ABSTRACT
Objective To screen the optimal RNA interference sequence of acute monocytic leukemia associated antigen 22 (MLAA-22) gene in order to study gene function of it. Methods MLAA-22 coding sequence (CDS) was cloned by reverse transcription polymerase chain reaction (RT-PCR) and the CDS was inserted in to pEGFP-N1-3FLAG vector to construct eukaryotic expression vector of MLAA-22. At the same time, four RNA interference sequences were designed and cloned to the vector. Expression vector and RNA interference vector were co-transfected into 293T cells, and the optimal RNA interference sequence was screened by fluorescence and Western blot analyses. Results MLAA-22 eukaryotic expression vector pEGFP-N1-3FLAG and four RNA interference vectors were successfully constructed. After co-transfected 293T cells, KD2 was selected as the optimal interference sequence of MLAA-22. Conclusion KD2 is an optimal interference sequence for targeting MLAA-22 antigen gene.
ABSTRACT
Th-2-biased immune responses are known to play a key role in the pathogenesis of atopic dermatitis. In particular, the macrophage-derived chemokine CCL22 is directly implicated in Th-2-associated skin inflammatory reactions, and its levels are significantly elevated in serum and are correlated with disease severity in atopic dermatitis. In this study, we tested the development of genetic therapeutic options to treat atopic dermatitis using bacteria expressing miRNA. We constructed a recombinant strain of Salmonella typhimurium expressing CCL22 miRNA (ST-miRCCL22) for the in vivo knockdown of CCL22. The CCL22 gene was downregulated with CCL22 miRNA in activated lymphocytes. In mice with a cutaneous disease similar to atopic dermatitis, interleukin-4 was inhibited and interferon-gamma was induced after treatments with ST-miRCCL22. Furthermore, CCL22 levels were suppressed in the atopic mice treated with ST-miRCCL22. These results suggest that ST-miRCCL22 may be an effective genetic agent for treating atopic dermatitis.