摘要
目的检测产肠毒素性大肠杆菌(Enterotoxigenic E.coli,ETEC)基因重组干酪乳杆菌(Lactobacillus casei,L.casei)贮存过程中质粒的稳定性及活菌数的变化。方法将ETEC K88重组干酪乳杆菌(pLA-K88/L.casei)接种于MRS培养基,转种WPC培养基,收集菌液,置4、15、37℃贮存。通过抗生素MRS(+)和MRS(-)平板计数菌落数的方法及PCR法检测重组质粒在4℃贮存1、15、30、60、90和120 d的稳定性;Dot ELISA法检测菌液外源蛋白免疫学活性的变化;平板计数法检测4、15、37℃贮存1、2、3、7、15、30、60、90、120、210和240 d的活菌数。结果在4℃贮存120 d内,重组质粒稳定性良好,目的基因片段无缺失,外源蛋白活性稳定,乳清蛋白对工程菌的稳定性及外源蛋白的免疫原性均无影响;工程菌在4℃贮存效果最好,存活时间最长,第240 d活菌数仍保持在9×106cfu/ml以上,15和37℃贮存期仅为90和15 d。结论 ETEC基因重组干酪乳杆菌于4℃贮存条件下,质粒及外源基因表达稳定。
Objective To observe the plasmid stability and viable count of recombinant Lactobacillus casei with entero-toxigenic E. coli （ETEC） gene during storage period. Methods Recombinant L. casei pLA-K88/L, casei was inoculated to MRS medium then transferred to WPC medium, and the bacterial liquid was collected and stored at 4, 15 and 37℃ respectively. The stabilities of recombinant plasmid after storage at 4 ℃ for 1, 15, 30, 60, 90 and 120 d were tested by colony counting on MRS（＋） and MRS（-） plates and PCR. The immune activity of foreign protein was determined by Dot ELISA. The viable counts of recombinant L. casei 1, 2, 3, 7, 15, 30, 60, 90, 120, 210 and 240 d after storage at 4, 15 and 37 ℃ were determined by plate counting. Results The recombinant plasmid showed high stability within 120 d after storage at 4 ℃. No target gene deletion was observed, while the activity of foreign protein was stable. Lactoalbumin showed no significant effect on the stability of recombinant L. casei or the immunogenicity of foreign protein. The recombinant L. casei stored at 4 ℃ survived for the longest time, of which the viable count 240 d after storage was more than 9 x 106 cfu/ml. However, the storage periods at 15 and 37 ℃ were only 90 and 15 d respectively. Conclusion The plasmid in recombinant L. casei with ETEC gene showed high stability at 4 ℃, while the foreign gene was expressed stably.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第2期222-224,229,共4页
Chinese Journal of Biologicals
基金
黑龙江省农垦总局科技攻关项目(HNK10A-08-03-03)
2012年黑龙江省大学生创新训练项目(YJSCX2011-248HLJ)
关键词
干酪乳杆菌
质粒稳定性
活菌数
Lactobacillus casei
Plasmid stability
Viable count
