小鼠白细胞介素17A原核表达系统的构建
发布时间：2018-10-18 20:44
【摘要】：目的 白细胞介素17(interleukin-17,IL-17)是近年来新发现的炎性细胞因子,具有多种生物学活性,是某些疾病发生和发展的重要因素之一,受到了人们的广泛关注。IL-17是一种主要由T细胞产生,通过表达与许多细胞上的受体发挥作用的蛋白质。人IL-17是一种同源二聚多肽,主要由活化CD4+记忆T细胞分泌产生。小鼠IL-17的总长度为158个氨基酸残基,其中21个氨基酸残基组成其前导信号序列。人IL-17与小鼠IL-17在氨基酸残基序列上有62.5%的相似性,与大鼠IL-17有58%的相似性。国内外的实验证实,IL-17在多种神经系统疾病中都有高表达。多发性硬化(multiple sclerosis,MS)患者体内IL-17表达水平增高,并且与疾病的严重程度有一定联系。IL-17A是IL-17家族中最主要的成员,参与免疫介导的炎症损伤,在全身炎症反应中发挥重要作用。如果能够制备出针对IL-17A的特异性抗体,并且在实验中证实此特异性抗体可以表现出突出的治疗效果,那么不但对于临床治疗多发性硬化提供一个新的治疗方向,更可能实现其在神经系统疾病中的广泛应用。 方法 本课题首先根据Genbank中小鼠IL-17A基因组序列,设计出针对小鼠IL-17A的引物,从提取的小鼠基因组中,利用PCR方法采用高保真DNA聚合酶使小鼠IL-17A基因片段大量复制扩增,片段长度为631bp,扩增片段为平滑末端,再与脱氧三磷酸腺苷相连接,将连接后的基因片段再与T克隆载体pMD19-T相连接,得到含有小鼠IL-17A基因片段的pMD19-T(+)/mIL-17A重组质粒,利用重组质粒对大肠埃希氏菌DH5a感受态细胞进行转化,并对经氨苄青霉素和指示培养基上蓝白斑筛选的阳性重组质粒分别进行PCR检测、双酶切检测和测序检测。双酶切检测结果较为理想的重组质粒,同时双酶切消化表达载体pET32a(+),将纯化后的目的片段相连接,得到重组表达载体pET32a(+)/mIL-17A,转化大肠埃希氏菌表达菌BL21(DE3),37℃,1mM IPTG,200rpm诱导目的蛋白在大肠埃希氏菌BL21(DE3)中表达。 结果 经SDS PAGE凝胶电泳和Western免疫印记检测后,有融合蛋白表达,分子量约53kDa,与预期的蛋白分子量大小(52.8kDa)基本一致,并且小鼠IL-17A融合蛋白大部分以可溶形式在大肠埃希氏菌BL21(DE3)中表达。 结论 实验结果证实,重组质粒pET32a(+)/小鼠IL-17A能够在大肠埃希氏菌BL21(DE3)中表达出目的蛋白,所获得的蛋白大小与预期相同。
[Abstract]:Objective Interleukin-17 (interleukin-17,IL-17) is a newly discovered inflammatory cytokine in recent years. It has many biological activities and is one of the important factors in the occurrence and development of some diseases. IL-17 is a protein that is mainly produced by T cells and functions with receptors on many cells. Human IL-17 is a homologous dimer polypeptide mainly produced by activated CD4 memory T cell secretion. The total length of mouse IL-17 is 158 amino acid residues, of which 21 amino acid residues constitute its leading signal sequence. There was 62.5% similarity between human IL-17 and mouse IL-17 in amino acid residues, and 58% similarity with rat IL-17. Experiments at home and abroad have confirmed that IL-17 is highly expressed in a variety of nervous system diseases. The expression of IL-17 in patients with multiple sclerosis (multiple sclerosis,MS) is increased and is related to the severity of the disease. IL-17A is the most important member of the IL-17 family and plays an important role in the systemic inflammatory response. If we can produce a specific antibody against IL-17A and prove in the experiment that the specific antibody can show outstanding therapeutic effect, it will not only provide a new therapeutic direction for the clinical treatment of multiple sclerosis. It is more likely to be widely used in nervous system diseases. Methods according to the mouse IL-17A genome sequence in Genbank, the primers for mouse IL-17A were designed. From the extracted mouse genome, the high fidelity DNA polymerase was used to replicate and amplify the mouse IL-17A gene fragment by PCR method. The length of the fragment was 631 BP, the amplified fragment was smooth terminal, and then connected with adenosine deoxytriphosphate. The recombinant plasmid pMD19-T () / mIL-17A containing mouse IL-17A gene fragment was obtained by ligating the ligated gene fragment with T-clone vector pMD19-T. Recombinant plasmids were transformed into Escherichia coli DH5a competent cells, and the positive recombinant plasmids screened by ampicillin and blue spot