Food-Grade Expression of Helicobacter pylori UreB Subunit in Lactococcus lactis and its Immunoreactivity

Abstract

Helicobacter pylori is the principal cause of chronic active gastritis, peptic ulcer, and gastric cancer. To develop an oral vaccine against H. pylori infection, we had expressed the H. pylori ureB gene (Genbank accession no. FJ436980) in nisin-controlled expression vectors using Lactococcus lactis NZ3900 as host. The ureB gene was amplified by PCR from a H.pylori strain MEL-Hp27. Then the ureB gene was fused translationally downstream of the nisin-inducible promoter nisA in a L. lactis plasmid pNZ8149. Lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade expression system employing L. lactis NZ3900. The conditions of UreB expression in this system were optimized by orthogonal experiment. The optimized conditions have been determined as follows: induction of expression was carried out at the cells density of OD(600) ≈ 0.4 with 25 ng/ml nisin, and harvest after 5 h. The maximum percentage of recombinant UreB was estimated to be 7% of total soluble cellular proteins and the yield was 12.9 μg/ml. Western blot demonstrated that the UreB protein was expressed in the L. lactis transformant and had favorable immunoreactivity. These results indicated that the lactococci-derived vaccines could be promising candidates as alternative vaccine strategies for preventing H. pylori infection.

An engineered food-grade Lactococcus lactis strain for production and delivery of heat-labile enterotoxin B subunit to mucosal sites

“…The bacterial cultivation conditions were as described previously [12, 14]. SPF BALB/c mice, aged 6 weeks, were purchased from Henan Experimental Animal Center (Zhengzhou, China).…”

“…The ltB gene fragment was collected by digestion of pMD19-T- ltB using Sph I and Xba I (TaKaRa, China), ligated with plasmid pNZ8149-SP (GenBank KY385376) , and used for transformation of L. lactis NZ3900 by electrophoration [12]. The recombinants were obtained by Elliker medium selection, and identified by restriction digestion and gene sequencing as reported before [12]. The recombinant NZ3900 strain carrying ltB gene was referred to as L. lactis NZ3900/pNZ8149-SP- ltB .…”

“…The expression of LTB in NZ3900/pNZ8149-SP- ltB was induced using 25 ng/ml nisin (Sigma, USA) as inducer under the conditions as previously described [12]. …”

“…The supernatant was collected and used as samples of the culture supernatant. Samples of bacterial cell lysates were processed as described before [12]. …”

“…Another study proved that LTB expressed in fusion with antigens in engineered Lactobacillus can significantly enhance the local and systemic immune responses to the antigens [10, 11]. Recently, increasing evidences supported that food-grade L. lactis expression systems, through expression and delivery of antigens/adjuvants, are promising oral vaccine vectors, particularly owing to their outstanding safety, avoidance of protein purification, reduced antigen degradation and efficient delivery of immunogens to the mucosal inductive sites [12, 13]. However, successful expression of LTB in food-grade L. lactis has not been reported to date.…”