缩短5′-UTR序列可以提高hGH基因在昆虫细胞中的表达

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.

中国猪瘟兔化弱毒(脾淋毒)基因组5′-UTR的克隆和二级结构分析

据已发表的 CSFV C-株序列 ,设计合成了 5′- UTR特异性 PCR引物对 P1- N 1及半套式下游引物N1′。从兔脾组织中提取总 RNA,应用 RT- PCR及 Half- nested PCR成功地扩增出了 CSFV5′- U TR,进一步将该片段克隆到 p MD- 18T载体质粒并进行了序列测定。与 Shimen株、AL D株、GPE-株、Holland C-株和 Russian C-株相应区段的比较分析表明 ,其同源性分别为 96 .2 %,95 .7%,96 .0 %,98.7%和 95 .4 %。 5′- U TR中发现的隐性 AUG起始密码子及茎环二级结构 ,可能在多聚蛋白前体的翻译以及病毒的复制中起着重要作用。