摘要
目的设计华游蛇血清PLIγ模拟肽并研究其对PLA2抑制作用。方法用RT-PCR方法克隆华游蛇PLIγ基因并测序进行序列比对。基于华游蛇PLIγ序列设计一个长度为19个氨基酸的多肽。利用Auto dock分子对接分析该19肽与sPLA2相互作用,运用琼脂糖平板法验证其对sPLA2的抑制效果,并通过小鼠实验进一步检测其抗sPLA2出血毒性。利用LPS诱导小鼠Raw264.7细胞炎性反应,检测细胞花生四烯酸含量以评价19肽对细胞sPLA2酶活性抑制作用。结果设计得到的华游蛇PLIγ模拟肽序列为PGLPLSYPNGGGGSVAFRS。该19肽较好地抑制蛇毒PLA2酶活性和出血毒性,并可以显著减少LPS诱导Raw264.7炎性细胞中花生四烯酸含量,IC50为86.3μmol/L。结论本文设计的华游蛇PLIγ模拟肽,具备了天然PLIγ的活性,具有抗细胞炎性反应和蛇毒出血毒作用。
Objective To design a simulating peptide based on Sinonatrix percarinata PLIγ sequence and to explore its inhibitory activity on PLA2. Methods Sinonatrix percarinata PLIγ gene was cloned by RT-PCR and aligned with other published PLIγsequences. Based on the results, a 19aa peptide was designed based on Sinonatrix per carinata PLIγ. Molecular docking was then achieved to investigate the interaction between the 19aa peptide and sPLA2 by Auto-dock software. Experimental validation was conducted by agarose plate method, and anti-hemor rhagic activity against snake venom sPLA2 on mice skin was tested as well. Arachidonic acid content in LPS in duced Raw264. 7 cells was assayed to evaluate the 19aa peptide inhibitory effect on mammal sPLA2 enzyme. Results The mimic 19aa peptide with the sequence as PGLPLSYPNGGGGSVAFRS, behaved dominant pharmaco logical inhibitive properties to enzymatic activity and hemorrhage arachidonic acid content in the LPS induced Raw264.7 inflammat toxicity of sPLA2. It also significantly reduced ory cells with an ICs0 of 86.3 μmol/L, probably by inhibiting the mammal sPLA2. Conclusions The 19aa peptide shows similar pharmacological properties withnatural Sinonatrix percarinata PLIγ, which indicates its anti-inflammatory and anti-hemorrhage potential.
出处
《基础医学与临床》
CSCD
北大核心
2014年第3期289-294,共6页
Basic and Clinical Medicine
基金
国家自然科学基金(31260209)
国家大学生创新实验项目(2012174)
江西省自然科学基金(20114BAB215016)
