新型重组人内毒素结合肽突变体的设计与构建

发布时间：2018-05-04 04:06

本文选题：内毒素结合肽 + 原核表达　；参考：《第三军医大学》2010年硕士论文

【摘要】： 研究背景和目的脂多糖结合蛋白(lipopolysaccharide binding protein, LBP)是一种介导LPS与其受体(CD14等)相结合的关键载体蛋白分子。其羧基末端将LPS递呈至受体CD14,并以LPS-LBP-CD14复合体形式启动TLR4-MD2-MyD88活化及跨膜信号转导,从而激活下游信号转导通路。多项研究表明:LBP、BPI和LALF等天然蛋白的衍生肽均具有中和内毒素能力,且可以通过阻断LBP-LPS的结合来抑制LPS诱发的体内外炎症反应。同时,它们具有一共同特点,即与LPS结合区域均富含正电荷及疏水性氨基酸残基。LPS分子结构中含有带负电荷的磷酸基团及疏水性长链脂肪酸,容易结合带正电荷及疏水性分子。因此,我们可以通过氨基酸替换来增加阳性电荷数和疏水性,从而增加这类小分子肽的拮抗内毒素活性。然而由于不同的氨基酸残基所带电荷及疏水性均不同,任意一个残基的替换都可能会使肽的电荷、疏水性乃至空间结构发生改变,所以,我们需了解此肽段中哪些氨基酸是与LPS结合的关键位点,并选择除这些关键氨基酸之外的位点进行突变,即把非关键氨基酸替换成疏水性、碱性氨基酸或重要结合位点氨基酸。LBP的NH2-末端部分(NH2-LBP)是LPS的结合部位,尤其是富含疏水性、碱性氨基酸的第108到133位氨基酸,与带有负电荷的磷酸基团及疏水性长链脂肪酸的内毒素分子有着高效亲和力。因此,本研究针对内毒素分子结构特征,在本课题组以往研究中已证实LBP108-133肽段,即内毒素结合肽(endotoxin binding peptide, EBP)具有一定中和内毒素活性能力的基础之上,为进一步提高其生物活性,我们对EBP基因片段进行改建,在构建表达载体的同时,选择可能影响其生物活性的氨基酸所对应的碱基进行突变,以期增强其抗内毒素活性,通过分子克隆、定点突变和表达纯化获得重组人内毒素结合肽突变体,为研究该肽段的功能奠定了基础。主要技术方法以本实验室已有的pET-30-LBP重组质粒为模板,采用PCR法,扩增EBP25基因,构建pET-30-EBP25融合表达载体并转化E.coli DH5α扩增。重组质粒经酶切和测序鉴定后,应用快速定点突变法将EBP25第2位缬氨酸、第5位谷氨酰胺、第15位苯丙氨酸、第18位天冬酰胺和第22位天冬氨酸所对应的碱基依次替换成赖氨酸、赖氨酸、色氨酸、赖氨酸、丙氨酸所对应的碱基,突变后重组质粒再经测序鉴定后,将表达量较高者转化至E.coli BL21(DE3) PlysS后经IPTG诱导表达,表达产物采用Western印迹进行鉴定后,用His-Tag亲和层析对融合蛋白进行纯化。结果 1.重组质粒pET-30-EBP25的构建:采用PCR技术,以pET-30-NH-LBP为模板,扩增得到产物大小约为96 bp的EBP25基因,将其克隆入原核表达载体pET30a(+),经酶切及测序鉴定,成功构建重组质粒。 2.重组质粒突变体pET-30-mEBP25的获得:采用快速定点突变法,以pET-30- EBP25为模板,依次将EBP25第2位缬氨酸、第5位谷氨酰胺、第15位苯丙氨酸、第18位天冬酰胺、第22位天冬氨酸所对应碱基分别替换为赖氨酸、赖氨酸、色氨酸、赖氨酸、丙氨酸所对应的碱基。 3.融合蛋白的表达及鉴定:构建好的五种重组质粒分别转化表达菌E.coli BL21(DE3) plysS,其中,EBP25及m1EBP25以包涵体形式获得高表达量,而其他三种突变体融合蛋白表达量低,甚至不表达。SDS-PAGE电泳结果表明,EBP25及m1EBP25重组阳性菌诱导后表达出约8 kD大小的新生融合蛋白带,与预期融合蛋白分子量相符。Western blot鉴定结果显示该蛋白与小鼠anti-His单克隆抗体有特异性结合能力。 4.融合蛋白的纯化及纯度鉴定:分离溶解包涵体,用Invitrogen公司的Ni-NTA agarose进行6×His标签融合蛋白的亲和层析纯化、透析复性,获得人EBP25和m1EBP25融合蛋白,经质谱检测m1EBP25融合蛋白的纯度为96.8%。结论本研究通过基因克隆等方法,构建pET-30-EBP25和突变后pET-30-m1-5EBP25融合表达载体,筛选出突变后表达量较高的重组菌,纯化出EBP25和mEBP25融合蛋白,为进一步研究其中和内毒素/脂多糖活性奠定了基础。
[Abstract]:Background and purpose of research
Lipopolysaccharide binding protein (LBP) is a key carrier protein molecule that mediates the combination of LPS and its receptor (CD14, etc.). Its carboxyl ends deliver LPS to receptor CD14, and activate the TLR4-MD2-MyD88 activation and transmembrane signal transduction in the form of LPS-LBP-CD14 complex, thus activating downstream signal transduction pathway. A number of studies have shown that natural proteins such as LBP, BPI and LALF have the ability to neutralize endotoxin and inhibit the internal and external inflammatory reactions induced by LPS by blocking the binding of LBP-LPS. At the same time, they have a common characteristic, that is, the.LPS molecular structure containing positive charge and hydrophobic amino acid residues in the LPS binding region is contained in the.LPS molecular structure. The negatively charged phosphate group and hydrophobic long chain fatty acids are easy to combine with positive charge and hydrophobic molecules. Therefore, we can increase the number of positive charges and hydrophobicity by amino acid replacement, thus increasing the antagonistic activity of this kind of small molecular peptide. However, the charge and hydrophobicity of different amino acid residues are both different. The substitution of any residue may change the charge, hydrophobicity and even the spatial structure of the peptide. So, we need to know which amino acids in the peptide segment are the key sites that bind to LPS and choose the sites other than these key amino acids to be mutated, that is, to replace the non critical amino acids to hydrophobic, alkaline amino acids or The NH2- terminal part of the important binding site amino acid.LBP (NH2-LBP) is the binding site of LPS, especially the 108th to 133 amino acids, which are rich in hydrophobic, alkaline amino acids, and have high