摘要
目的构建携带大鼠SOCS3基因的重组腺病毒载体并转染大鼠血管平滑肌细胞,评价其转染效果,为血管增殖性疾病转基因治疗的实验研究奠定基础。方法用BamHⅠ和EcoRⅠ将目的基因pUC57-Simple-rSOCS3及腺病毒穿梭载体pYr-adshuttle-4(带GFP标记)行双酶切。回收酶切质粒的目的片段进行连接,产物转化至DH5α感受态。提取质粒酶切鉴定正确后测序。通过LR体外同源重组将rSOCS3表达框构建至pAd/PL-DEST腺病毒表达载体上。将腺病毒线性化后转染HEK293细胞包装腺病毒,PCR鉴定此腺病毒中是否含有rSOCS3基因。放大培养并收集病毒,TCD法测定病毒滴度。将此病毒感染大鼠血管平滑肌细胞,荧光显微镜观察感染效率,real-time PCR及免疫印迹检测SOCS3mRNA及蛋白表达。结果 rSOCS3腺病毒穿梭质粒酶切鉴定正确,测序与rSOCS3基因库中的序列完全相符。PCR鉴定pAd/PL-DEST腺病毒表达载体成功携带目的基因rSOCS3。TCD法测定的最终滴度为2×1010pfu/mL。荧光显微镜观察此病毒感染血管平滑肌细胞的效率在80%以上,real-time PCR及免疫印迹检测结果显示血管平滑肌细胞中SOCS3mRNA及蛋白表达明显上调。结论成功构建携带大鼠SOCS3基因的腺病毒载体,并在血管平滑肌细胞中高表达,为血管增殖性疾病转基因治疗的实验研究奠定了基础,具有一定的应用价值。
Objective To construct the recombinant adenovirus vector carrying rat SOCS3 gene and assess its transfection efficiency in vascular smooth muscle cells（VSMCs）.Methods The rat SOCS3 plasmid（pUC57-Simple-rSOCS3）and advenovirus shuttle plasmid（pYr-adshuttle-4）containing green fluorescent protein（GFP）reporter gene were cleaved by restriction endonuclease BamHⅠ and EcoR Ⅰ.The target gene fragments were connected together to generate a recombinant plasmid pYr-adshuttle-4-rSOCS3 that was transfected into E.coli DH5α.The plasmid was confirmed to be constructed as expectation by enzyme digestion and sequencing.The plasmid pYr-adshuttle-4-rSOCS3 and type 5 adenovirus backbone plasmid pAd/PL-DEST were reconstructed by homologous recombination processes to obtain rat SOCS3 recombinant adenovirus vector which named pYrAd-rSOCS3.The plasmid pYrAd-rSOCS3 was linearized by PacⅠ and subsequently transfected into HEK293 cells for packaging and amplification.After purification,virus titer was determined by tissue culture infectious dose 50（TCID50）.Polymerase chain reaction（PCR）was used to confirm the existence of recombinant rat SOCS3 gene.The primary rat VSMCs were infected by pYrAd-rSOCS3.After 24 h,the expression of GFP was observed under the fluorescent microscopy.SOCS3 mRNA and protein expression was detected by using real-time PCR and Western blot.Results Restriction endonuclease and PCR analysis demonstrated that the recombinant adenovirus vector was constructed correctly.hEF1a and CMV promoter existed in the expression vector.The virus titer reached 2×1010 pfu/mL.Transfection efficiency of recombinant adenovirus in VSMCs was more than 80%.Real-time PCR and Western blot revealed that SOCS3 mRNA and protein expression was up-regulated significantly in the infected VSMCs.Conclusion In this study,we successfully constructed a recombinant adenovirus vector that carries rat SOCS3 gene and can be helpful for further research on the effects and mechanisms of the SOCS3 gene on the vascular proliferation diseases.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2012年第5期556-561,共6页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
