高表达毒力因子ROP18的转基因刚地弓形虫株的建立及鉴定

中国寄生虫学与寄生虫病杂志

高表达毒力因子ROP18的转基因刚地弓形虫株的建立及鉴定

蒋宗儒1，安然1，杨杰2，万俐娟1，都建1，3 *

1 安徽医科大学基础医学院生化与分子生物学教研室；2 安徽医科大学第一临床学院；3 安徽病原生物学省级实验室，合肥 230032

出版日期:2015-02-28 发布日期:2015-05-04
基金资助:
国家自然科学基金资助项目（No. 81271864，30801329）；教育部霍英东教育基金会高等院校青年教师基金（No. 131033）；安徽省杰出青年基金资助项目（No. 10040606Y19，1308085JGD11）；国家级大学生创新创业训练计划（No. 201310366008）

Construction and Identification of Transgenic Strain of Toxoplasma gondii High-expressing Virulence Factor ROP18

JIANG Zong-ru1，AN Ran1，YANG Jie2，WAN Li-juan1，DU Jian1，3 *

1 Department of Biochemistry and Molecular Biology，Anhui Medical University，Hefei 230032，China；2 The First Clinical Medical School，Anhui Medical University，Hefei 230032，China；3 Anhui Provincial Laboratory of Microbiology and Parasitology，Anhui Medical University，Hefei 230032，China

Online:2015-02-28 Published:2015-05-04
Supported by:
Supported by the National Natural Science Foundation of China（No. 81271864，30801329），the Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China（No. 131033），the Fund of Anhui Province for Outstanding Youths（No. 10040606Y19，1308085JGD11）and the National College Students′ Innovative Entrepreneurial Training Plan（No. 201310366008）

摘要/Abstract

摘要：

目的构建并鉴定高表达毒力因子ROP18的转基因刚地弓形虫（Toxoplasma gondii）虫株。方法 RT-PCR扩增刚地弓形虫RH株速殖子的ROP18基因片段。将纯化的PCR产物克隆至pCR-Blunt Ⅱ-Topo载体构建 pROP18质粒，PCR扩增ROP18基因序列。将纯化的PCR产物亚克隆至pTUB8-mycGFPPftail-Ty1载体，构建pTUB8-ROP18-Ty1质粒。将质粒电穿孔转染刚地弓形虫RH株，刚地弓形虫悬液转移到长有贴壁细胞的培养瓶中培养，用含25 μg/ml霉酚酸和50 μg/ml黄嘌呤的DMEM培养基筛选高表达ROP18的刚地弓形虫RH株。免疫荧光和蛋白质印迹（Western blotting）检测ROP18在转基因刚地弓形虫株中的表达。吉氏染色比较弓形虫RH株和高表达ROP18的转基因RH株弓形虫分别感染人包皮成纤维细胞（HFF细胞）的增殖情况。20只昆明小鼠均分为2组，每鼠分别腹腔内注射1×103高表达ROP18弓形虫RH株和1×103弓形虫RH株，统计小鼠存活率。结果 RT?鄄PCR扩增出1 665 bp的ROP18基因序列。经酶切和测序鉴定证明pTUB8-ROP18-Ty1质粒构建成功。Western blotting分析结果显示，高表达ROP18 RH株可表达相对分子质量（Mr）为56 000的蛋白，与ROP18-Ty1蛋白预期结果一致。免疫荧光结果发现，ROP18-Ty1定位于弓形虫前端的棒状体。吉氏染色结果表明，高表达ROP18 RH株弓形虫感染HFF细胞6、12和24 h后的速殖子数量分别为100.0±16.9、476.0±31.1和860.0±52.3，均显著高于RH株弓形虫（88.0±16.9，300.0±11.3，675.0±35.4）（P<0.05）。弓形虫毒力实验结果显示，RH株弓形虫感染小鼠在第8天均存活，第14天存活率下降至30%（3/10），至第16天全部死亡；高表达ROP18 RH株弓形虫感染小鼠在第5天均存活，第8天存活率下降至30%（3/10），至第9天全部死亡。结论构建了高表达毒力因子ROP18的转基因弓形虫虫株。

关键词: 弓形虫, ROP18基因, 转基因虫株

Abstract:

Objective To construct a transgenic strain of Toxoplasma gondii high-expressing ROP18. Methods The gene sequence of encoding ROP18 was amplified by RT-PCR with RNA of T. gondii RH strain. The purified PCR product was subcloned into pCR-Blunt Ⅱ-Top vector to construct pROP18. The gene sequence of encoding ROP18 was amplified from pROP18， and subcloned into pTUB8-mycGFPPftail-Ty1. The recombinant plasmid pTUB8-ROP18-Ty1 was electroporated into T. gondii RH strain. Stable transgenic cells were selected in the presence of 25 μg/ml mycophenolic acid and 50 μg/ml xanthine， and parasite clones were isolated by limiting dilution after drug selection. The expression of ROP18 in transgenic parasites was detected by immunofluorescence analysis and Western blotting. Giemsa assay was used to detect the proliferation rate of RH strain and the transgenic strain of T. gondii high-expressing ROP18. Twenty mice were divided into two groups. Each mouse in RH strain group was intraperitoneally injected with 1×103 RH strain, and that of transgenic strain group received 1×103 transgenic high-expressing ROP18 strain. Results The full-length sequence of ROP18 gene（1 665 bp） was amplified by RT-PCR. The recombinant plasmid pTUB8-ROP18-Ty1 was identified by restriction enzyme digestion and sequencing methods. Western blotting analysis showed that the transgenic high-expressing ROP18 strain expressed ROP18-Ty1（Mr 56 000）. Immunofluorescence assay showed that ROP18-Ty1 was localized in the rhoptry of T. gondii. Giemsa assay confirmed that ROP18 protein enhanced the proliferation of T. gondii. On the 6th， 12th， and 24th hour after HFF cells infected with T. gondii， the number of tachyzoites in transgenic strain group was 100.0±16.9， 476.0±31.1， and 860.0±52.3， respectively， higher than that of RH strain group （88.0±16.9， 300.0±11.3， 675.0±35.4）（P<0.05）. In RH strain group， all the mice were survival on the 8th day post-infection， the survival rate on the 14th day was 30% （3/10）， and all died on the 16th day. In transgenic strain group， all the mice were survival on the 5th day post-infection， the survival rate on the 8th day was 30%（3/10）， and all died on the 9th day. Conclusion The transgenic high-expressing ROP18 strain of T. gondii is constructed.

Key words: Toxoplasma gondii, ROP18 gene, Transgenetic parasite

蒋宗儒1，安然1，杨杰2，万俐娟1，都建1，3 *. 高表达毒力因子ROP18的转基因刚地弓形虫株的建立及鉴定[J]. 中国寄生虫学与寄生虫病杂志.

JIANG Zong-ru1，AN Ran1，YANG Jie2，WAN Li-juan1，DU Jian1，3 *. Construction and Identification of Transgenic Strain of Toxoplasma gondii High-expressing Virulence Factor ROP18[J]. Chinese Journal of Parasitology and Parasitic Diseases.

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