人β防御素3和植物des-pGlu1-Brazzein融合蛋白的纯化、活性分析及其乳糖诱导表达条件优化  

Purification and Activity Analysis of a Fusion Protein of Human β Defensin 3 and Plant des-pGlu1-Brazzein and Optimization of Lactose Inductive Conditions

作　　者：李春丽[1,2] 阮晖[1] 玛丽平[1] 何国庆[1] 

机构地区：[1]浙江大学生物系统工程与食品科学学院,杭州310019 [2]河南农业大学牧医工程学院,郑州450002

出　　处：《农业生物技术学报》2008年第6期1035-1041,共7页Journal of Agricultural Biotechnology

基　　金：河南省教育厅基础研究项目(No.2004922043)资助

摘　　要：对表达乳糖诱导工程菌株BL-pET-hBD3-Brazzein（Bra）的人β防御素3（humanβ defensin,hBD3）和植物des-pGlu1-Brazzein融合蛋白进行了纯化和活性分析,并对工程菌株的乳糖诱导表达条件进行了优化。对目的蛋白的活性分析表明,hBD3-Bra融合蛋白有一定甜味,但是杀菌活性很弱,经凝血酶切割后,重组hBD3有明显抑菌活性,des-pGlu1-Brazzein的甜度大约为蔗糖的600倍。菌株生长和目的蛋白表达结果显示：0.5%～5.0%范围内高乳糖浓度会抑制菌株生长（P〈0.01）,但对目的蛋白表达无显著影响（P〉0.05）;诱导时间延长对菌株生长有利（P〈0.01）,对目的蛋白表达无显著影响（P〉0.05）;温度对菌株生长和目的蛋白表达均有显著影响（P〈0.01）。进一步研究表明,菌株最优生长温度是30～32℃,蛋白表达最优温度是30℃,乳糖和IPTG的诱导结果无显著差异（P〉0.05）。The recombinant protein composed of human 13 defensin 3 and des-pGlu-brazzein （hBD3-Bra） expressed heterologously by the engineered strain Escherichia coli BL21 - pET-hBD3-Bra was purified and its activity was analyzed, as well as its lactose inductive conditions were optimized. The purified hBD3- Bra showed weak antimicrobial activity and tasted little sweet. After cleaved by thrombin, the released recombinant hBD3 had high antimicrobial activity and the des-pGlul-Brazzein was 600 times sweeter than sucrose. The influences of three factors, lactose concentration, inductive time and inductive temperature, on strain growth and yield ofhBD3-Bra were analyzed. The results indicated that the strain growth was inhibited by higher lactose concentration （P 〈 0.01）, however, the interested protein expression was not affected by 0.5%-5.0% of lactose （P 〉 0.05）. Biomass was improved along with time increase（P 〈 0.01）, while the interested protein yield did not increase obviously （P 〉 0.05）. Both strain growth and interested protein expression were influenced by temperature （P 〈 0.01）, the optimal temperature for strain growth was 30 -32℃ and for interested protein expression 30 ℃. As an inducer, Lactose was as good as IPTG （P 〉 0.05）.

关 键 词：人Β防御素3 植物甜蛋白 乳糖 优化 

分 类 号：S188[农业科学—农业基础科学]