Objective To construct a recombinant lactobacillus (Lb.) strain excreting the human chorionic gonadotropin beta-subunit (hCGβ) Methods The hCGβ cDNA was ligated to the signal peptide sequence of S-layer protein fr...Objective To construct a recombinant lactobacillus (Lb.) strain excreting the human chorionic gonadotropin beta-subunit (hCGβ) Methods The hCGβ cDNA was ligated to the signal peptide sequence of S-layer protein from Lb. brevis and then cloned into down-stream of lactose-inducible promoter of an integrative plasmid, pIlac. After electroporation into Lb. 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 genome. Radioimmunoassay (RIA) was used to determine the level of hCGβ in the supernatant and the cell lysate. Results The hCGβ was integrated into the genome of Lb. casei CECT5276. The highest concentration of hCGβ in the culture supernatant amounted to 440 mIU/mL 21 h after lactose induction. About 2/3 of the abstract Objective proteins were excreted into the supernatant. Conclusion We have obtained stable and efficient hCGβ excretion in Lb. casei, which was inducible by lactose.展开更多
文摘Objective To construct a recombinant lactobacillus (Lb.) strain excreting the human chorionic gonadotropin beta-subunit (hCGβ) Methods The hCGβ cDNA was ligated to the signal peptide sequence of S-layer protein from Lb. brevis and then cloned into down-stream of lactose-inducible promoter of an integrative plasmid, pIlac. After electroporation into Lb. 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 genome. Radioimmunoassay (RIA) was used to determine the level of hCGβ in the supernatant and the cell lysate. Results The hCGβ was integrated into the genome of Lb. casei CECT5276. The highest concentration of hCGβ in the culture supernatant amounted to 440 mIU/mL 21 h after lactose induction. About 2/3 of the abstract Objective proteins were excreted into the supernatant. Conclusion We have obtained stable and efficient hCGβ excretion in Lb. casei, which was inducible by lactose.
