BPI-LL37抗菌融合蛋白质表达载体的构建与功能鉴定

Expression vector construction and antibacterial efficacy of BPI-LL37 antibacterial fusion protein

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-increasing protein,BPI）和LL37基因的c DNA中扩增并修饰获得需要的r BPI21和LL37活性片断,构建r BPI21-LL37/LL37-r BPI21融合蛋白的表达载体,构建的表达载体（p LVX-Tight-Puro）上的BPI和LL37基因之间通过柔性片段（GGSGG）连接。之后使用Poly JetTM体外DNA转染试剂转染人肺腺癌细胞A549。通过Western blot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（p LVX-Tight-Puro-BPI-LL-37）构建成功;Western blot检测结果表明融合蛋白r BPI21-LL37/LL37-r BPI21能被真核细胞A549细胞表达,融合蛋白大小介于25～30×10^3;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r BPI21-LL37/LL37-r BPI21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。

Objective To construct the expression vector of bactericidal/permeability-increasing protein （BPI） -LL37 antibacterial fusion protein and prepare the antibacterial fusion protein, so as to explore a novel clinical intervention strategy for curing sepsis. Methods PCR was used to obtain rBPI21 and LL37 active segments from eDNA of BPI gene and LL37 gene. The rBPI21 and LL37 DNA segments were linked with a flexible linker （GGSGG） and cloned into expression vector pLVX-Tight-Puro, and then the recombinant vector was transferred into A549 cells using PolyJetTM in vitro DNA transfeetion reagent. The expression of the antibacterial fusion protein in the A549 cells was identified by Western blotting, and the antibacterial efficacy was assayed by bacterial inhibitory test. Results Expression vector pLVX-Tight-Puro-BPI-LL-37 was successfully constructed and identified by DNA electrophoresis after restriction enzyme digestion and DNA sequencing. Western blot assay showed that rBPI_21-LL37/LL37-rBPI_21 fusion protein [ （25 - 30） × 10^3 ] was stably expressed in the A549 cells. The bacterial inhibition test showed that the fusion protein could inhibit bacterial growth. Conclusion The expression vector pLVX-Tight-Puro-BPI-LL-37 is successfully constructed and introduced into human somatic cells to secrete fusion protein with antibacterial activity, and the fusion protein may be potential drug for treating sepsis.