine 2000 Regent将pEGFP-CCL17和pEGFP-C1质粒转染于SUVEC细胞中,转染48h显微镜下观察到典型的带有绿色荧光标记的细胞形态,进一步用Western-blot方法验证CCL17基因得到了成功表达。以上结果初步掌握了猪体内CCL17基因在体外表达的条件和特点,为进一步对猪体内CCL17蛋白的免疫学特性、功能的研究奠定了基础。
Chemokines are generated by a wide variety of cells and function as activators and chemoattractants for leukocytes while the organism is defending and eliminating invading pathogen as well as other non-self molecules. Chemokines also act as a chemoattractant to guide the migration of cells. Cells that are attracted by chemokines follow a signal of increasing chemokine concentration towards the source of the chemokine, thereby immune cells are attracted to the site of immune response and participate in immunomodulation and immunopathological reactions. Finally, chemokine superfamily play significant roles in both the physiological and pathological modulation especially in the autoimmune and inflammatory diseases.
Chemokine ligand 17, aka( thymus and activation-regulated chemokine ,TARC) is the first recognized member in CC subfamiliy with super selective chemotactic capability to recruit Th2 cells from peripheral blood to the site of inflammation. Current perspectives focused on the role of CCL17 and its receptor in autoimmune diseases. However, the pathogenesis and immune mechanism of CC17 remains to be explained thoroughly. This study has cloned the porcine CCL17 gene and performed both prokaryotic and eukaryotic expression. The results are as follows.
(1)We obtained an EST sequence with accession number of DB794536 from porcine EST database in NCBI. Using blast tools and ORF Finder on NCBI, an ORF of this sequence containing the intact CCL17 gene was found. Designing primers based on this sequence, we cloned porcine CCL17 gene using RT-PCR method and validated the existence of this gene. Results suggested that the ORF contained 309 nucleotides, coding 102 amino acids.
(2)CCL17 gene was amplified and cloned to its prokaryotic vector pET-32a(+), and then the recombinant plasmids were transformed to E.Coli BL21 (DE3). By the challenge of variant concentrations of IPTG and other conditions, SDS-PAGE analysis showed that the target protein was overexpressed in E. coli BL21 (DE3) with the optimal condition of 28℃, 0.5mol/L IPTG and 4-6 induction hours
(3)CCL17 fragment was ligated into eukaryotic expression vector pEGFP-C1 which contained an enhanced green fluorescence reporter gene, thus recombinant plasmid pEGFP- CCL17 was constructed. The recombinant plasmid and the control plasmid pEGFP-C1 were respectively transfected to SUVEC cells by Lipofectamine 2000 Regent. Positively transfected clones were observed with inversion fluorescence microscope, and CCL17 gene expression were further verified by western-blot. From the results above, conditions and profiles of CCL17 gene expression in vitro were acquired preliminarily. And the findings may offer some references to the further revealing of its immunological properties, detection kit preparation and functional exploration.
引文
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