人α防御素__(期刊论文)

人α防御素的稳定表达和活性鉴定

http://www.100md.com 《第四军医大学学报》 2005年第2期

人α防御素,,人α防御素；CHOdhfr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 αdefensins

    LIU Juan1 ,ZHAI Song1, DU DeWei1,WANG LinXu1,WANG ShaoYang1,WANG DinCheng2, SUN YongTao1

    1Center of Diagnosis & Treatment for Infectious Diseases of PLA, 2Department of Clinical Laboratory, Tangdu Hospital, Fourth Military Medical University, Xian 710038, China

    【Abstract】 AIM: To prepare secretary human αdefensin 1 and αdefensin 3 and to determine the functional characterization of their antimicrobial activities. METHODS: Eukaryotic expression vector pcDNA31/V5HisTOPOHNP1 and pcDNA3.1/V5HisTOPOHNP3 were cotransfected with plasmid pDCH1P11 (carrying dhfr gene ) into CHOdhfr- 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 αdefensin1 ranged from 1885 ~ 4746 mg/L·48 h per 106 cells, while that of αdefensin3 was 1689 ~3557 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. KB disc agar diffusion test found obvious bacteriastatic diffusion on MH plate of E.coli. CONCLUSION: Human αdefensin 1 and αdefensin 3 are effectively secreted, which are of high antimicrobial activities. Our results lay the basis for the development of antiHIV1 activity by recombinant αdefensin 1 and 3 . ......

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