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目的 构建含小鼠次级淋巴组织趋化因子 (SLC)cDNA的重组腺相关病毒载体。 方法 以C5 7BL 6J小鼠的淋巴组织为材料抽提总RNA ,用RT PCR方法克隆小鼠SLC的成熟肽基因。PCR产物回收后经EcoRⅠ和XhoⅠ双酶切 ,插入pAAV IRES hrGFP质粒 ,用磷酸钙法 ,与pAAV RC和pHelper三者共同转染HEK2 93细胞 ,包装成重组的病毒颗粒 ,并通过绿色荧光蛋白 (GFP)报告基因荧光检测及抽提病毒DNA ,进行PCR扩增等进一步证实重组病毒的形成。 结果 约 5 0 0bp的小鼠SLC成熟肽基因被成功克隆 ,序列分析表明 ,所克隆的SLC基因与基因库注册的相同 ,重组载体的酶切及测序鉴定表明SLC基因被定向插入。重组载体转染HEK2 93细胞 ,经荧光显微镜和病毒DNA的PCR检测 ,证实已完成对重组病毒的包装 ,并表达GFP。 结论 成功构建了SLC成熟肽基因重组腺相关病毒载体。
Objective To construct a recombinant adeno-associated virus vector containing murine secondary lymphoid chemokine (SLC) cDNA. Methods The total RNA was extracted from lymphoid tissue of C5 7BL 6J mice and the mature peptide gene of mouse SLC was cloned by RT PCR. The PCR product was digested with EcoR I and Xho I and inserted into the pAAV IRES hrGFP plasmid. The recombinant plasmid was co-transfected with pAAV RC and pHelper into HEK2 93 cells by calcium phosphate method and packaged into recombinant virus particles. GFP) reporter gene fluorescence detection and extraction of viral DNA, PCR amplification further confirmed the formation of recombinant virus. Results The mouse SLC mature peptide of about 500bp was successfully cloned. Sequence analysis showed that the cloned SLC gene was identical to that of the gene bank. The restriction endonuclease digestion and sequencing of the recombinant vector indicated that the SLC gene was inserted. Recombinant vector was transfected into HEK2 93 cells. PCR and fluorescence microscopy were used to confirm the packaging of recombinant virus and express GFP. Conclusion SLC mature peptide gene recombinant adeno-associated virus vector was successfully constructed.