本文選題：內(nèi)毒素結(jié)合肽 + 原核表達(dá)　； 參考：《第三軍醫(yī)大學(xué)》2010年碩士論文

【摘要】： 研究背景和目的 脂多糖結(jié)合蛋白(lipopolysaccharide binding protein, LBP)是一種介導(dǎo)LPS與其受體(CD14等)相結(jié)合的關(guān)鍵載體蛋白分子。其羧基末端將LPS遞呈至受體CD14,并以LPS-LBP-CD14復(fù)合體形式啟動TLR4-MD2-MyD88活化及跨膜信號轉(zhuǎn)導(dǎo),從而激活下游信號轉(zhuǎn)導(dǎo)通路。多項(xiàng)研究表明:LBP、BPI和LALF等天然蛋白的衍生肽均具有中和內(nèi)毒素能力,且可以通過阻斷LBP-LPS的結(jié)合來抑制LPS誘發(fā)的體內(nèi)外炎癥反應(yīng)。同時(shí),它們具有一共同特點(diǎn),即與LPS結(jié)合區(qū)域均富含正電荷及疏水性氨基酸殘基。LPS分子結(jié)構(gòu)中含有帶負(fù)電荷的磷酸基團(tuán)及疏水性長鏈脂肪酸,容易結(jié)合帶正電荷及疏水性分子。因此,我們可以通過氨基酸替換來增加陽性電荷數(shù)和疏水性,從而增加這類小分子肽的拮抗內(nèi)毒素活性。然而由于不同的氨基酸殘基所帶電荷及疏水性均不同,任意一個(gè)殘基的替換都可能會使肽的電荷、疏水性乃至空間結(jié)構(gòu)發(fā)生改變,所以,我們需了解此肽段中哪些氨基酸是與LPS結(jié)合的關(guān)鍵位點(diǎn),并選擇除這些關(guān)鍵氨基酸之外的位點(diǎn)進(jìn)行突變,即把非關(guān)鍵氨基酸替換成疏水性、堿性氨基酸或重要結(jié)合位點(diǎn)氨基酸。LBP的NH2-末端部分(NH2-LBP)是LPS的結(jié)合部位,尤其是富含疏水性、堿性氨基酸的第108到133位氨基酸,與帶有負(fù)電荷的磷酸基團(tuán)及疏水性長鏈脂肪酸的內(nèi)毒素分子有著高效親和力。因此,本研究針對內(nèi)毒素分子結(jié)構(gòu)特征,在本課題組以往研究中已證實(shí)LBP108-133肽段,即內(nèi)毒素結(jié)合肽(endotoxin binding peptide, EBP)具有一定中和內(nèi)毒素活性能力的基礎(chǔ)之上,為進(jìn)一步提高其生物活性,我們對EBP基因片段進(jìn)行改建,在構(gòu)建表達(dá)載體的同時(shí),選擇可能影響其生物活性的氨基酸所對應(yīng)的堿基進(jìn)行突變,以期增強(qiáng)其抗內(nèi)毒素活性,通過分子克隆、定點(diǎn)突變和表達(dá)純化獲得重組人內(nèi)毒素結(jié)合肽突變體,為研究該肽段的功能奠定了基礎(chǔ)。 主要技術(shù)方法 以本實(shí)驗(yàn)室已有的pET-30-LBP重組質(zhì)粒為模板,采用PCR法,擴(kuò)增EBP25基因,構(gòu)建pET-30-EBP25融合表達(dá)載體并轉(zhuǎn)化E.coli DH5α擴(kuò)增。重組質(zhì)粒經(jīng)酶切和測序鑒定后,應(yīng)用快速定點(diǎn)突變法將EBP25第2位纈氨酸、第5位谷氨酰胺、第15位苯丙氨酸、第18位天冬酰胺和第22位天冬氨酸所對應(yīng)的堿基依次替換成賴氨酸、賴氨酸、色氨酸、賴氨酸、丙氨酸所對應(yīng)的堿基,突變后重組質(zhì)粒再經(jīng)測序鑒定后,將表達(dá)量較高者轉(zhuǎn)化至E.coli BL21(DE3) PlysS后經(jīng)IPTG誘導(dǎo)表達(dá),表達(dá)產(chǎn)物采用Western印跡進(jìn)行鑒定后,用His-Tag親和層析對融合蛋白進(jìn)行純化。 結(jié)果 1.重組質(zhì)粒pET-30-EBP25的構(gòu)建:采用PCR技術(shù),以pET-30-NH-LBP為模板,擴(kuò)增得到產(chǎn)物大小約為96 bp的EBP25基因,將其克隆入原核表達(dá)載體pET30a(+),經(jīng)酶切及測序鑒定,成功構(gòu)建重組質(zhì)粒。 2.重組質(zhì)粒突變體pET-30-mEBP25的獲得:采用快速定點(diǎn)突變法,以pET-30- EBP25為模板,依次將EBP25第2位纈氨酸、第5位谷氨酰胺、第15位苯丙氨酸、第18位天冬酰胺、第22位天冬氨酸所對應(yīng)堿基分別替換為賴氨酸、賴氨酸、色氨酸、賴氨酸、丙氨酸所對應(yīng)的堿基。 3.融合蛋白的表達(dá)及鑒定:構(gòu)建好的五種重組質(zhì)粒分別轉(zhuǎn)化表達(dá)菌E.coli BL21(DE3) plysS,其中,EBP25及m1EBP25以包涵體形式獲得高表達(dá)量,而其他三種突變體融合蛋白表達(dá)量低,甚至不表達(dá)。SDS-PAGE電泳結(jié)果表明,EBP25及m1EBP25重組陽性菌誘導(dǎo)后表達(dá)出約8 kD大小的新生融合蛋白帶,與預(yù)期融合蛋白分子量相符。Western blot鑒定結(jié)果顯示該蛋白與小鼠anti-His單克隆抗體有特異性結(jié)合能力。 4.融合蛋白的純化及純度鑒定:分離溶解包涵體,用Invitrogen公司的Ni-NTA agarose進(jìn)行6×His標(biāo)簽融合蛋白的親和層析純化、透析復(fù)性,獲得人EBP25和m1EBP25融合蛋白,經(jīng)質(zhì)譜檢測m1EBP25融合蛋白的純度為96.8%。 結(jié)論 本研究通過基因克隆等方法,構(gòu)建pET-30-EBP25和突變后pET-30-m1-5EBP25融合表達(dá)載體,篩選出突變后表達(dá)量較高的重組菌,純化出EBP25和mEBP25融合蛋白,為進(jìn)一步研究其中和內(nèi)毒素/脂多糖活性奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

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