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目的 构建重组人前脑啡肽原基因(hPPE)腺相关病毒载体(AAV)。方法 构建质粒骨架上含有新霉素抗性基因表达盒的重组腺相关病毒(rAAV)载体质粒pSNAV-hPPE并酶切鉴定,转染金黄地鼠胚胎肾细胞(BHK 21),后用G418筛选培养形成稳定携带rAAV载体的混合细胞株BHK／SH1;再用HSV1-rc／△UI2感染、包装此细胞株并收获病毒载体rAAV-hPPE,并行DNA探针点杂交测定病毒滴度。结果 酶切鉴定pSNAV-hPPE重组成功,病毒滴度为2.5×1012v.g.／ml。结论 获得的rAAV-hPPE滴度高,感染性好,可以用于转基因镇痛的实验研究。
Objective To construct recombinant adeno-associated proenkephalin gene (hPPE) adeno-associated virus vector (AAV). Methods Recombinant adeno-associated virus (rAAV) vector plasmid pSNAV-hPPE containing neomycin resistance gene expression cassette was constructed and digested with restriction endonucleases. The BHK 21 cells were transfected with BHK 21 and then screened with G418 The mixed cell line BHK / SH1 stably carrying the rAAV vector was formed. The cell line was infected with HSV1-rc / △ UI2 and the virus vector rAAV-hPPE was harvested. The virus titer was determined by DNA probe dot blotting. Results The recombinant plasmid pSNAV-hPPE was identified by restriction enzyme digestion and the virus titer was 2.5 × 1012v.g / ml. Conclusion The rAAV-hPPE obtained has high titer and good infectivity and can be used in the experimental study of transgenic analgesia.