【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)。更多还原

【Abstract】 The recombinant protein composed of human β 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-pGlu1-Brazzein was 600 times sweeter than sucrose. The influences of three factors, lactose concentration, inductive time and inductive temperature, on strain growth and yield of hBD3-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).更多还原