CAJ | 학술논문

　　基于四膜虫小核rDNA改造得到的pD5H8载体，在转染进接合生殖时期的四膜虫后能发育成9000~10000拷贝的大核rDNA，因此pD5H8载体是四膜虫异源基因表达的研究热点；但其载体上匮乏合适的克隆位点和无启动子结构阻碍了该载体的进一步发展。本研究在四膜虫启动子HSP70-2和终止子HSP70-1之间，通过稀有酶切位点I-Sc e玉导入细菌绿色荧光蛋白(HGFP)这一筛选标记，由此组成的DNA片段(HSP70-25' UTR-I-Sce玉-HGFP-I-Sce玉-HSP70-13' UTR)整合进pD5H8，将之改造成pD5H8-HGFP表达载体。来自真核生物的绿色荧光蛋白(GFP)藉由I-Sc e玉酶切位点一步导入pD5H8-HGFP表达载体后，在四膜虫中大量表达且具有生物学活性。因此，改造后的pD5H8-HGFP表达载体拥有一步法引入异源基因，几乎无酶切位点的限制，有效的筛选标记，高效的异源基因表达能力，以及环境友好、便捷可控等诸多优点，这对于深入开展四膜虫基因功能的过表达研究和开发异源基因在四膜虫这一真核表达系统中的应用奠定了技术基础。
　　기우사막충소핵rDNA개조득도적pD5H8재체，재전염진접합생식시기적사막충후능발육성9000~10000고패적대핵rDNA，인차pD5H8재체시사막충이원기인표체적연구열점；단기재체상궤핍합괄적극륭위점화무계동자결구조애료해재체적진일보발전。본연구재사막충계동자HSP70-2화종지자HSP70-1지간，통과희유매절위점I-Sc e옥도입세균록색형광단백(HGFP)저일사선표기，유차조성적DNA편단(HSP70-25' UTR-I-Sce옥-HGFP-I-Sce옥-HSP70-13' UTR)정합진pD5H8，장지개조성pD5H8-HGFP표체재체。래자진핵생물적록색형광단백(GFP)자유I-Sc e옥매절위점일보도입pD5H8-HGFP표체재체후，재사막충중대량표체차구유생물학활성。인차，개조후적pD5H8-HGFP표체재체옹유일보법인입이원기인，궤호무매절위점적한제，유효적사선표기，고효적이원기인표체능력，이급배경우호、편첩가공등제다우점，저대우심입개전사막충기인공능적과표체연구화개발이원기인재사막충저일진핵표체계통중적응용전정료기술기출。
Vector of pD5H8, modified from rDNA of Tetrahymena small nuclear, can develop into rDNA of large nuclear with 9 000~10 000 copies after introducing into conjugation cells, which make it to be an ideal expression vector of exogenous genes. However, both insufficient appropriate clone sites and lacking promoter region hinder the further application of pD5H8. In this study, a bacterium green fluorescence protein gene (HGPF) was inserted between the 5' UTR (untranslated regions) of HSP70-2 gene and the 3' UTR of HSP70-1 gene, and these three DNA fragment were connected by the rare restriction enzyme site of I-SceⅠ. The constructed DNA fragment (HSP70-2 5' UTR-I-SceⅠ-HGFP-I-SceⅠ-HSP70-1 3' UTR) was then integrated into the pD5H8 vector, which became an expression vector of pD5H8-HGFP. The HGFP fragment of pD5H8-HGFP vector could be one-step replaced by an eukaryote green fluorescence protein (GFP) gene via restriction enzyme site of I-Sc eⅠ, and the recombinant vector was able to express a large number of products with biological activity in Tetrahymena. Therefore, the modified vec-tor of pD5H8-HGFP had many advantages, such as one-step introduction of exogenous gene, unlimited restriction enzyme sites, effective selection marker, efficient expression level of exogenous gene, environment-friend and con-venient controllable, which will facilitate the research of gene over-expression, and pave the way for wildly applica-tion of exogenous genes expression in Tetrahymena.