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“九五”国家科技攻关计划(961204601)

作品数:2 被引量:17H指数:1
相关作者:张宝珠俞新大赵东明郜鹏耿朝晖更多>>
相关机构:南开大学更多>>
发文基金:“九五”国家科技攻关计划更多>>
相关领域:生物学更多>>

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用Bac-to-Bac杆状病毒系统表达人生长激素被引量:16
2002年
利用Bac to Bac杆状病毒载体表达系统将人生长激素 (humangrowthhormone ,hGH)基因cDNA克隆至转移载体pFastBac1中 ,得到pFastBac hGH ,再将其转化进入含穿梭载体Bacmid的受体菌DH10Bac中 ,发生转座作用 ,得到含hGH基因的重组穿梭载体rBacmid hGH .纯化DNA ,直接转染培养的昆虫细胞Sf9,得到重组病毒rAcV Bac hGH .经酶切PCR及Southern杂交鉴定 ,hGH基因正确地插入病毒基因组的多角体蛋白基因启动子下 ,SDS PAGE测得产物蛋白分子量为 2 2kD左右 .用免疫化学发光法测得转染上清中hGH表达水平可达 18μg ml ,与用传统的BEVS表达hGH相比 ,转染上清中hGH表达水平提高 4 0
耿朝晖郜鹏刘莹赵东明张宝珠俞新大李建民
关键词:BAC-TO-BAC系统昆虫细胞HGH杆状病毒生长激素
缩短5′-UTR序列可以提高hGH基因在昆虫细胞中的表达被引量:1
2002年
The regulation of foreign gene expression in Insect-Baculovirus Expression System is very complex. In this report, the effect of 5′-UTR in the expression of hGH gene in cultured Sf9 cells was examined. A 18 bp length in the end of 5′-UTR of hGH (human Growth Hormone, hGH) cDNA including a stem-loop structure was deleted by PCR. The truncated hGH cDNA, Δ1hGH was cloned in pFastBac1, named pFast-Bac-Δ1hGH. After transforming into E.coli. DH10Bac, which have a shuttle vetor-Bacmid, the Δ1hGH was integrated into Bacmid by site-specific transposition, and an expression vector, rBacmid-Δ1hGH DNA was acquired. By transfecting the cultured Sf9 cells with the recombinant expression vector DNA, pure recombinant virus, rAcV-Bac-Δ1hGH was obtained, and hGH gene was expressed. Immuno-blot and Chemiluminescent assay revealed that the expressed hGH had normal immunological activity, the ammount of hGH expression level in Sf9 cell supernatant infected with rAcV-Bac-Δ1hGH containing the truncated 5′UTR was four to five times higher than that infected with rAcV-Bac-hGH.
耿朝晖刘莹郜鹏赵东明李澍俞新大张宝珠
关键词:昆虫细胞
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