Recombinant Adeno-associated Virus Vector Inhibits The Expression Of IL-5 In T Lymphocytes Of Asthmatic Rats

ObjectiveTo investigate effects of IL-5 antisense oligodeoxynucleotides of different sequences on the expression of IL-5 and IL-5 mRNA in CD4⁺ T lymphocytes of asthmatic rats.MethodsThrough liposome, different antisense oligodeoxynucleotides( AS-1 to translation initiation, AS-2 to Exon-2, AS-3 to translation terminal) and blank control were transfected in CD4⁺ T lymphocytes of asthmatic rats which were obtained from rats blood by gradient of Ficoll and immunomagnetic beads. Cultured for 4 hours in 6-well plates, these T cells were mixed with ConA. After 28 hours, IL-5 in supernatant of cells culture and IL-5 mRNA in T lymphocytes were determined with ELISA and semi-quantitative RT-PCR respectively.Results(1) Relative ratio A of absorbance(IL-5/β-actin) of AS-1, AS-2 and AS-3 are 0.4259 ± 0.0468, 0.4330 ± 0.0847 and 0.4710 ± 0.0823 respectively, and lower significantly than A of control group (1.0288 ±0.1233) (P<0.01), but not different distinctly among them (P > 0.05);(2) Contents of IL-5 in supernatant of culture of AS-1, AS-2 and AS-3 are 5.3063 ± 0.6151 (pg/ml) ,4.7345±1.2285 (pg/ml) and 5.9465 + 1.4520 (pg/ml) respectively, and lower significantly than that of control group (12.1633 ±3.4257pg/ml) (P<0.01) , but not different distinctly among them (P>0.05) . ConclusionIL-5 antisense oligodeoxynucleotides can inhibit the expression of IL-5 and IL-5 mRNA in CD4⁺ T lymphocytes of asthmatic rats; the inhibitory effects of three kinds of antisense oligodeoxynucleotides are not different significantly.Part 2 Construction and transfection of eukaryotic antisense IL-5 expressing vector plasmid of recombinant adeno-associated virusObjectiveTo construct eukaryotic antisense IL-5 expressing vector plasmid of recombinant adeno-associated virus (pasIL-5/rAAV), and explore effects of this plasmid transfection on IL-5 mRNA and protein in CD4⁺ T lymphocytes of asthmatic rats.MethodsThe pasIL-5/rAAV was constructed by gene recombination technique. Through liposome, the plasmid was transfected in CD4⁺ T lymphocytes of asthmatic rats that were obtained from rats' blood by gradient of Ficoll and immunomagnetic beads. Then IL-5 mRNA in T lymphocytes and IL-5 protein in supernatant of cells culture were determined with semi-quantitative RT-PCR and ELISA respectively.Results(1) It was verified by partial nucleotide sequencing analysis and restriction endonuclease digestion that the constructed pasIL-5/rAAV was correct;(2) Relative ratio A of absorbance (IL-5/β-actin) of plasmid group is 0.2683 ±0.0600, and lower significantly than A of control group (0.5358 ±0.1222) (n=6,P< 0.01) ;(3) Contents of IL-5 in supernatant of culture of plasmid group is 5.9437 ± 0.7088 (pg/ml) , and lower significantly than that of control group (8.1117± 0.8840pg/ml) (n=6,P<0.01) .ConclusionConstruction of pasIL-5/rAAV is successful, and transfection of this plasmid can inhibit the expression of IL-5 mRNA and protein in CD4⁺ T lymphocytes of asthmatic rats. Results of this study provide the data of further studying the antisense IL-5 rAVV vector and gene therapy of asthmatic.Part 3 Effects of recombinant adeno-associated virus vectorcarrying antisense IL-5 on the expression of I IL-5 mRNA andprotein in CD4⁺ T lymphocytes of asthmatic ratsObjectiveTo construct recombinant adeno-associated virus vector carrying antisense IL-5 gene (rAAV-asIL5), and explore effects of this virus transfection on IL-5 mRNA and protein in CD4⁺ T lymphocytes of asthmatic rats.MethodsThe eukaryotic antisense IL-5 expressing vector plasmid of recombinant adeno-associated virus (pasIL-5/rAAV) was constructed by gene recombination technique. The rAAV-asIL5 particles were produced by co-transfection of pasIL-5/rAAV, pXX2, and pXX6 in package cell 293 through phosphate calciumdeposit, and the titers of rAAV-asIL5 were measured by Southern blot; the rAAV-asIL5 particles were transfected in CD4⁺ T lymphocytes of asthmatic rats that were obtained from rats' blood by gradient of Ficoll and immunomagnetic beads. Then IL-5 mRNA in T lymphocytes and IL-5 protein in supernatant of cells culture were determined with semi-quantitative RT-PCR and ELIS A respectively.Results(1) The rAAV-asIL5 was constructed and identified success. The titers of rAAV-asIL5 were 1.3 × 10¹¹ virus particles/ml;(2) Relative ratio A of absorbance (IL-5/ β -actin) of rAAV group is 1.0515 ±0.1477, and lower significantly than A of control group (1.4271 ±0.1655) (n=6, P <0.01) ;(3) Contents of IL-5 in supernatant of culture of rAAV group is 12.0840± 1.4769 (pg/ml) , and lower significantly than that of control group (15.3590± 1.2685pg/ml) (n=6,P<0.01) .ConclusionConstruction of rAAV-asIL5 is successful, and transfection of this virus can inhibit the expression of IL-5 mRNA and protein in CD4⁺ T lymphocytes of asthmatic rats. Results of this study can provide the data of further studying gene therapy of asthmatic.SummaryRecombinant adno-associated virus into which antisense IL-5 sequence was inserted was constructed successfully by the technique of genetic engineering. We discovered that rAAV could inhibit the expression of IL-5 mRNA and protein in CD4⁺ T lymphocytes of asthmatic rats. Our experimental data can be provided to asthmatictic gene therapy.