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目的 构建及验证荧光素酶标记ROP18过表达弓形虫ROP18?Ty1?LUC RH株,为研究弓形虫毒力及相关宿主免疫调控机制奠定良好基础.方法 利用PCR技术扩增出目的片段ROP18?Ty1和带ROP18?Ty1同源序列接头的质粒骨架,通过重组克隆系统构建既能表达ROP18?Ty1又含荧光素酶标记的质粒P?ROP18?Ty1?LUC.将质粒P?ROP18?Ty1?LUC和筛选质粒P?TUB?CAT?SAG1通过电转染的方法转染到RH株弓形虫,经过氯霉素筛选和极限稀释得到目的弓形虫ROP18?Ty1?LUC RH株.构建成功的弓形虫ROP18?Ty1?LUC RH株在基因、蛋白、动物水平进行验证.结果 利用重组克隆系统成功构建了质粒P?ROP18?Ty1?LUC,利用双酶切实验和测序验证了序列的正确性.以弓形虫ROP18?Ty1?LUC RH株基因组为模板,用特异引物能PCR扩增出1704 bp大小的ROP18?Ty1和1644 bp大小的LUC基因;用特异抗体anti?Ty1通过Western blot检测外源蛋白ROP18?Ty1的表达,在56000 Mr处有单一条带;用荧光素酶报告基因检测系统检测LUC的表达,利用多标记酶标仪检测到荧光的发射;通过活体成像系统检测ROP18?Ty1?LUC RH弓形虫株在小鼠体内的增殖,与对照组相比感染了ROP18?Ty1?LUC RH弓形虫株的小鼠体内photon值明显更多(P<0.05);最后小鼠毒力实验也验证了ROP18?Ty1?LUC RH弓形虫毒力比RH?LUC弓形虫的毒力强(P<0.05).结论 成功构建和验证荧光素酶标记的ROP18过表达弓形虫ROP18?Ty1?LUC RH株,可以在动物个体水平更客观统计ROP18对弓形虫增殖速率的影响.“,”Objective This study was aimed to construct and verify the ROP18?Ty1?LUC RH strain Toxoplasma gondii(T. gondii),which overexpresses both ROP18?Ty1 and luciferase,and lay the foundation to understand the regulatory mecha?nism of Toxoplasma virulence and host immunity mediated by ROP18. Methods The ROP18?Ty1 gene and plasmid frame?work were amplified by PCR. P?ROP18?Ty1?LUC plasmid and P?TUB?CAT?SAG1 selection plasmid were cotransfected into RH T. gondii. ROP18?Ty1?LUC RH T. gondii was obtained by chloramphenicol selection and limiting dilution. The features of transgenic parasite ROP18?Ty1?LUC RH were verified at gene and protein level and in vivo. Results P?ROP18?Ty1?LUC plasmid was constructed by recombinant clone system and verified by restriction enzyme digestion and sequencing. PCR ap?proach was used to amplify the exogenous ROP18?Ty1 and LUC genes,and fragments of 1704 bp ROP18?Ty1 and 1644 bp LUC were amplified using ROP18?Ty1?LUC RH DNA as template. Western blot with anti?Ty1 was used to detect the exoge?nous ROP18?Ty1 expression,and a single band of 56000 Mr was observed. Luciferase reporter assay system was used to de?termine the activity of luciferase and high luciferase activity was detected in ROP18?Ty1?LUC RH T. gondii. Moreover,re?sults from in vivo imaging analysis showed that the proliferation rate of ROP18?Ty1?LUC RH T. gondii in C57BL/6J mice was significantly faster than that of WT RH(P<0.05),and the survival rate of ROP18?Ty1?LUC RH?infected KM mice was signifi?cantly lower than that of WT RH?infected mice(P<0.05). Conclusion Toxoplasma gondii ROP18?Ty1?LUC RH strain was successfully constructed and verified,it is benefits to objective study ROP18 influence proliferation rate of T. gondii in vivo.