[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].


CONCLUSIONS
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.
Gao
Bao-an
BA
Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Xiong
Wei-ning
WN
Xu
Yong-jian
YJ
Zhang
Zhen-xiang
ZX
Cao
Yong
Y
Tang
Yi-jun
YJ
Ye
Tao
T
Du
Chun-ling
CL
D000818:Animals; D001249:Asthma / Q000276:immunology / Q000628:therapy*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D015848:Interleukin-5 / Q000096:biosynthesis* / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D015848:Interleukin-5; D012333:RNA, Messenger