摘要
目的构建表达人神经生长因子β基因片段的重组腺病毒Ad CMV-h NGF-β。方法以健康人白细胞中DNA为模板,扩增h NGF-β基因片段,导入p GEM-T-Easy质粒,获得重组质粒p GEM-T/h NGF-β。经酶切鉴定后与复制缺陷型腺病毒穿梭质粒p ACCMV-p Lp A构建重组腺病毒穿梭质粒p ACCMV-h NGF-β。酶切鉴定后再与辅助包装质粒PJM17共转染293细胞,经同源重组后,构建复制缺陷型重组腺病毒Ad CMV-h NGF-β。对Ad CMV-h NGF-β进行基因鉴定,并测定滴度及在He La细胞中的表达。结果顺利克隆出h NGF-β基因片段,构建的重组质粒p GEM-T/h NGF-β和重组腺病毒穿梭质粒p ACCMVh NGF-β,经酶切鉴定实际结果与理论推导符合。构建的复制缺陷型重组腺病毒Ad CMV-h NGF-β滴度为1.2×1012pfu/ml,并能在He La细胞中表达。结论以h NGF-β基因片段为基础成功构建了重组腺病毒Ad CMV-h NGF-β,测试表明该重组病毒具有高滴度活性和良好的感染人类细胞、表达目的蛋白的能力。
Objective To construct the recombinant adenovirus AdCMV-hNGF-β.Methods With healthy human leukocyte DNA as a template,PCR was used to amplify hNGF-β coding region fragments.The gene fragments of hNGF-β were inserted into pGEM-T-Easy vector to form the recombinant plasmid pGEM-T/hNGF-β.The pGEM-T/hNGF-β and replicated-defective adenovirus shuttle plasmid pACCMV-pLpA were constructed into adenovirus shuttle plasmid pACCMV-hNGF-β.And then the pACCMV-hNGF-β and auxiliary packaging plasmid PJM17 were co-transfected into the 293 cells.The recombinant adenovirus AdCMV-hNGF-β was constructed after homologous recombination in 293 cells.The gene of AdCMV-hNGF-β was identified,and the hNGF-β expression in HeLa cells infected with AdCMV-hNGF-β was tested by immunohistochemistry.Results The hNGF-β gene fragments were successfully cloned from blood leukocyte DNA.The recombinant plasmid pGEM-T/hNGF-β and the recombinant adenovirus shuttle plasmid pACCMV-hNGF-β were also successfully constructed,and identified consistent with the theoretical ones.The titer of AdCMV-hNGF-β was 1.2 × 1012 pfu/ml,and the hNGF-β was expressed in the HeLa cells.Conclusion On this basis of the gene fragments of hNGF,the recombinant adenovirus AdCMV-hNGF-β is successfully constructed.The results suggest AdCMV-hNGF-β can express hNGF-β and have the effect of viral infection and high titer of activity and the ability of infecting human cell and expressing the target protein.
出处
《山西医科大学学报》
CAS
2014年第11期1013-1017,1117,共6页
Journal of Shanxi Medical University
