反义IL-5载体构建及其对IL-5 mRNA和蛋白表达的影响

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。

Effects of recombinant adeno-virus vector carrying interleukin-5 antisense on the expression of interleukin-5 mRNA and protein in CD4+ T lymphocytes of asthmatic rats

OBJECTIVE: To construct recombinant adeno-associated virus vector carrying antisense interleukin-5 (IL-5) gene (rAAV-asIL-5), and to explore the effects of this virus transfection on IL-5 mRNA and protein in CD(4)(+) T lymphocytes of asthmatic rats. METHODS: The eukaryotic antisense IL-5 expressing vector plasmid of recombinant adeno-associated virus (pasIL-5/rAAV) was constructed by gene recombination technique. The rAAV-asIL-5 particles were produced by co-transfection of pasIL-5/rAAV, pXX2, and pXX6 in package cell 293 through phosphate calcium deposit, and the titers of rAAV-asIL-5 were measured by Southern blot. The rAAV-asIL-5 particles were transfected into CD(4)(+) T lymphocytes obtained by gradient of Ficoll and immunomagnetic beads from the peripheral blood of asthmatic rats. Then IL-5 mRNA in T lymphocytes and IL-5 protein in supernatant of cell culture were determined with semi-quantitative RT-PCR and ELISA respectively. RESULTS: (1) The rAAV-asIL-5 was constructed and identified, and the titer of rAAV-asIL-5 was 1.3 x 10(11) virus particles/ml. (2) The relative ratio A of absorbance (IL-5/beta-actin) of rAAV group was 1.0515 +/- 0.1477, which was significantly lower than that of the control group (1.4271 +/- 0.1655) (n = 6, P < 0.01). (3) The protein level of IL-5 in supernatant of culture of rAAV group was (12.0840 +/- 1.4769) ng/L, significantly lower than that of the control group (15.3590 +/- 1.2685) ng/L, n = 6, P < 0.01]. CONCLUSION: Construction of rAAV-asIL-5 was successful, and transfection of this virus was capable of inhibiting the expression of IL-5 mRNA and protein in CD(4)(+) T lymphocytes of asthmatic rats. The results of this study provide experimental data for further study of gene therapy for asthma.