[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.