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...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.展开更多
钙蛋白酶抑制蛋白(Calpastatin,CAST)专一抑制钙蛋白酶(Calpain,CAPN)活性,参与调节牛肉的嫩化及肌内蛋白的水解,其编码基因是肉质性状的主要候选基因。本研究根据牛CAST基因5'UTR区(GenBank No.AY834771)和3'UTR区(Gen Bank No...钙蛋白酶抑制蛋白(Calpastatin,CAST)专一抑制钙蛋白酶(Calpain,CAPN)活性,参与调节牛肉的嫩化及肌内蛋白的水解,其编码基因是肉质性状的主要候选基因。本研究根据牛CAST基因5'UTR区(GenBank No.AY834771)和3'UTR区(Gen Bank No.DQ991097)序列设计两对引物,用PCR-SSCP方法对鲁西黄牛和渤海黑牛共305个样本进行了单核苷酸多态性分析。结果显示,CAST基因5'UTR和3'UTR区存在PCR-SSCP多态,在5'UTR区存在6437(C/T)、6477(G/A)和6614(G/T)3个SNP位点,表现为AA、AB、BB、BC和AD 5种基因型;在3'UTR区存在359(T/C)和366(A/G)2个SNP位点,表现为EE、EF、FF和EG 4种基因型。经检验表明,鲁西黄牛和渤海黑牛均处于非Hardy-Weinberg平衡状态;群体遗传多态性分析表明这两种牛的多态信息含量均为中度多态。展开更多
文摘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.
