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目的使内毒素结合肽(EBP)及其突变体mEBP在E．coli DH5α中表达,纯化后鉴定其抗内毒素／脂多糖(LPS)活性。方法(1)将含PinpointXa3-EBP及其突变体PinpointXa3-mEBP的工程菌DH5α活化,加入异丙硫半乳糖苷(IPTG)诱导其表达生物素融合蛋白。分离纯化表达产物,因子Xa酶切融合蛋白分离目的肽EBP和mEBP。采用亲和层析及反相高效液相色谱法纯化目的肽,用氨基末端10个氨基酸残基序列分析法鉴定突变体mEBP。(2)分离正常人外周血单核细胞(PBMC),用5 mg／L异硫氰酸荧光素(FITC)-LPS+不同浓度EBP或mEBP(均为2．0、5．0、12．5 mg／L)刺激PBMC,检测其平均荧光强度。用1 mg／L LPS+3种浓度(同前)EBP或mEBP刺激PBMC,5 h后取上清液,检测肿瘤坏死因子(TNF)α和白细胞介素(IL)6浓度。(3)将25只昆明小鼠分为正常对照组(5只):腹腔注射等渗盐水0．2 ml;模型组(5只):小鼠造成20％TBSAⅢ度烧伤,伤后腹腔注射LPS(1 mg／kg);治疗组(15只):小鼠同前致伤后,腹腔注射多黏菌素B(PMB,5 mg／kg)或EBP或mEBP(后两者均为10 mg／kg)。6 h后检测各组小鼠血清TNF-α、IL-6浓度及肝组织中TNF-αmRNA的表达水平。结果(1)纯化后的目的肽EBP、mEBP纯度均达98％以上,mEBP氨基末端10个氨基酸残基序列分析符合预期结果。(2)随着mEBP或EBP浓度的增加,PBMC表面的平均荧光强度逐渐减弱;且在同一浓度下,加入mEBP后平均荧光强度的减弱程度较加入EBP明显。与用1 mg／L LPS刺激PBMC比较,加入1 mg／L LPS+12．5 mg／L EBP以及1 mg／L LPS+3种浓度mEBP刺激后, PBMC培养上清液中IL-6及TNF-α的水平均明显降低(P<0．01)。(3)与模型组比较,治疗组小鼠血清IL-6、TNF-α水平均明显降低(P<0．01),其中10 mg／kg mEBP治疗组两指标低于等浓度EBP治疗组(P<0．05)。(4)小鼠肝组织TNF-αmRNA表达水平:正常对照组相对灰度值为0．25,模型组为0．93,10 mg／kg mEBP治疗组为0．51,10 mg／kg EBP治疗组为0．77,5 mg／kg PMB治疗组为0．43。结论具有抗LPS活性的小分子肽可通过原核表达获得;EBP及mEBP均具有抗LPS活性,其中mEBP拮抗作用更强。
Objective To make endotoxin binding peptide (EBP) and its mutant mEBP in E. coli DH5α, purified and identified its anti-endotoxin / lipopolysaccharide (LPS) activity. Methods (1) DH5α, an engineering strain containing PinpointXa3-EBP and its mutant PinpointXa3-mEBP, was activated and isopropylthiogalactoside (IPTG) was added to induce its expression of biotin fusion protein. The expressed product was isolated and purified. Factor Xa was used to digest the fusion protein EBP and mEBP. The target peptide was purified by affinity chromatography and reverse-phase high-performance liquid chromatography, and the mutant mEBP was identified by amino terminal amino acid sequence analysis. (2) PBMCs were isolated from healthy volunteers. Fluorescein isothiocyanate (FITC) -LPS + different concentrations of EBP or mEBP (2.0, 5.0, 12.5 mg / L) stimulated PBMC to detect the average fluorescence intensity. PBMCs were stimulated with 1 mg / L LPS + 3 concentrations of EBP or mEBP for 3 h, and then supernatants were harvested for detection of tumor necrosis factor (TNF) α and interleukin (IL) 6 concentrations. (3) 25 Kunming mice were divided into normal control group (n = 5): intraperitoneal injection of isotonic saline 0.2 ml; model group (n = 5): mice were inflicted with 20% TBSA third degree burns and intraperitoneal injection of LPS (1 mg / kg). In the treatment group (15 mice), intraperitoneal injection of polymyxin B (PMB, 5 mg / kg) or EBP or mEBP (10 mg / kg). After 6 h, the levels of TNF-α and IL-6 in serum and TNF-αmRNA expression in liver tissues were detected. Results (1) The purity of purified EBP and mEBP reached more than 98%, and the sequence analysis of amino terminal 10 amino acids of mEBP was in line with the expected results. (2) With the increase of mEBP or EBP concentration, the average fluorescence intensity of PBMC surface gradually weakened; at the same concentration, the decrease of average fluorescence intensity after addition of mEBP was more obvious than that of EBP. Compared with stimulation with 1 mg / L LPS, IL-6 and TNF in PBMC culture supernatants were stimulated by 1 mg / L LPS + 12.5 mg / L EBP and 1 mg / L LPS + 3 concentrations of mEBP -α levels were significantly lower (P <0.01). (3) Compared with the model group, the levels of IL-6 and TNF-α in serum of the mice in the treatment group were significantly decreased (P <0.01), and those in the 10 mg / kg mEBP treatment group were lower than those in the EBP-treated group P <0.05). (4) The expression level of TNF-αmRNA in liver of mice: the relative gray value of normal control group was 0.25, the model group was 0.93, the treatment group of mEBP was 0.5 mg / kg and the treatment group was 0.5 mg / kg of EBP Group 0.77,5 mg / kg PMB treatment group 0.43. Conclusion Small molecule peptide with anti-LPS activity can be obtained by prokaryotic expression. Both EBP and mEBP have anti-LPS activity, of which mEBP antagonism is stronger.