affinity with the endotoxin molecules with negative charged phosphate groups and hydrophobic long chain fatty acids. In our previous research, we have confirmed that the LBP108-133 peptide segment, endotoxin binding peptide (EBP), has the ability to neutralize the activity of endotoxin. In order to further improve its biological activity, we restructure the EBP gene fragment, and choose to influence its biological activity while constructing the expression vector. The amino acids corresponding to the bases were mutated to enhance their anti endotoxin activity. The recombinant human endotoxin binding peptide mutants were obtained by molecular cloning, fixed point mutation and expression purification, which laid the foundation for the study of the function of the peptide.
Main technical methods
Using the recombinant plasmid of pET-30-LBP in our laboratory as a template, the EBP25 gene was amplified by PCR, and the pET-30-EBP25 fusion expression vector was constructed and transformed into E.coli DH5 alpha. The recombinant plasmid was identified by enzyme digestion and sequencing, and the EBP25 second valine, fifth glutamine, Fifteenth phenylalanine and eighteenth asparagine were used for rapid fixed-point mutation. The bases corresponding to amides and twenty-second aspartic acids are replaced by lysine, lysine, tryptophan, lysine, and alanine, and then the recombinant plasmid is then sequenced and then transformed to E.coli BL21 (DE3) PlysS after IPTG induced expression, and the expression product is identified by Western blot and H Is-Tag affinity chromatography was used to purify the fusion protein.
Result
1. construction of recombinant plasmid pET-30-EBP25: using PCR technology and using pET-30-NH-LBP as the template, the EBP25 gene of about 96 BP was amplified and cloned into the prokaryotic expression vector pET30a (+). The recombinant plasmid was successfully constructed by enzyme digestion and sequencing.
2. recombinant plasmid mutant pET-30-mEBP25: using fast fixed point mutation method and using pET-30- EBP25 as template, EBP25 second valine, fifth bit glutamine, Fifteenth phenylalanine, eighteenth asparagine and twenty-second aspartic acid were replaced with lysine, lysine, tryptophan, lysine, alanine, respectively. The corresponding base.
The expression and identification of 3. fusion protein: five recombinant plasmids were constructed to transform the expression bacteria E.coli BL21 (DE3) plysS respectively. Among them, EBP25 and m1EBP25 were highly expressed in inclusion body form, while the other three mutant fusion proteins were low in expression and even did not express.SDS-PAGE electrophoresis results. After EBP25 and m1EBP25 recombinant positive bacteria were induced. A new fusion protein band of about 8 kD was expressed, which was consistent with the molecular weight of the expected fusion protein.Western blot identification results showed that the protein had a specific binding ability to the murine anti-His monoclonal antibody.
4. fusion protein purification and purity identification: separation and dissolution of inclusion body, purified by affinity chromatography of 6 x His label fusion protein by Invitrogen company's Ni-NTA agarose, dialytic renaturation, obtained human EBP25 and m1EBP25 fusion protein, and the purity of m1EBP25 fusion protein was 96.8%. by mass spectrometry to be 96.8%.
conclusion
In this study, the fusion expression vector of pET-30-EBP25 and pET-30-m1-5EBP25 was constructed by gene cloning and other methods. The recombinant strain with high expression after mutation was screened and the fusion protein of EBP25 and mEBP25 was purified, which laid the foundation for further study and the activity of endotoxin / lipopolysaccharide.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R346

【参考文献】