表达H9N2禽流感病毒NP蛋白的巨型艾美耳球虫转染载体的构建及体外转染
onstruction of transfection vector expressing AIV nucleoprotein and its transient expression in Eimeria maxima
目的：构建表达禽流感NP蛋白的鸡巨型艾美耳球虫双框载体并验证其功能。方法：从禽流感病毒（H9N2北京株）中扩增得到NP基因，在本实验室已构建好的表达红绿荧光蛋白的双框载pMEAAssRA基础上，采用Age I，Not I进行双酶切，用NP基因片断取代红荧光基因片断，得到表达NP蛋白的pMEAAssNPmA双框载体。将构建好的质粒经核穿孔方法转染巨型艾美耳球虫子孢子，并在MDBK细胞中进行培养。结果：经酶切和PCR鉴定及DNA测序，确认表达NP蛋白的双框载体构建完全正确。核转24h后可在荧光显微镜下观察到表达荧光的子孢子。结论：成功构建表达NP蛋白的球虫转染载体，为下一步表达NP抗原的转基因巨型艾美耳球虫的构建奠定了基础。
Objective: Construct transfection vector expressing H9N2 avian influenza virus nucleoprotein by a double-cassette expression vector strategy. Method: The NP gene amplified from H9N2 virus (Beijing strain) was cloned into Age I and Not I digested pMEAAssRA vector, generating the pMEAAssNPmA plasmid. Then the linearized vector was transfected into the sporozoites of Eimeria maxima via Nucleofector transfection. Results: The double-cassette expression vector was sucessfully constructed and sporozoites expressing yellow fluorescent protein were observed. Conclusion: NP gene from the H9N2 virus can be expressed by E. maxima, encouraging the development of transgenic E. maxima expressing genes from influenza virus.
刘贤勇、索勋、谢丽、索静霞、李媛、殷光文
分子生物学生物工程学遗传学
NP蛋白双框载体巨型艾美耳球虫体外转染
NP proteindouble-cassette vectorEimeria maximatransient expression
刘贤勇,索勋,谢丽,索静霞,李媛,殷光文.表达H9N2禽流感病毒NP蛋白的巨型艾美耳球虫转染载体的构建及体外转染[EB/OL].(2012-09-17)[2025-08-11].http://www.paper.edu.cn/releasepaper/content/201209-177.点此复制
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