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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 the recombinant adenovirus vector p Ad-4-h MASP-2 by using p Yr-adshuttle-4 plasmid. Methods The gene of mannan-binding lectin associated serine protease-2 (h MASP-2) was amplified by PCR and double digested with Bam HⅠand Eco RⅠ, then ligated with p Yr-adshuttle- 4 plasmid pYr-ads-4-h MASP-2 was constructed and cloned into vector pAd-PL MAS-2. The recombined adenoviral vector pAd-4 -H MASP-2 was transfected into HEK293 cells to obtain the recombinant adenovirus r Adh MASP-2. The recombinant adenovirus AdH MASP-2 was identified by enzyme digestion, PCR amplification and gene sequencing, and the virus titer was determined. Mice infected with Ad-h MASP-2 were infected with h MASP-2 gene by real-time fluorescence quantitative PCR. Results The recombinant shuttle plasmid pYr-ads-4-h MASP-2 was confirmed by double enzyme digestion and sequencing. The recombinant adenovirus vector pAd-4-h MASP-2 was obtained by homologous recombination, and the titer was amplified Was 1.5 × 109 PFU / ml recombinant adenovirus particles r Ad-h MASP-2, which was expressed in mouse lung tissue. Conclusion The recombinant adenoviral vector p Ad-4-h MASP-2 and recombinant adenovirus Ad-h MASP-2 were successfully obtained and provided an effective transgenic vector for studying the activity of h MASP-2 in vitro and in vivo.