乳酸乳球菌中冷休克蛋白基因的高效表达及其抗冻保护作用

Expression of cold-shock-protein genes from Lactococcus lactis and analysis of the cryoprotection function

【目的】微生物在适应外界环境急剧降温的条件下都会发生应激反应,产生一系列蛋白质被称为冷休克蛋白。冷休克蛋白对乳酸菌适应低温环境和增强抗冻能力方面发挥着重要作用。本文目的是为了研究乳酸乳球菌中冷休克蛋白CspC、CspD的作用。【方法】将冷休克蛋白CspC、CspD基因分别重组到质粒pNZ8148,转化乳酸乳球菌NZ9000后,加入Nisin诱导,对表达产物进行SDS-PAGE电泳分析,比较重组菌与空白菌在30℃条件下菌体生长差异及反复冻融活菌数的差异。【结果】得出CspC、CspD的相对分子量分别为7.0、6.2kDa。【结论】CspC使菌体更加迅速的恢复了生长;冷休克蛋白CspD增强了菌体的抗冻存活率(增加了30～40倍)。

[Objective] Bacteria are able to adapt to temperatures far below their optimum growth temperatures, and a set of proteins （cold shock proteins, CSPs） are strongly induced in response to a rapid decrease in growth temperature. We studied the key functions in cryoprotection against damage caused by freezing of these proteins. [Methods] NcoI-HindlII CspC and CspD fragments were cloned respectively between NcoI and HindlII in pNZ8148, the recombinants plasmid were subsequently transformed by electroporation into Lactobacillus lactisNZ9000. Overproduction of CSPs was achieved by the addition of different concentrations of nisin to cultures and was analyzed by SDS-PAGE. In order to study the cryoprotection of CspC and CspD, the growth curves including the control strain and CSP-overproducing strains were developed. The number of colony-forming units was determined just before freezing and after four consecutive freeze-thaw operations. [Results] The 7 kDa cold-shock protein CspD and 6.2 kDa cold-shock protein CspC were identified respectively. [Conclusion] The results indicate that CspC improves the recovery of cells and CspD increases the viability after freezing （30-40 folds）.