摘要
目的构建携带EGFP标志人白细胞介素10(hIL-10)基因腺病毒载体。方法以pSNAV2.0-hIL-10重组质粒为模板,PCR扩增hIL-10基因,获得hIL-10cDNA片段,并酶切连接到带有含强绿色荧光蛋白基因(EGFP)标记的pDC316-IRES-EGFP-lacZalpha载体质粒上,PmeI线性化重组质粒pDC316-hIL-10-IRES-EGFP,与腺病毒包装系统AdMax转染293包装细胞,包装产生复制缺陷型重组腺病毒,经反复感染293细胞扩增病毒后,进行离子交换法纯化病毒,并测定病毒颗粒数、滴度。结果经PCR鉴定、限制性酶切分析及序列测定,证明已正确构建重组穿梭质粒pDC316-hIL-10-IRES-EGFP和重组腺病毒质粒Ad-hIL-10-IRES-EGFP。扩增纯化后,测得重组腺病毒颗粒数为3.2×1011VP/ml,OD260/OD280值约为2.0,滴度为1.1×1010TCID50/ml。结论成功构建了重组腺病毒质粒Ad-hIL-10-IRES-EGFP,为进一步研究hIL-10基因奠定实验基础。
Objective To construct the recombinant adenovirus vector carrying human interleukin-10（hIL-10） marked enhanced green fluourescent protein（EGFP）. Methods The hIL-10 gene was constructed by PCR with pSNAV 2.0-hIL-10 recombinant plasmid as template, enzyme digestion and ligation and inserted the obtained hIL-10 cDNA fragment into shuttle vector pDC316- IRES-EGFP-lacZalpha linearization with PmeI and supercoil adenovirus skeleton plasmid pAdMax for homologous recombination. The 293 cells were transfected with the obtained recombinant adenovirus vector Ad-hIL-10-IRES-EGFP in mediation of liposme and propagated for producing defective recombinant adenovirus by repeatedly infecting 293 cells and purifying virus by ion exchange method. The virus particles were counted and the purity was titered. Results Both recombinant shuttle plasmid pDC316-hIL-10-IRES-EGFP and recombinant adenovirus vector Ad-hIL-10-IRES-EGFP were correctly constructed as proved by PCR, restriction analysis and sequencing. After propagation and purification,the virus particle count, OD260/OD280 and titer of recombinant adenovirus were 3.2 ×10^11VP/ml,2.0 and 1.1 ×10^10 TCID 50/ml,respectively. Conclusion Recombinant adenovirus vector Ad-hIL-10-IRES-EGFP has been successfully constructed, which may provide a foundation for the further study on adenovirus vector mediated immunosuppressive therapy with hIL-10 gene.
出处
《临床麻醉学杂志》
CAS
CSCD
北大核心
2009年第1期58-60,共3页
Journal of Clinical Anesthesiology
基金
福建省科技厅青年人才创新基金项目(项目编号2007F3029)
福建省科技厅自然科学基金项目(项目编号2008J0091)
