犬贾第虫病毒转染载体介导的绿色荧光蛋白在犬贾第虫体内的表达

中国寄生虫学与寄生虫病杂志 ›› 2007, Vol. 25 ›› Issue (1): 8-40.

犬贾第虫病毒转染载体介导的绿色荧光蛋白在犬贾第虫体内的表达

陈丽凤1,2;李建华1;刘全3;赵月平1;曹利利1;张西臣1

1 吉林大学畜牧兽医学院，长春130062；2 河北科技师范学院, 秦皇岛 066000;3 军事医学科学院军事兽医研究所，长春130062

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-02-28 发布日期:2007-02-28
通讯作者: 张西臣

Giardia canis Virus Transfection Vector-Mediated Expression of Green Fluorescent Protein in the Parasite

CHEN Li-feng1;LI Jian-hua1;LIU Quan2;ZHAO Yue-ping1;CAO Li-li1;ZHANG Xi-chen1

1 College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China; 2 Hebei Normal University of Science ＆ Technology, Qinhuangdao 066000, China; 3 Veterinary Institute, Academy of Military Medical Sciences, Changchun 130062, China

Received:1900-01-01 Revised:1900-01-01 Online:2007-02-28 Published:2007-02-28
Contact: ZHANG Xi-chen

摘要/Abstract

摘要： 目的构建犬贾第虫病毒（Giardia canis virus，GCV）转染载体。方法根据GCV基因组（DQ238861）的转录起始位点、复制起始位点及包装位点的序列特征和表达外源基因的顺式作用元件，将绿色荧光蛋白（GFP）基因替换GCV基因部分编码区，构建GCV基因与 GFP基因的嵌合体，并置T7启动子之下。用T7 RNA聚合酶体外转录后经电穿孔转染犬贾第虫滋养体，并用荧光显微镜检测GFP表达情况，间接ELISA测定转染后GFP的表达量。结果构建了犬贾第虫病毒重组质粒pGCV634/GFP/GCV2174, 经SacⅠ和NotⅠ双酶切得到约2.0和3.5 kb两条带，与预计值相符。由其介导的绿色荧光蛋白在犬贾第虫体内得到了高效表达，其表达量在转染后第1天达高峰(A₄₉₀=1.8)；以后随时间的延长而逐渐下降，14 d后绿色荧光信号基本消失。结论成功构建了犬贾第虫病毒转染载体，为贾第虫细胞基因表达调控的研究提供了方法。

关键词: 犬贾第虫病毒, 转染载体, 绿色荧光蛋白

Abstract: Objective To construct Giardia canis virus (GCV) transfection vector. Methods According to transcriptional start site, replication origin and packaging site of GCV genome (DQ238861), a system was developed for the expression of a foreign gene in this organism by flanking the green fluorescent protein (GFP) gene with the fragments of GCV positive-strand RNA. The transcript of the construct was synthesized in vitro with T7 RNA polymerase and used to transfect GCV-infected trophozoites by electroporation. Results The recombinant plasmid pGCV634/GFP/GCV2174 was constructed. The expression of green fluorescent protein mediated by GCV transfection vector in Giardia canis peaked at 1 d after electroporation(A₄₉₀=1.8), and slowly decreased until 14 d post-transfection. Conclusion The engineered GCV vector can be successfully used to introduce and efficiently express a heterologous gene in the eukaryotic microorganism.

Key words: Giardia canis virus, Transfer vector, Green fluorescent protein

陈丽凤;李建华;刘全;赵月平;曹利利;张西臣. 犬贾第虫病毒转染载体介导的绿色荧光蛋白在犬贾第虫体内的表达[J]. 中国寄生虫学与寄生虫病杂志, 2007, 25(1): 8-40.

CHENLi-feng;LIJian-hua;LIUQuan;ZHAOYue-ping;CAOLi-li;ZHANGXi-chen. Giardia canis Virus Transfection Vector-Mediated Expression of Green Fluorescent Protein in the Parasite[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2007, 25(1): 8-40.

[1]	赵楠, 李青, 胡薇. 慢病毒介导的绿色荧光蛋白基因在日本血吸虫体内的成功表达[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(3): 213-217.
[2]	辛玥1，赵楠1，李青1*，胡薇1,2. 慢病毒介导的绿色荧光蛋白基因在日本血吸虫体内的表达和检测[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(6): 8-517-521.
[3]	李学荣1, 2 *, 吴银娟1, 2, 尚梅1, 2, 李晔1, 2, 徐劲1, 2, 余新炳1, 2, ATHAR Chishti3. 恶性疟原虫信号肽肽酶-绿色荧光蛋白突变株的建立及其在疟原虫体内的表达分析[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(4): 2-253-257.
[4]	吴亮, 袁异玮, 姜旭淦, 傅行礼, 逯兆喜, 涂国华, 李礼, 陈盛霞, 周红. 刚地弓形虫绿色荧光蛋白突变株的构建[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(6): 8-439-442.
[5]	魏超君, 卢思奇, 曹利静, 田喜凤. 蓝氏贾第鞭毛虫alpha-8贾第素特异性锤头状核酶-GCV重组载体的构建[J]. 中国寄生虫学与寄生虫病杂志, 2011, 29(5): 1-321-326.
[6]	曹利静;冯宪敏;魏超君;王凤云;张西臣;卢思奇. 蓝氏贾第鞭毛虫丙酮酸激酶特异性锤头状核酶-GCV重组载体的构建[J]. 中国寄生虫学与寄生虫病杂志, 2010, 28(2): 4-102.
[7]	付雍;丁艳;周桃莉;陈继德;彭小红;徐文岳. 蚊期和红内期持续表达绿色荧光蛋白的重组约氏疟原虫[J]. 中国寄生虫学与寄生虫病杂志, 2009, 27(6): 8-491.
[8]	陈丽凤;;李建华;邹亚学;赵月平;曹利利;张西臣. 犬贾第虫病毒转染载体介导的锤头状核酶对KRR1基因体外转录体切割效果的研究[J]. 中国寄生虫学与寄生虫病杂志, 2007, 25(4): 3-284.
[9]	陈丽凤;李建华;张西臣;刘全;赵月平;曹利利. 犬贾第虫携病毒株体外纯培养的建立[J]. 中国寄生虫学与寄生虫病杂志, 2006, 24(4): 5-265.
[10]	司进;朱荫昌;曹利民;王晓婷;梁幼生;管晓虹. 弓形虫主要表面抗原1单链抗体与绿色荧光蛋白的融合表达及其生物活性初步观察[J]. 中国寄生虫学与寄生虫病杂志, 2006, 24(3): 1-165.
[11]	陆惠萍;孙树汉. 绿色荧光蛋白及其在分子寄生虫学研究中的应用[J]. 中国寄生虫学与寄生虫病杂志, 1997, 15(1): 17-53.