小鼠β防御素1与甲型流感病毒M2e融合基因表达及其免疫特性研究  

Expression and immunity of mBD_1-M-2e fusion gene

作　　者：张强[1,2] 丰锋[1] 左斌[1] 巩天祥[1] 李婉宜[1] 王保宁[1] 李明远[1] 

机构地区：[1]四川大学华西基础医学与法医学院微生物室,成都610041 [2]四川省遂宁市第一人民医院内一科,遂宁629000

出　　处：《中国人兽共患病学报》2010年第4期299-303,共5页Chinese Journal of Zoonoses

基　　金：国家自然科学基金项目(30671964)资助

摘　　要：目的构建mBD1与M2e融合基因的真核表达载体,并检测其在MDCK细胞中的表达情况,初步探讨其免疫特性。方法利用PCR方法分别扩增mBD1与M2e基因片段,再通过重叠延伸PCR技术(SOE-PCR)将mBD1与M2e通过一段多肽接头Gly4Ser连接为融合基因mBD1-M2e。将该融合基因插入真核表达载体pcDNA3.1(+)获得重组质粒pcDNA3.1(+)/mBD1-M2e,经酶切、PCR及测序鉴定正确后,用脂质体转染MDCK细胞,通过免疫荧光、RT-PCR产物序列分析检测融合蛋白表达情况,最后采用MTT法检测mBD1-M2e融合蛋白刺激淋巴细胞增殖能力。结果经鉴定后证实构建真核表达质粒pcDNA3.1(+)/mBD1-M2e正确,该重组质粒能在MDCK细胞中稳定表达,该细胞培养上清液能刺激淋巴细胞增殖。结论本研究成功构建mBD1-M2e融合基因的真核表达质粒,并证实其融合蛋白能在MDCK细胞中稳定表达,这一结果为进一步研究新型高效通用的流感病毒核酸疫苗奠定了坚实的基础。The objective of this present study was to construct eukaryotic expression vector for mouse β-defensin 1 （mBD1） and influenza A virus M2e fusion gene, as well as study the expression and immunity of fused protein mBD1-M2e in MDCK cells. The mBD1 and M2e genes were amplified from plasmids pcDNA3.1 （＋）/mBD1 and pcDNA3.1 （＋）/M2e respectively. And a polypeptide linker Gly4Ser was used to splice mBD1 and M2e genes by SOE-PCR to construct mBD1-M2e fusion gene. Then the mBD1-M2e fragment was inserted into eukaryotic expressing plasmid pcDNA3.1 （＋） to construct pcDNA3.1 （＋） /mBD1-M2e. Recombinant plasmid was identified by restriction enzymes digestion, PCR and sequencing analysis, then the pcDNA3.1 （＋）/mBD1-M2e was transfected into MDCK cells by PolyFect Transfection Reagent. The expression of mBD1-M2e gene was detected by immunofluorescence assay, RT-PCR and lymphocytes multiplication test with MTT. Expression vector pcDNA3.1 （＋）/mBD1-M2e was constructed successfully in this study. The fused protein could be detected in MDCK cells transfected with pcDNA3.1 （＋）/mBD1-M2e and could stimulate lymphocyte proliferation. The construction of the eukaryotic expression vectors for mBD1-M2e fusion gene, pcDNA3.1 （＋）/ mBD1-M2e, was completely successful and the mBD1-M2e fusion protein could be expressed in MDCK cells stably. This result established a solid foundation for further exploration in efficient and broad-spectrum DNA vaccine against influenza A virus.

关 键 词：甲型流感病毒 Β防御素 融合基因 重叠延伸PCR 免疫荧光 

分 类 号：R373.3[医药卫生—病原生物学]