山羊SKIP基因重組腺病毒載體的構建

摘要：試驗利用Gateway技術構建含山羊SKIP基因的重組腺病毒載體。首先采用PCR擴增山羊SKIP基因連接入pcDNA3.1（+），將酶切回收的SKIP產物片段克隆至腺病毒穿梭載體pYr-adshuttle-2 獲得重組質粒pYr-adshuttle-2-SKIP。從該重組質粒上，利用LR同源重組將SKIP-EGPF表達框轉移至腺病毒骨架質粒pAd/PL-DEST，獲得重組腺病毒質粒pAd-SKIP。該質粒經pacI 線性化后轉染HEK293A細胞包裝病毒，獲得重組腺病毒rAd-SKIP。重組腺病毒分別經酶切和PCR 等方法進行鑒定。熒光顯微鏡觀察轉染效果。結果證實腺病毒載體中含有SKIP基因的目的片段，熒光顯微鏡發現該重組腺病毒能在C2C12細胞中高效表達。表明成功構建了同時表達SKIP蛋白和綠色熒光蛋白的重組腺病毒，為下一步開展關于SKIP基因的功能研究奠定了基礎。

關鍵詞：重組腺病毒；山羊SKIP基因；基因過表達；感染細胞

中圖分類號：Q78 文獻標識碼：A 文章編號：0439-8114（2013）21-5258-04

Construction of A Recombinant Adenovirus Vector pAd-SKIP

XIONG Qi，ZHANG Nian，SUO Xiao-jun，LI Xiao-feng，LIU Yang，CHEN Ming-xin

（Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding / Institute of Animal Husbandry and Veterinary， Hubei Academy of Agricultural Sciences， Wuhan 430064，China）

Abstract： In this study， the Gateway technology was used to construct a recombinant adenovirus vector pAd-SKIP. The SKIP gene was amplified first by PCR and inserted into pcDNA3.1（+） vector， then cloned into adenovirus adshuttle vector pYr-adshuttle-2 to obtain recombinant plasmid pYradshuttle-2-SKIP. The expression cassette of SKIP-EGPF was transferred to pAd/PL-DEST with LR recombinant reaction to acquire recombinant adenovirus plasmid pAd-SKIP-EGFP. The correct clone was linearized and tranformed into 293A cells. Recombinant adenovirus pAd-SKIP was obtained and identified. The transfection efficiency was observed under the fluorescent microscope. The results confirmed that the recombinant adenovirus vector contained goat SKIP gene. This vector was found to infect C2C12 cells efficiently. It indicated that the recombinant adenovirus expressing SKIP gene and green fluorescence was successfully constructed， laying the foundation for further study.

Key words： recombinant adenovirus vector； goat SKIP gene； gene overexpression； infected cells

5′肌醇磷酸酶（SKIP）由Ijuin等[1]首先發現并分離，在動物各組織中廣泛表達，尤其在心臟骨骼肌和腎臟中高表達，因此SKIP被稱為骨骼肌和腎臟富含的5′肌醇磷酸酶。SKIP被鑒定為通過水解PI（3，4，5）P3為PI（3，4）P2而調控胰島素信號的5′-脂質磷酸酶。PI（3，4，5）P3的下游PI（3，4，5）P3/Akt信號在成肌分化進程中有重要作用[2-4]。SKIP雜合突變小鼠比目魚肌和股四頭肌的質量顯著提高[5]也提示SKIP具有調節骨骼肌纖維發育的功能。因此有必要進一步研究該基因在體內外參與骨骼肌生長發育的調控機制。本試驗采用基因工程技術，成功構建了SKIP基因重組腺病毒，為下一步在體內外研究SKIP對成肌分化的作用機制以及SKIP基因的功能奠定了基礎。

1 材料與方法