摘要
目的 :探索乳酸杆菌携带表达 h CGβ避孕疫苗的抗生育作用。方法 :用 PCR从短小乳酸杆菌染色体中扩增得到 S-层蛋白的信号肽序列 ,将其与 h CGβ基因连接后克隆到乳酸杆菌整合型表达载体 p Ilac的 lac启动子下游 ,得到质粒 p Ilac h CGβ,电穿孔转化干酪氏乳酸杆菌 Lb. caseiCECT5 2 76。挑选转化后的耐药菌落 ,在含红霉素培养基中培养 ,抽提其染色体 DNA,PCR及测序鉴定证实 h CGβ基因整合到 Lb.casei的基因组中。h CG放免检测上清及细胞裂解液中的 h CGβ。结果 :h CGβ基因已经整合到 Lb.casei的基因组中 ;细菌产生的目的蛋白约有 3/ 4分泌进入培养上清 ,而加入诱导剂乳糖 2 1 h后 ,上清中的蛋白浓度达到峰值 ,为 440 m IU/ m L。重组菌在无选择压力条件下培养 5 0代后仍可分泌 h CGβ。结论 :h CGβ在乳酸杆菌中得到了稳定的。
Objective:To construct a recombinant lactobacillus strain excreting the human chorionic gonadotropin beta subunit (hCGβ). Methods:The cDNA of hCGβ was ligated to the signal peptide sequence of S layer protein from Lactobacillus brevis and then cloned into down stream of lactose inducible promoter of an integrative plasmid pIlac. After electroporation of the plasmid into Lactobacillus casei CECT5276, PCR using the genomic DNA of the recombinant lactobacillus as template was performed to confirm whether the hCGβ gene had been integrated into the chromosome. Radioimmunology assay(RIA) was used to detect the level of hCGβ in the supernatant and cell lysate. Results:The hCGβ gene had been integrated into the genome of Lb casei CECT5276 .The highest concentration of hCGβ in the culture supernatant amounted to 440 mIU/mL, 21 hours after lactose induction. About 3/4 of the objective protein was secreted into the supernatant. The recombinant strain could secret the objective to 250 mIU/mL after passaged for 50 generations under no selection pressure of erythromycin. Conclusion: We have got stable and efficient hCGβ excretion in Lb. casei that were inducible by lactose.
出处
《生殖与避孕》
CAS
CSCD
北大核心
2003年第3期129-134,T001,共7页
Reproduction and Contraception
关键词
乳酸杆菌
HCGΒ
电穿孔转化
Lactobacillus casei, human chorionic gonadotropin beta subunit, electroporation
