人源溶菌酶基因在杆状病毒系统中的表达

目的在杆状病毒系统中表达人源溶菌酶(Human lysozyme,hLY)基因,为其临床应用和大规模生产奠定基础。方法将人工合成的含信号肽和不含信号肽的溶菌酶基因克隆至重组杆状病毒表达系统的载体中,构建重组穿梭质粒,转染sf9细胞,SDS-PAGE和Western blot验证蛋白的表达,溶菌试验验证表达产物的溶菌酶活性。结果重组克隆质粒PFastBacHTA-signal-lysozyme和PFastBac HTA-lysozyme的PCR产物分别可见444和393 bp的目的基因条带,测序结果证实重组克隆质粒构建正确。PCR结果显示Lysozyme-1、Lysozyme-2、slysozyme-1、slysozyme-2、slysozyme-3、slysozyme-4号重组穿梭质粒可扩增出目的条带。sf9-PHLY和sf9-PHSLY的表达产物经12%SDS-PAGE分析,分别可见相对分子质量约17 500和19 300的特异蛋白条带;表达的两种重组蛋白均可与鼠抗6×His单抗特异性结合;表达产物具有溶菌酶活性。结论成功在杆状病毒表达系统中表达了hLY基因,为其临床应用和大规模生产奠定了基础。

Expression of human lysozyme gene in baculovirus system

Objective To express human lysozyme(hLY)gene in baculovirus system.Methods The signal peptide-containing and signal peptide-free hLY genes were cloned into the vector in baculovirus system,and the constructed recombinant shuttle plasmids were transfected to sf9 cells.The expressed protein was identified by SDS-PAGE and Western blot,and determined for lysozyme activity by bacteriolysis test.Results The target gene bands,at lengths of 444 and 393 bp,were amplified from recombinant cloning plasmids PFastBac HTA-signal-lysozyme and PFastBac HTA-lysozyme by PCR,respectively.Sequencing results showed that the recombinant cloning plasmids were constructed correctly.Target gene bands were amplified from the recombinant shuttle plasmids for Lysozyme-1,Lysozyme-2,slysozyme-1,slysozyme-2,slysozyme-3 and slysozyme-4 by PCR.The expressed products of sf9-PHLY and sf9-PHSLY showed specific protein bands with relative molecular masses of about 17 500 and 19 300 on 12% SDSPAGE profile,respectively.Both the expressed recombinant proteins showed specific binding to mouse monoclonal antibody against 6 × His as well as lysozyme activity.Conclusion The hLY gene was successfully expressed in baculovirus system,which laid a foundation of clinical application and large-scale production of hLY.