[乳酸乳球菌冷休克蛋白基因的表达及冷冻保护功能分析]
[Expression of cold-shock-protein genes from Lactococcus lactis and analysis of the cryoprotection function].
作者信息
Yinghua Zhang, Yuting Lei, Guicheng Huo
机构信息
Key Lab of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, China.
出版信息
Wei Sheng Wu Xue Bao. 2008 Sep;48(9):1203-7.
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-HindIII CspC and CspD fragments were cloned respectively between NcoI and HindIII 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).
目的
细菌能够适应远低于其最适生长温度的环境，并且一组蛋白质（冷休克蛋白，CSPs）会在生长温度迅速下降时被强烈诱导产生。我们研究了这些蛋白质在冷冻保护中防止冷冻损伤的关键功能。
方法
分别将NcoI-HindIII CspC和CspD片段克隆到pNZ8148的NcoI和HindIII之间，随后通过电穿孔将重组质粒转化到乳酸乳球菌NZ9000中。通过向培养物中添加不同浓度的乳链菌肽来实现CSPs的过量表达，并通过SDS-PAGE进行分析。为了研究CspC和CspD的冷冻保护作用，绘制了包括对照菌株和CSP过量表达菌株的生长曲线。在冷冻前和连续四次冻融操作后测定菌落形成单位的数量。
结果
分别鉴定出7 kDa的冷休克蛋白CspD和6.2 kDa的冷休克蛋白CspC。
结论
结果表明，CspC提高了细胞的复苏能力，CspD提高了冷冻后的活力（提高30-40倍）。
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