Construction And Use Of Expression System In Lactic Acid Bacteria

Lactic acid bacteria (LAB), a group of G⁺ bacteria that can ferment carbohydrates to produce large quantities of lactic, are considered GRAS (Generally Regarded As Safe) organisms that could safely be used for the expression of foreign proteins. Mucosa system plays an important role in whole immune responses of our body. Most pathogens enter the body through mucosal surfaces, and the development of vaccine protective at such sites should be a very effective means. The attenuated pathogenic vectors may impair their immunogenicity and raises questions about the safety. Probiotics, such as Lactobacillus and Lactococcus, offer an original alternative as antigen delivery vehicles. Administered orally or nasally route, it would not necessitate the professional health care infrastructure required for injectable preparations. LAB, promising new general vaccine vector, survive the low pH in stomach and entry into intestines mucosa. Lc. lactis has been used successfully to induce secretary and protective systemic responses against pathogen orally or nasally in some reports. As commensal bacteria, some Lactobacillus strains maybe cause immune tolerance.The enhanced green fluorescent protein (EGFP) as a reporter was expressed in different vectors induced by two promoters in Lc. lactis, H⁺ induced promoter P170 and nisin-induced promoter P_nisA. P170 is strongly activated at pH below 6.5 in the transition to stationary phase, without addiing an exogenous inducer. The growth phase is separated from the protein production phase. P_nisA is strongly induced by nisin, widely used as food preservative because of its broad host spectrum. P_nisA is regulated by a phosphorylase NisK and a response regulator NisR. Studies with increasing the concentration of nisin showed a linear dose-curve of foreign protein expressed. The gene of egfp was placed behind the two inducible promoters P170 and P_nisA in intracellular or extracellular vectors. The results showed that the expression of EGFP can be induced by addition of the amounts of nisin (10 ng/ml) to the culture medium, not so efficient expression induced by P170 intracellularly or extracellularly. After the gene of protective antigen (PA) of B. anthracis pa was placed behind the promoter P_nisA, the expression of PA was obtained both intracellular and extracellular by the same methods. By enzyme-linked immunosorbent assay (ELISA) we found that PA was expressed extracellularly after 12 hours culture and no more expressed after 16 hours. Using Western Blotting (WB), we found PA was expressed intracellulary. SDS-PAGE showed that PA was expressed after 4 hours induced by nisin added, and after 16 hours induced, PA was expressed no more than before.In order to avoid the erythromycin resistance gene {ery) and and lose of plasmid, a suicide crossover vector based on pMD18-T was constructed. Because the histidinol phosphate phosphatase is coded by hisB and one gene located elsewhere in the Lc. lactis, hisB is used as a homologous crossover sequence for recombination in the genome of Lc. lactis. As pMD18-T doesn't contain an origin of replication in Lc. lactis, it and its derivates are suicide vectors in Lc. lactis. After hisB and ery were inserted in pMD18-T, named pHEC, followed digested by SnaB I, PnisA - PA was ligased to the pHEC, named pHEC- PnisA- PA, which is suicide vector without signal peptide for Lc. lactis. Single crossover mutant Lc. lactis1 .2030S and double crossover mutant Lc. lactis1. 2030D were obtained, and SDS-PAGE showed the expression of PA. The expression vectors of PA with 6 His-tag on the N or C point for Lc. lactis1. 2030 were botained, and PA with 6 His-tag on the N point was expressed by pSIP300-PA-His in Lb. plantarum.The result verified that construction of live bacteria vaccine using the vector pHEC and hisB as homologous crossover sequence was possible, and all vectors constructed in this experiment were acceptable for expression in Lc. lactis or Lb. plantarum. The strategy of developing live bacterial vaccine based on LAB provided a new way of the delivery of antigens in vivo.