人α防御素稳定表达及活性鉴定.docVIP

人α防御素稳定表达及活性鉴定 　 作者：刘娟　翟嵩　杜德伟　王临旭　王少扬　汪定成　孙永涛 【关键词】 人α防御素 　　Expression and functional characterization of human αdefensins 　　【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#12539;48 h per 106 cells, while that of αdefensin3 was 1689 ~3557 mg/L#12539;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 . 　　【Keywords】 human αdefensin; CHOdhfr;methotrexate 　　【摘要】 目的： 制备有较高生物学活性的分泌型人α防御素1与α防御素3,并初步鉴定其抗菌活性；方法： 将真核表达质粒pcDNA31/V5HisTOPOHNP1,3与含有dhfr基因的质粒pDCH1P11共转染入CHOdhfr，ELISA检测到培养上清中重组蛋白的表达，对阳性表达的细胞进行G418筛选，对其中9个表达量较高的阳性克隆进行氨甲喋呤（MTX）加压扩增, ELISA法、RTPCR法及间接免疫荧光（IFA）分析蛋白表达情况，并同步测定其体外抗菌活性.结果： α防御素1的表达量是1885~4746 mg/L#12539;48 h#12539;106个细胞； α防御素3的表达量是1689~3557 mg/L#12539;48h#12539;106 个细胞.用RTPCR从稳定表达的细胞株中扩增出全长约303 bp的片段；IFA 显示稳定转染的细胞胞质核周质中有强的绿色荧光；KB纸片琼脂扩散法显示α防御素1,3在大肠埃希菌MH平板上形成了明显的抑菌圈.结论： 成功高效地表达了分泌型的α防御素1,3，并具有良好的生物学活性，为进一步探讨α防御素1,3对HIV1的抑制作用提供了物质基础. 　　【关键词】 人α防御素；CHOdhfr细胞；甲氨