东北农业大学学报
東北農業大學學報
동북농업대학학보
JOURNAL OF NORTHEAST AGRICULTURAL UNIVERSITY
2012年
12期
26-31
,共6页
祁光宇%刘萍%刘斌%王凡%武发菊%杜平%董金杰%黄银君%牟克斌%刘学荣
祁光宇%劉萍%劉斌%王凡%武髮菊%杜平%董金傑%黃銀君%牟剋斌%劉學榮
기광우%류평%류빈%왕범%무발국%두평%동금걸%황은군%모극빈%류학영
T7RNA聚合酶%EGFP和RED荧光基因%猪睾丸细胞%ST/T7RNA细胞系
T7RNA聚閤酶%EGFP和RED熒光基因%豬睪汍細胞%ST/T7RNA細胞繫
T7RNA취합매%EGFP화RED형광기인%저고환세포%ST/T7RNA세포계
T7 RNA polymerase%EGFP and RED fluorescent gene%swine testis cell%ST/ T7 RNA cell line
根据GenBank中登录的T7RNA聚合酶基因参考序列,设计合成了1对特异性引物扩增对T7RNA聚合酶基因进行扩增,将测序正确的T7RNA聚合酶基因和真核表达载体pIRES2-EGFP双酶切后进行连接构建pIRES2-EGFP-T7RNA RNA质粒.再将构建正确的pIRES2-EGFP-T7RNA质粒经用脂质体法转染猪睾丸细胞,通过G418筛选和单细胞克隆化,同时构建pET-32a-RED原核表达质粒载体,用其检测T7启动子控制下的红色荧光蛋白的表达.结果表明,建立的ST/T7RNA细胞系经20次传代仍然能稳定表达T7 RNA聚合酶.结果显示,成功建立能稳定表达T7 RNA聚合酶的猪睾丸细胞系,为猪瘟病毒反向遗传操作平台奠定了基础.
根據GenBank中登錄的T7RNA聚閤酶基因參攷序列,設計閤成瞭1對特異性引物擴增對T7RNA聚閤酶基因進行擴增,將測序正確的T7RNA聚閤酶基因和真覈錶達載體pIRES2-EGFP雙酶切後進行連接構建pIRES2-EGFP-T7RNA RNA質粒.再將構建正確的pIRES2-EGFP-T7RNA質粒經用脂質體法轉染豬睪汍細胞,通過G418篩選和單細胞剋隆化,同時構建pET-32a-RED原覈錶達質粒載體,用其檢測T7啟動子控製下的紅色熒光蛋白的錶達.結果錶明,建立的ST/T7RNA細胞繫經20次傳代仍然能穩定錶達T7 RNA聚閤酶.結果顯示,成功建立能穩定錶達T7 RNA聚閤酶的豬睪汍細胞繫,為豬瘟病毒反嚮遺傳操作平檯奠定瞭基礎.
근거GenBank중등록적T7RNA취합매기인삼고서렬,설계합성료1대특이성인물확증대T7RNA취합매기인진행확증,장측서정학적T7RNA취합매기인화진핵표체재체pIRES2-EGFP쌍매절후진행련접구건pIRES2-EGFP-T7RNA RNA질립.재장구건정학적pIRES2-EGFP-T7RNA질립경용지질체법전염저고환세포,통과G418사선화단세포극륭화,동시구건pET-32a-RED원핵표체질립재체,용기검측T7계동자공제하적홍색형광단백적표체.결과표명,건립적ST/T7RNA세포계경20차전대잉연능은정표체T7 RNA취합매.결과현시,성공건립능은정표체T7 RNA취합매적저고환세포계,위저온병독반향유전조작평태전정료기출.
Based on the published gene sequence of T7 RNA polymerase (T7 RNAP) in GenBank, a pair of specific primers was designed for amplifying the complete gene of T7 RNAP from E.coli BL21 (DE3) cells, the sequencing of the T7 RNA polymerase gene and the eukaryotic expression vector pIRES2-EGFP by BglⅡ and EcoRⅠ restriction enzyme digestions and inserted into the eukaryotic expression vector pIRES2-EGFP to construct the recombinant plasmid pIRES2-EGFP-T7 RNAP. ST cell line stably expressing T7 RNA polymerase was established by using liposome method, G418 selection and cell cloning, at the same time construction of prokaryotic expression vector pET-32a-RED for detection of the red fluorescent protein expression under the control of T7 promoter. The results showed that the ST line of stably expressing T7 RNAP was successfully established. The ST line of stably expressing the T7 RNAP gene could provide a good platform for reverse genetic manipuLation of porcine RNA viruses in vitro.