on indicated medium were detected by PCR, double enzyme digestion and sequencing, respectively. The recombinant plasmid was digested by double enzyme digestion, and the purified target fragment was ligated into the expression vector pET32a (), which was digested by double enzyme digestion, and the recombinant plasmid was digested by double enzyme digestion. The recombinant expression vector pET32a () / mIL-17A, was obtained to transform BL21 (DE3) into Escherichia coli. The target protein was induced by 1mM IPTG,200rpm at 37 鈩，
本文编号：2280318
[Abstract]:Objective Interleukin-17 (interleukin-17,IL-17) is a newly discovered inflammatory cytokine in recent years. It has many biological activities and is one of the important factors in the occurrence and development of some diseases. IL-17 is a protein that is mainly produced by T cells and functions with receptors on many cells. Human IL-17 is a homologous dimer polypeptide mainly produced by activated CD4 memory T cell secretion. The total length of mouse IL-17 is 158 amino acid residues, of which 21 amino acid residues constitute its leading signal sequence. There was 62.5% similarity between human IL-17 and mouse IL-17 in amino acid residues, and 58% similarity with rat IL-17. Experiments at home and abroad have confirmed that IL-17 is highly expressed in a variety of nervous system diseases. The expression of IL-17 in patients with multiple sclerosis (multiple sclerosis,MS) is increased and is related to the severity of the disease. IL-17A is the most important member of the IL-17 family and plays an important role in the systemic inflammatory response. If we can produce a specific antibody against IL-17A and prove in the experiment that the specific antibody can show outstanding therapeutic effect, it will not only provide a new therapeutic direction for the clinical treatment of multiple sclerosis. It is more likely to be widely used in nervous system diseases. Methods according to the mouse IL-17A genome sequence in Genbank, the primers for mouse IL-17A were designed. From the extracted mouse genome, the high fidelity DNA polymerase was used to replicate and amplify the mouse IL-17A gene fragment by PCR method. The length of the fragment was 631 BP, the amplified fragment was smooth terminal, and then connected with adenosine deoxytriphosphate. The recombinant plasmid pMD19-T () / mIL-17A containing mouse IL-17A gene fragment was obtained by ligating the ligated gene fragment with T-clone vector pMD19-T. Recombinant plasmids were transformed into Escherichia coli DH5a competent cells, and the positive recombinant plasmids screened by ampicillin and blue spot on indicated medium were detected by PCR, double enzyme digestion and sequencing, respectively. The recombinant plasmid was digested by double enzyme digestion, and the purified target fragment was ligated into the expression vector pET32a (), which was digested by double enzyme digestion, and the recombinant plasmid was digested by double enzyme digestion. The recombinant expression vector pET32a () / mIL-17A, was obtained to transform BL21 (DE3) into Escherichia coli. The target protein was induced by 1mM IPTG,200rpm at 37 鈩，
本文编号：2280318
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