CAJ | 학술논문

目的构建干扰素λ1表达载体PCI-dhfr-λ1和连接增强子SP163的表达载体PCI-dhfr-SP163-λ1,并在CHO(dhfr-)细胞中表达.方法在合成的干扰素λ1基因中引入相应酶切位点,并通过融合PCR获得连接增强子SP163-λ1基因,测序正确后将该基因插入表达载体PCI-dhfr,构建重组表达载体PCI-dhfr-λ1和PCI-dhfr-SP163-λ1.将所构建的重组表达载体用脂质体法转入CHO(dhfr-)细胞,通过间接免疫荧光法和Western Blot鉴定了λ1蛋白的表达,应用细胞病变抑制法初步鉴定了λ1蛋白的抗病毒活性.结果成功构建了干扰素λ1真核表达载体PCI-dhff-λ1和PCI-dhfr-SP163-λ1.免疫荧光结果显示分别转染了两种表达载体的CHO(dhfr-)细胞均能表达干扰素λ1蛋白,SP163增强子明显增强了干扰素λ1蛋白的表达,Western Blot结果显示转染SP163的CHO(dhfr-)细胞表达干扰素λ1蛋白.通过细胞病变抑制实验,在转染细胞48h后的细胞培养液中检测到了干扰素λ1的抗病毒活性.结论本研究成功的在CHO(dhfr-)细胞中表达了干扰素λ1,表达的蛋白具有抗病毒活性,为进一步建立稳定表达干扰素λ1的细胞系提供了条件.
목적 구건간우소λ1표체재체PCI-dhfr-λ1화련접증강자SP163적표체재체PCI-dhfr-SP163-λ1,병재CHO(dhfr-)세포중표체.방법 재합성적간우소λ1기인중인입상응매절위점,병통과융합PCR획득련접증강자SP163-λ1기인,측서정학후장해기인삽입표체재체PCI-dhfr,구건중조표체재체PCI-dhfr-λ1화PCI-dhfr-SP163-λ1.장소구건적중조표체재체용지질체법전입CHO(dhfr-)세포,통과간접면역형광법화Western Blot감정료λ1단백적표체,응용세포병변억제법초보감정료λ1단백적항병독활성.결과 성공구건료간우소λ1진핵표체재체PCI-dhff-λ1화PCI-dhfr-SP163-λ1.면역형광결과현시분별전염료량충표체재체적CHO(dhfr-)세포균능표체간우소λ1단백,SP163증강자명현증강료간우소λ1단백적표체,Western Blot결과현시전염SP163적CHO(dhfr-)세포표체간우소λ1단백.통과세포병변억제실험,재전염세포48h후적세포배양액중검측도료간우소λ1적항병독활성.결론 본연구성공적재CHO(dhfr-)세포중표체료간우소λ1,표체적단백구유항병독활성,위진일보건립은정표체간우소λ1적세포계제공료조건.
Objective To construct the eukaryotic expression vector PCI-dhfr-λ1 and PCI-dhfrSP163-λ1 which linked the enhancer SP163 with interferon λ1.Then express the interferon λ1 in CHO (dhfr-) cells.Methods Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon λ1.After sequenced,λ1 and SP163-λ1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-λ1 and PCI-dhfr-SP163-λ1 which was constructed successfully confirming by sequencing.Then the expressing vectors were transfected into CHO(dhfr-) cells using liposome transfection method and interferon λ1 protein was assayed with indirect immunofluorescence and Western Blot.Using cytopathic effect inhibition evaluated the antiviral activity of interferon λ1.Results Successfully constructing the eukaryotic expression vectors of interferon λ1 and the vectors could express interferon λ1.The result of immunofluorescence showed the enhancer developed the expression of interferon λ1.Detecting the interferon λ1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot.The cytopathic effect inhibition showed the expressed interferon λ1 has the antiviral activity.Conclusion Successfully expressed the interferon λ1 in CHO(dhfr-) cells and the protein possesses antiviral activity,which may supply a valuable basis for building the stable cell line of interferon λ1.