VP22与EGFP、3种弓形虫抗原融合表达重组质粒的构建与鉴定  

Construction and Identification of Cell Penetrating Peptides VP22 Gene Fused with Enhanced Green Fluorescent Protein and Three Antigens of Toxoplasma gondii

作　　者：王娜[1,2] 刘彦双 刘宁 金超 张喜路 金月[2] 薛书江[1] WANG Na LIU Yan-shuang LIU Ning JIN Chao ZHANG Xi-lu JIN Yue XUE Shu-jiang(Agricultural College, Yanbian University, Yanji 133002, China Longjing Animal Husbandry Station, Longjing 133400, China Longjing Livestock Authority, Longjing 133400, China Meihekou Animal Husbandry Station, Meihekou 135000, China Dalian Jinzhou New Area District Animal Health Supervision, Dalian 116100, China)

机构地区：[1]延边大学农学院,延吉133002 [2]龙井市畜牧站,龙井133400 [3]龙井市畜牧业管理局,龙井133400 [4]梅河口市畜牧总站,梅河口135000 [5]大连市金州新区动物卫生监督所,大连116100

出　　处：《中国畜牧兽医》2016年第11期2859-2865,共7页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然基金(31460657);吉林省科技厅青年科研基金(20150520129JH)

摘　　要：为了探索新型弓形虫疫苗的传递系统,本试验分别构建了细胞渗透肽VP22与增强型绿色荧光蛋白(EGFP)融合表达的重组质粒(pcDNA-VP22-EGFP),以及细胞渗透肽VP22与3种弓形虫抗原融合表达的重组质粒(pcDNA-VP22-SAG1、pcDNA-VP22-GRA4、pcDNA-VP22-AMA1)。经PCR鉴定、酶切鉴定和序列测定后,将重组质粒pcDNA-VP22-EGFP转染COS7细胞,通过荧光显微镜和RT-PCR检测,验证VP22-EGFP基因在COS7细胞中的表达情况。PCR鉴定、酶切鉴定和序列测定结果显示所有重组质粒均构建正确。转染72h后的细胞,在荧光显微镜下成功观察到绿色荧光。RT-PCR扩增得到了大小为1 449bp的目的条带,与预期结果一致。结果表明,重组质粒构建成功。To study a novel vaccine of Toxoplasma gondii,the recombinant plasmids that cell penetrating peptides VP22 gene fused with one enhanced green fluorescent protein（EGFP）（pcDNAVP22-EGFP）and three antigens of T.gondii（pcDNA-VP22-SAG1,pcDNA-VP22-GRA4 and pcDNA-VP22-AMA1）were constructed,respectively.The recombinant plasmids were identified by PCR,restriction endonuclease digestion and DNA sequencing.The pcDNA-VP22-EGFP plasmid was transfected into COS7 cells,and the expression of VP22-EGFP in COS7 cells was confirmed by fluorescence microscope and RT-PCR.The results showed that all recombinant plasmid were constructed correctly.Green fluorescence was observed successfully under the fluorescence microscope in transfected cells after 72 h.The gene fragment of 1 449 bp was obtained by RTPCR.The results indicated that the recombinant plasmids were constructed successfully.

关 键 词：细胞渗透肽VP22 弓形虫 真核表达 

分 类 号：S85[农业科学—兽医学]