摘要
目的重组抗菌肽LL-37并探讨其治疗内毒素血症的疗效。方法提取人肺腺上皮SPC-A-1细胞总RNA,RT-PCR扩增出LL-37肽cDNA,构建pGEX-1λT-LL-37重组质粒并转染大肠杆菌JM 109,培养转染细菌并诱导表达融合蛋白,裂解细菌,亲和层析出融合蛋白,裂解融合蛋白生成LL-37肽,高压液相色谱法提纯LL-37;建立BALB/C小鼠内毒素血症模型并观察LL-37的疗效。结果重组肽核苷序列5′-端比天然序列多4个密码子,N-端多4个氨基酸残基。小、大剂量LL-37治疗组(Ⅰ组、Ⅱ组)及内毒素血症组(Ⅲ组)6h存活率分别为62.5%、87.5%、0;Ⅰ、Ⅱ组平均存活时间和最长存活时间均长于Ⅲ组;Ⅰ、Ⅱ组24h后小鼠存活率有差异,量效关系明显。结论重组抗菌肽LL-37可用于内毒素血症的治疗。
Objective： To recombine LL-37 peptide and explore its effects on treating experimental endotoxima. Methods：Total RNA was extracted from human pulmonary gland epithelial cell line SPC-A-1,and a cDNA encoding mature LL-37 peptide was amplified by RT-PCR. The recombinant expression vector pGEX-1λT-LL-37 was constructed and transfected escherichia coli JM109. Transfected bacteria was cultured, and fusion protein expression was induced. The bacteria was lysised by lysozyme and using the freeze/thawn method. Fusion protein was purified by using affinity chromatography and cleaved by thrombin, then produced LL-37peptide. Purified LL-37 was obtained by using high-pressure liquid chromatograph （HPLC）. The sensitivity of BALB/C mice for Lipopolysaccharide （LPS） was induced by employing Actinomycin D. BALB/C mice were divided randomly into three groups （n = 8）,endotoxima control group （Ⅲ）,LL-37 （Ⅱ） and LL-37 （Ⅰ） recombinant peptide treated groups. The mice were bred and observed in same 48 hours,and the survival rates of BALB/C mice were calculated. Results：Four additional codons in 5＇-terminal coding sequence of recombint peptide were observed compared with naive sequence,which were GGA,TCC,TTT and GCC. In accordingly,four amino acid residues in the N-terminal of recombint peptide were observed, which were glycine, serine, phenylalanine and alaine. The actinomycin-sensitized BALB/C mice endotoxima model were constructed. Survival rates of endotoxima（Ⅲ）,LL-37（Ⅱ） and LL-37（Ⅰ） recombinant peptide subgroups were 0,87.5%,62.5% respectively. Both mean surrival time and longest survival time in treated group by LL-37 was longer than that in control group. There were a clear relationship between the dose of LL-37 and survival rate. Conclusion： LL-37 recombinant peptide is effective on treating endotoxima.
出处
《山东医药》
CAS
北大核心
2005年第29期1-3,共3页
Shandong Medical Journal
基金
国家自然科学基金资助项目(39970932)
