论文部分内容阅读
目的构建含反义IL-5的重组腺相关病毒(rAAV)asIL-5,观察rAAV asIL-5对哮喘大鼠CD+4T淋巴细胞IL-5mRNA和蛋白表达的影响。方法用基因重组方法构建反义IL5rAAV真核表达载体质粒pasIL-5/rAAV,磷酸钙沉淀法将真核表达载体质粒pasIL-5/rAAV、包装质粒pXX2、辅助质粒pXX6共转染入病毒包装细胞293细胞中,合成rAAV asIL5,Southernblot测重组病毒的滴度;将rAAV asIL5转染经密度梯度法和免疫磁珠法分离的哮喘大鼠CD+4T淋巴细胞,用半定量RT PCR、ELISA分别检测转染后细胞内IL5mRNA及细胞培养上清液中IL-5蛋白的表达水平。结果(1)成功构建并鉴定了rAAV asIL-5,滴度为1.3×1011病毒颗粒/ml;(2)病毒转染孔的相对吸光度值为1.0515±0.1477,低于对照孔(1.4271±0.1655,P<0.01);(3)细胞培养上清液中IL-5的含量病毒转染孔为(12.0840±1.4769)ng/L,低于对照组[(15.3590±1.2685)ng/L,P<0.01]。结论rAAV asIL-5能够抑制哮喘大鼠CD+4T淋巴细胞IL5mRNA和蛋白表达,为研究哮喘的基因治疗提供了实验依据。
Objective To construct antisense IL-5 recombinant adeno-associated virus (rAAV) asIL-5 and investigate the effect of rAAV asIL-5 on the expression of IL-5 mRNA and protein in CD + 4 T lymphocytes of asthmatic rats. Methods The antisense IL5rAAV eukaryotic expression vector plasmid pasIL-5 / rAAV was constructed by gene recombination method. The eukaryotic expression vector plasmid pasIL-5 / rAAV, packaging plasmid pXX2 and helper plasmid pXX6 were cotransfected into virus packaging cells 293 cells, the rAAV asIL5 was synthesized and the titers of the recombinant viruses were measured by Southern blot. The rAAV asIL5 was transfected into CD + 4T lymphocytes of asthmatic rats isolated by density gradient and immunomagnetic beads method. Semi-quantitative RT-PCR and ELISA were used to detect IL-5 mRNA expression in the transfected cells and IL-5 protein expression in cell culture supernatants. Results (1) The rAAV asIL-5 was successfully constructed and identified with a titer of 1.3 × 1011 virus particles / ml. (2) The relative absorbance value of the virus transfection wells was 1.0515 ± 0.1477, lower than that of the control wells (1.4271 ± 0.1655, P <0.01). (3) The content of IL-5 in cell culture supernatant was (12.0840 ± 1.4769) ng / L, which was lower than that in control group [(15.3590 ± 1.2685) ng / L, P <0.01 ]. Conclusion rAAV asIL-5 can inhibit the expression of IL-5mRNA and protein in CD + 4T lymphocytes of asthmatic rats, which provides an experimental basis for studying the gene therapy of asthma.