p LA-PEDV-S1质粒拷贝数与发酵重组乳酸菌S1表达量之间的相关性研究  

Reciprocity p LA-PEDV-S1 Plasmid Copy Number and S1 Expression Level of Recombinant Lactobacilli in Fermentation

作　　者：窦速林 刘卫贞 侯喜林[2] 张冰冰[1] 王桂华[1] 余丽芸[1,2] DOU Su-lin;LIU Wei-zhen;HOU Xi-lin;ZHANG Bing-bing;WANG Gui-hua;YU Li-yun(Coll.of Sci.＆ Technol;Coll.of Anim.Sci.＆ Technol.,Heilongjiang Bayi Agric.Uni.,Daqing 163319)

机构地区：[1]黑龙江八一农垦大学生命科学技术学院,黑龙江大庆163319 [2]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《微生物学杂志》2018年第5期27-33,共7页Journal of Microbiology

基　　金：黑龙江省垦区科研课题(HNK135-04-06-04)

摘　　要：探究质粒拷贝数以及目标蛋白S1表达量与发酵时间的关系,从而确定放大生产p LA-PEDV-S1/Lactobacillus casei的最佳发酵时间。通过发酵重组干酪乳杆菌,绘制重组干酪乳杆菌的生长曲线,确定其生长的最佳时期。将p LA-PEDV-S1/L. casei分别接种至添加抗生素和不添加抗生素的MRS培养基中传代培养,进行稳定性实验。使用荧光定量PCR方法检测重组干酪乳杆菌中质粒的拷贝数,使用流式细胞术检测乳酸菌表达的目标蛋白。重组菌在7 h达到生长顶点,传至120代时外源质粒并无丢失情况出现。质粒拷贝数在9 h达到峰值29. 34,表达目标蛋白的重组菌在7 h达到峰值97. 98%。结果显示在细菌生长的对数生长期末期,质粒拷贝数最高且S1表达量最多;在平台期随着发酵时间的增加,质粒拷贝数逐渐降低,S1表达量也相应减少。发酵的最佳时间为7～10 h,质粒拷贝数与S1表达量之间存在着正相关性。In order to investigate the relationship among fermentation time, the plasmid copy numbers （PCNs） and the expression level of the S1 target protein, so as to determine the optimal time for the amplification of the fermentation production of pLA-PEDV-S1/ Lactobacillus casei（L. casei） . The growth curves were drawn according to the fermentation of the recombinant L. casei , to determine its optimal growth period. Thereafter, the pLA-PEDV-S1/ L.casei was cultured on MRS media with and without antibiotic respectively for generation passing culture to carry out the stability experiment. The PCNs in recombinant L. casei was determined by fluorescent quantitative PCR, and the objective protein of lactic acid bacterium expression was determined by flow cytometry. The recombinant strain reached the growth peak at 7 h and the exotic plasmids were not lost when passing through 120 th generation at all. And the PCNs reached to a peak value of 29.34 at 9 h, while the bacteria expressed S1 protein reached to a peak value of 97.98% at 7 h. The above experimental results showed that the peak of the PCNs and S1 protein expression occurred in the end of the logarithmic growth phase; in the platform period of bacterial growth, the PCNs and the recombinant bacteria decreased as the fermentation time increased, and the expression level of S1 was correspondingly decreased. The optimal fermentation time was at 7-10 h, there existed a positive reciprocity between PCN and the S1 expression level.

关 键 词：重组干酪乳杆菌 荧光定量PCR 质粒拷贝数 S1蛋白的表达量 

分 类 号：Q939.117[生物学—微生物学]