小鼠白細(xì)胞介素17A原核表達(dá)系統(tǒng)的構(gòu)建
發(fā)布時(shí)間：2018-10-18 20:44
【摘要】：目的 白細(xì)胞介素17(interleukin-17,IL-17)是近年來(lái)新發(fā)現(xiàn)的炎性細(xì)胞因子,具有多種生物學(xué)活性,是某些疾病發(fā)生和發(fā)展的重要因素之一,受到了人們的廣泛關(guān)注。IL-17是一種主要由T細(xì)胞產(chǎn)生,通過(guò)表達(dá)與許多細(xì)胞上的受體發(fā)揮作用的蛋白質(zhì)。人IL-17是一種同源二聚多肽,主要由活化CD4+記憶T細(xì)胞分泌產(chǎn)生。小鼠IL-17的總長(zhǎng)度為158個(gè)氨基酸殘基,其中21個(gè)氨基酸殘基組成其前導(dǎo)信號(hào)序列。人IL-17與小鼠IL-17在氨基酸殘基序列上有62.5%的相似性,與大鼠IL-17有58%的相似性。國(guó)內(nèi)外的實(shí)驗(yàn)證實(shí),IL-17在多種神經(jīng)系統(tǒng)疾病中都有高表達(dá)。多發(fā)性硬化(multiple sclerosis,MS)患者體內(nèi)IL-17表達(dá)水平增高,并且與疾病的嚴(yán)重程度有一定聯(lián)系。IL-17A是IL-17家族中最主要的成員,參與免疫介導(dǎo)的炎癥損傷,在全身炎癥反應(yīng)中發(fā)揮重要作用。如果能夠制備出針對(duì)IL-17A的特異性抗體,并且在實(shí)驗(yàn)中證實(shí)此特異性抗體可以表現(xiàn)出突出的治療效果,那么不但對(duì)于臨床治療多發(fā)性硬化提供一個(gè)新的治療方向,更可能實(shí)現(xiàn)其在神經(jīng)系統(tǒng)疾病中的廣泛應(yīng)用。 方法 本課題首先根據(jù)Genbank中小鼠IL-17A基因組序列,設(shè)計(jì)出針對(duì)小鼠IL-17A的引物,從提取的小鼠基因組中,利用PCR方法采用高保真DNA聚合酶使小鼠IL-17A基因片段大量復(fù)制擴(kuò)增,片段長(zhǎng)度為631bp,擴(kuò)增片段為平滑末端,再與脫氧三磷酸腺苷相連接,將連接后的基因片段再與T克隆載體pMD19-T相連接,得到含有小鼠IL-17A基因片段的pMD19-T(+)/mIL-17A重組質(zhì)粒,利用重組質(zhì)粒對(duì)大腸埃希氏菌DH5a感受態(tài)細(xì)胞進(jìn)行轉(zhuǎn)化,并對(duì)經(jīng)氨芐青霉素和指示培養(yǎng)基上藍(lán)白斑篩選的陽(yáng)性重組質(zhì)粒分別進(jìn)行PCR檢測(cè)、雙酶切檢測(cè)和測(cè)序檢測(cè)。雙酶切檢測(cè)結(jié)果較為理想的重組質(zhì)粒,同時(shí)雙酶切消化表達(dá)載體pET32a(+),將純化后的目的片段相連接,得到重組表達(dá)載體pET32a(+)/mIL-17A,轉(zhuǎn)化大腸埃希氏菌表達(dá)菌BL21(DE3),37℃,1mM IPTG,200rpm誘導(dǎo)目的蛋白在大腸埃希氏菌BL21(DE3)中表達(dá)。 結(jié)果 經(jīng)SDS PAGE凝膠電泳和Western免疫印記檢測(cè)后,有融合蛋白表達(dá),分子量約53kDa,與預(yù)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腸埃希氏菌BL21(DE3)中表達(dá)。 結(jié)論 實(shí)驗(yàn)結(jié)果證實(shí),重組質(zhì)粒pET32a(+)/小鼠IL-17A能夠在大腸埃希氏菌BL21(DE3)中表達(dá)出目的蛋白,所獲得的蛋白大小與預(yù)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，
本文編號(hào)：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，
本文編號(hào)：2280318
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