重组腺病毒载体pAd-4-hMASP-2的构建  

Construction of recombinant adenovirus vector pAd-4-hMASP-2

作　　者：张国超[1] 雒艳萍[1] 陆小铃 王倩[1] 董信芳[1] 曹明强[1] 于红娟[1] 马兴铭[1,2] 

机构地区：[1]兰州大学基础医学院,甘肃兰州730000 [2]甘肃省新药临床前重点实验室,甘肃兰州730000

出　　处：《中国生物制品学杂志》2016年第11期1141-1145,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金项目(31370912);兰州大学中央高校基本科研业务费专项资金(lzujbky-2014-146)资助

摘　　要：目的利用p Yr-adshuttle-4质粒构建重组腺病毒载体p Ad-4-h MASP-2。方法应用PCR技术扩增甘露糖凝集素相关丝氨酸蛋白酶-2(human mannan-binding lectin associated serine protease-2,h MASP-2)基因,经Bam HⅠ和Eco RⅠ双酶切后与p Yr-adshuttle-4质粒连接,构建穿梭质粒p Yr-ads-4-h MASP-2,在LR酶作用下与腺病毒骨架载体p Ad/PL-DEST进行体外同源重组,获得重组腺病毒载体p Ad-4-h MASP-2,转染HEK293细胞,获得重组腺病毒r Adh MASP-2,应用酶切、PCR扩增及基因测序进行鉴定,并测定病毒滴度。将r Ad-h MASP-2感染小鼠,实时荧光定量PCR法检测h MASP-2基因m RNA在小鼠肺组织中的表达。结果重组穿梭质粒p Yr-ads-4-h MASP-2经双酶切及测序证实构建正确,通过同源重组获得了重组腺病毒载体p Ad-4-h MASP-2,扩增出滴度为1.5×109 PFU/ml的重组腺病毒颗粒r Ad-h MASP-2,其能在小鼠肺组织中表达。结论成功获得重组腺病毒载体p Ad-4-h MASP-2和重组腺病毒颗粒r Ad-h MASP-2,为体内外研究h MASP-2的活性提供了有效的转基因载体。Objective To construct recombinant adenovirus vector p Ad-4-h MASP-2 by using p Yr-adshuttle-4. Methods Human mannan-binding lectin associated serine protease-2（h MASP-2）gene was amplified by PCR, digested with Bam HⅡand Eco RⅡ, and inserted into plasmid p Yr-adshuttle-4. The constructed shuttle plasmid p Yr-ads-4-h MASP-2 and p Ad /PL-DEST adenovirus backbone vector was reconstructed through homologous recombination in mediation of LR enzyme.The obtained plasmid p Ad-4-h MASP-2 was transfected to HEK293 cells for packing. The obtained recombinant adenovirus r Ad-h MASP-2 was identified by restriction analysis, PCR and gene sequencing, and determined for titer. BALB / c mice were infected with the recombinant adenovirus, and the expression of h MASP-2 m RNA in lung was determined by realtime fluorescent quantitative PCR. Results The shuttle plasmid p Yr-4-h MASP-2 was constructed correctly as proved by restriction analysis and sequencing. Recombinant adenovirus vector p Ad-4-h MASP-2 was obtained by homologous recombination, based on which the recombinant adenovirus particles with a titer of 1. 5 × 10^9 PFU / ml was amplified, and expressed in the lung tissues of mice. Conclusion Both recombinant adenovirus vector p Ad-4-h MASP-2 and recombinant adenovirus r Ad-h MASP-2 were successfully obtained, which provided an effective transgenic vector for in vitro and in vivo studies on h MASP-2.

关 键 词：甘露糖凝集素相关丝氨酸蛋白酶-2 同源重组 腺病毒 

分 类 号：Q782[生物学—分子生物学]