内毒素结合肽及其突变体的表达纯化和抗内毒素／脂多糖活性研究

Study on the expression,purification and the anti-endotoxin activity of human endotoxin binding peptide and its mutant

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 express endotoxin binding peptide and its mutant in E coli DH5α and detect the antiendotoxin activity of the purified peptides. Methods （ 1 ） E coli DH5αt containing pinpointXa3- EBP and pinpointXa3-mEBP was activated by IPTG to express biotin fusion protein. The fusion proteins were purified, and then digested by factor Xa for isolation of EBP and mEBP. The target peptide was purified with affinity chromatography and reversed-phase HPLC. Sequences of 10 amino acids at N-terminal were used for identification of mEBP. （2） PBMCs were isolated from blood of normal people, and they were stimulated with 5 mg/L FITC-LPS plus 2.0,5.0 and 12.5 mg/L EBP or mEBP. Then the mean fluorescent intensity was detected. PBMC was also stimulated with 1 mg/L LPS plus 2.0, 5.0 and 12.5 mg/L EBP or mEBP for 5 hours for the detection of the TNF-α and IL-6 level in the supernatant. （3） Thirty-five Kunming mice were randomized into normal control （ n = 5, with intraperitoneal injection of 0.2 ml isotonic saline） , model group（n = 5, with intraperitoneal injection of LPS and 20% TBSA full-thickness burns） , and treatment group （ n = 15, with intraperitoneal injection of 5 mg/kg PMB or 10 mg/kg EBP or mEBP after burns）. The serum contents of TNF-α and IL-6, and TNF-α mRNA level in hepatic tissue in each group were determined 6 hours after treatment. Results （ 1 ） EBP and mEBP were obtained after Xa digestion of biotin fusion protein, with purity reaching above 98%. The sequence of 10 amino acid at N-terminal was in accord with what expected. （2） The fluorescent intensity was decreased followed by an increase in mEBP or EBP concentration. Compared with normal PMBC, IL-6 and TNF-α level in the supernatant were obviously lowered in 1 mg/L LPS＋12.5 mg/L EBP group and 1 mg/L LPS＋2.0 , 5.0, 12.5 mg/L mEBP groups （ P 〈 0. 01 ）. （3） The serum level of IL-6 and TNF-α in the therapeutic groups were obviously lower than that in model group （ P 〈0. 01 ） , and the levels of these cytokines were significantly lower in 10 mg/kg mEBP group than that in 10 mg/kg EBP group （ P 〈0.01 ） , but they were similar to that in 5 mg/kg PMB treatment group （ P 〉0.05）. （4） Relative optical density of TNF-α mRNA in control, model, 10 mg/kg mEBP, 10 mg/kg EBP and 5 mg/kg PMB groups was 0. 25, 0. 93, 0. 51, 0. 77 and 0. 43, respectively. Conclusion Endotoxin binding peptides can be obtained by procaryon expression. Both EBP and mEBP have anti-LPS activity, but mEBP is more effective.