人α防御素的稳定表达和活性鉴定
人α防御素,,人α防御素;CHOdhfr细胞;甲氨喋呤,0引言,1材料和方法,2结果,3讨论
Expression and functional characterization of human αdefensinsLIU Juan1 ,ZHAI Song1, DU DeWei1,WANG LinXu1,WANG ShaoYang1,WANG DinCheng2, SUN YongTao1
1Center of Diagnosis & Treatment for Infectious Diseases of PLA, 2Department of Clinical Laboratory, Tangdu Hospital, Fourth Military Medical University, Xian 710038, China
【Abstract】 AIM: To prepare secretary human αdefensin 1 and αdefensin 3 and to determine the functional characterization of their antimicrobial activities. METHODS: Eukaryotic expression vector pcDNA31/V5HisTOPOHNP1 and pcDNA3.1/V5HisTOPOHNP3 were cotransfected with plasmid pDCH1P11 (carrying dhfr gene ) into CHOdhfr- cells respectively and the recombinant protein was verified by ELISA. The stable expression cell strains were screened by selective medium containing G418 and then serially passaged in methotraxate (MTX) for gene amplification. The expressions were analyzed by ELISA, RT-PCR and IFA, and the bacteriastatic activities were assayed in vitro. RESULTS: The expression level of αdefensin1 ranged from 1885 ~ 4746 mg/L·48 h per 106 cells, while that of αdefensin3 was 1689 ~3557 mg/L·48 h. 303 bp segments were amplified from stably tranfectant clones and strong fluorescence was visible in cell plasma around the nuclear in the stably transfectant cells by IFA. KB disc agar diffusion test found obvious bacteriastatic diffusion on MH plate of E.coli. CONCLUSION: Human αdefensin 1 and αdefensin 3 are effectively secreted, which are of high antimicrobial activities. Our results lay the basis for the development of antiHIV1 activity by recombinant αdefensin 1 and 3 . ......
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