| Helicobacter pylori is a Gram-negative microaerophilic bacterium with a infection rate of more than 50% in population. Hp infection was confirmed to be closely related with diseases, such as gastritis and gastric carcinoma. Development and application of vaccine were hopefully the most efficacious way to control the prevalence of H. pylori infection owing to the increased drug resistance, greater side effects, unable prevention of repeated infections accompanying application of antibiotic therapy for treatment of this infection. Although there are few vaccine candidates that have been reported for clinical trials, they are thought to be deficient in terms of immune effect and research design. The recent appearance of a large amount of basic researches on anti-H. pylori vaccine formulation also suggests that further researches of H. pylori vaccines are still required.H. pylori mainly colonizes human gastric mucosa suggesting that it is rational for vaccines to be administrated orally. As a vector delivering oral vaccines, Lactococcus lactis possesses better safety than conventional attenuated Salmonella, and it has been proved that the vaccine antigens like urease delivered by L. lactis can produce protection in vaccinated mice against challenge with H. pylori, indicating that L. lactis is a potential vehicle for anti-H. pylori oral vaccines.Lpp20 protein of H. pylori is a highly conserved lipoprotein located on the outer membrane of bacteria, which is the main component of the bacterial antigens recognized by anti-H. pylori serum antibodies and have strong immunogenicity. Vaccination of mice and guinea pigs with Lpp20 containing outer membrane vesicles in combination with cholera toxin B(CTB) produced remarkable immune protection against H. pylori attack, suggesting that Lpp20 could be an ideal vaccine candidate antigen. Currently, the immune effect has not been demonstrated for vaccination of animals with Lpp20 delivered by L. lactis.Escherichia coli heat-labile enterotoxin(LT) is a biogenic mucosal immune adjuvant, the subunit B of LT(LTB) is more commonly used for its nontoxicity as compared with the subunit A. Low efficiency is existing in current preparation of natural LTB, whereas purification of proteins is required and effect of endotoxin are difficultly been diminished in separation of recombinant LTB from Escherichia coli gene engineering strains. The bacteria can be applied in mucosal immunization without purification of the recombinant proteins supposing that LTB could be made expressed in L. lactis. Construction of LTB expression system in L. lactis is of importance for development of mucosal immune adjuvant.Therefore, this study was designed to construct two L. lactis expression systems expressing H. pylori Lpp20 and E. coli LTB, respectively, by using the food-grade L. lactis expression system established previously by our research team, and the recombinant L. lactis strains were used to orally vaccinate mice, and evaluation of the effects of vaccination on the systematic and local mucosal immune reaction and protection against H. pylori challenge for constructing basis for development of H. pylori oral vaccines.Method 1. The lpp20 and lt B gene fragments were obtained by PCR amplification, ligated with the cloning vector p MD19-T, respectively, and used for transformation of E. coli DH5α. Plasmids were extracted from the recombinant strains, and the cloned gene fragments were transferred into L. lactis strain NZ3900 using p NZ8149-SP as expression vector through electroporation. Elliker medium was adopted for screening of the positive transformants. 2. Expression of Lpp20 and LTB in L. lactis recombinant strains was induced using nisin. Cellular proteins and supernate of the bacterium cultivating medium was separated as samples. Expression of recombinant proteins and their immunoreactivity was identified by Western blot analysis with mouse anti-L. lactis serum and antiLTB antibodies as the primary antibodies. 3. BALB/c mice were randomly divided into four groups, namely Lpp20 group, Lpp20+LTB group, NZ3900 group and PBS group, which were treated by gavage with bacterial liquid of NZ3900/p NZ8149-SP-lpp20, mixture of NZ3900/ p NZ8149-SP-lpp20 and NZ3900/p NZ8149-SP-lt B, NZ3900/p NZ8149-SP and PBS, respectively. Half of the mice were used for collecting samples of the sera and intestinal feces, which were tested for specific serum Ig G and fecal SIg A antibodies employing ELISA methods. The remain of the mice were challenged with H. pylori 11637, and then gastric tissues of mice were separated as samples for assaying urease activity. 4. The software package SPSS 21.0 was applied for statistical analysis of the experimental data. The one-way analysis of variance was employed to compare the measurement data, which were expressed as `x ± s. The pairwise comparisons of mean values were carried out through the least significant deviation methods(LSD). The difference was considered as significant at P <0.05.Results 1. The products of PCR amplification of lpp20 and lt B genes were 548 bp and 334 bp respectively in length as expected. 2. p NZ8149-SP were ligated with lpp20 and lt B gene fragments respectively, and then used to transform L. lactis NZ3900. Two recombinant strains were obtained by screening of the bacterial colonies, and identified as correctly constructed by PCR, enzyme digestion and gene sequencing. 3. As demonstrated by SDS-PAGE, Lpp20 was mainly expressed as a cytoplasmic protein while LTB as a secreted protein in the recombinant L. lactis strains induced. Western blots analysis resulted in that Lpp20 was detected both in bacterial somatic proteins and supernatant of culture medium, and LTB in the later. 4. Animal experiments: for serum Ig G antibody, the difference between groups of Lpp20 and NZ3900, Lpp20+LTB and NZ3900, Lpp20 and PBS, Lpp20+LTB and PBS, Lpp20 and Lpp20+LTB was statistically significant(P<0.05); for intestinal SIg A antibody, the difference between the groups of Lpp20+LTB and Lpp20, Lpp20+LTB and NZ3900, Lpp20+LTB and PBS was considered as significant(P<0.05);the difference between the group Lpp20 and NZ3900, Lpp20 and PBS was insignificant(P>0.05);as for urease activity of gastric tissues, the difference between group Lpp20 and NZ3900, Lpp20 and PBS, Lpp20+LTB and NZ3900, Lpp20+LTB and PBS was of significance(P<0.05) while the difference between group Lpp20 and Lpp20+LTB was insignificant(P>0.05).Conclusion 1. L. lactis NZ3900/p NZ8149-SP-lpp20 and NZ3900/p NZ8149-SP-lt B have been constructed successfully for expressing Lpp20 and LTB, respectively, and the recombinant proteins were detected and identified as antigenic proteins. 2. L. lactis NZ3900/p NZ8149-SP-lpp20 is immunogenic, oral vaccination of mice with which can irritate both systematic and mucosal immune reaction, and reduce H. pylori colonization level of stomach. The strain is of application potential in vaccine development. 3. L. lactis NZ3900/p NZ8149-SP-lt B can upregulate mucosal immune response evoked with oral vaccines and be novel sources of the mucosal immune adjuvant. |
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