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BPI-LL37抗菌融合蛋白质表达载体的构建与功能鉴定

, PP. 589-593

Keywords: 抗菌蛋白,融合蛋白,杀菌/通透性增加蛋白,LL,抗菌,脓毒症

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Abstract:

目的构建杀菌/通透性增加蛋白和LL37抗菌蛋白的融合蛋白(BPI-LL37)表达载体,获得针对脓毒症治疗的多效抗菌融合蛋白,为脓毒症治疗提供新的措施。方法通过PCR方法从杀菌/通透性增加蛋白(bactericidal/permeability-increasingprotein,BPI)和LL37基因的cDNA中扩增并修饰获得需要的rBPI21和LL37活性片断,构建rBPI21-LL37/LL37-rBPI21融合蛋白的表达载体,构建的表达载体(pLVX-Tight-Puro)上的BPI和LL37基因之间通过柔性片段(GGSGG)连接。之后使用PolyJetTM体外DNA转染试剂转染人肺腺癌细胞A549。通过Westernblot检测真核细胞A549表达融合蛋白的水平,通过抑菌试验验证此融合蛋白的分泌与抗菌能力。结果经限制内酶切酶切、DNA电泳和DNA测序证明融合蛋白表达载体(pLVX-Tight-Puro-BPI-LL-37)构建成功;Westernblot检测结果表明融合蛋白rBPI21-LL37/LL37-rBPI21能被真核细胞A549细胞表达,融合蛋白大小介于25~30×103;抑菌试验进一步验证此融合蛋白具有抑制细菌生长的生物学效应。结论构建的两种rBPI21-LL37/LL37-rBPI21表达载体能够成功转染入人的体细胞,并表达和分泌具有抗菌活性的融合蛋白,该融合蛋白有潜力成为治疗脓毒症的新药。

References

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