Construction And Immune Protective Effect Of Recombinant Lactobacillus Acidophilus Expressing Helicobacter Pylori Adhesin Hp0410

1、Research background and proposalHelicobacter pylori lead to chronic gastritis and stomach ulcer and it is also closely related to gastric adenocarcinoma and stomach mucosa related B cell lymphoma. H. pylori infects about 50% of the world’s population and up to 64% of the China’s population, in which 10%-20% are patients. H. pylori often infect the children under the age of 10. it is not easy to eradicate and lead to chronic gastritis and gastric ulcer. Therefore, How to prevent Hp infection is the focus problem, which is concerned by a large number of researchers.Triple therapy, consisting of two antibiotics, clarithomycin and amoxicillin or metronidazole incombination with a proton pump inhibitor (PPI) has become the first-line option.However, about 20% of patients can not be effectively eradicated.The high cost, patients’noncompliance and antibiotic resistance are the most reasons. About 75%～80% of patients relapse quickly. Some scholars also believe that Hp can be continuously supplied to the stomach by Hp existing in the patient’s mouth, which is another important reason of Hp recurrent infections. Vaccination against H. pylori is therefore one of the most effective ways to defeat Hp. Presently, the hot points of vaccine research is protein vaccines, DNA vaccines, whole inactivated vaccine and live attenuated bacterial vaccine. Evidence suggests that protein vaccines, DNA vaccines can not stimulate effective Mucosal vaccination. Whole inactivated vaccine and live attenuated bacterial vaccine could induce Mucosal vaccination, but also cause adverse effects. To date, there still is no a safe and effective vaccine.Recently, Lactic acid bacteria (LAB)have largely been concerned as mucosal vaccine delivery vehicles. The system have an advantage over traditional vaccine,which can colonize in the respiratory tract, gastrointestinal tract, urinary tract and other mucosal epithelial cells and induce a strong mucosal immune response. To date, several bacterial and viral antigens have already been produced in L. lactis,L. lactis harboring recombinants could be expressed HPV16 L1 and tetanus toxin fragment C (TTFC),which could stimulate a systemic and mucosal immune responses. After vaccine intranasal immunization, Recombinant LAB expressed Streptococcus pneumoniae Psp that may stimulate a strong mucosal IgA response, so that the body required the ability to resist deadly attack from Streptococcus pneumoniae. Dengue virus capsid protein (EDIII) expressed by MG1363 can induce mucosal immunity. This report make recombinant In this study,Lactobacillus acidophilus as oral vaccination producer strains prevent gastric infection.It allows a direct contact between the antigen and the immune system. In addition, vaccine vectors offer several practical advantages, including avoidance of culturing pathogens, no need to purify antigenic components and the low risk of infection, which is advantageous, particularly for children, the elderly or immunocompromised individuals.Adhesin would be an ideal protective antigen of Hp. The sIgA antibodies against adhesin could prevent Hp adhering and colonizationing in gastric and duodenal mucosa. Hp0410 is a putative homologue protein of HpaA,which may probably well be the subunit of N-acetylneuraminyl-lactosebinding haemagglutinin (NLBH). Hp0410 have also many advantages such as having a signal peptide and high antigenicity, being highly conserved, as well as low protein homology with other species. It may become the one of the rational vaccine candicates of Hp.Our main purpose is to build recombinant Lactobacillus acidophilus with efficient, stable, constitutive expression of Hp0410.After immuning SPF grade C57BL/6 the mice with recombinants, attack the immuned mice with highly pathogenic Hp SSI strains that aim to elicit specific immune in mice in particular mucosal immunity and study the protective effect of the recombinants against Hp infection. 2、Methods1. The primers were used to amplify the hp0410 from the extraction of Hp NCTC11639’ genomic DNA via PCR. The production were coloned into PMD19-S-T and sequenced. Then the fragment was digested and coloned into expression vector pMG36e, resulting in pGM36e-hp0410.2. The Lactobacillus acidophilus was transformed by electroporation with pMG36e-hp0410. Positive transformants could be identified via Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nitrate silver staining and western blot, which were performed to detect the purpose protein.At last, detect the stability of pMG36e-hp0410 in vitro.3. SPF grade female C57BL/6 mice were immunized orally with recombinants expressing Hp0410 to detected specific IgG and specific SIgA antibodies in serum and Gastric mucosa. Meanwhile, detect the live time of recombinants in mice’body.4. Feed SPF grade female C57BL/6 mice on NaHCO3 to damage gastric mucosa. Then the injured mice were attacked with the highly pathogenic strain SS1 Hp and were sacrificed after 4,8,12weeks. The gastric tissue removed of mice were embedded in paraffin, sliced to do HE staining which aimed to identify whether the model was successful.5. SPF grade Female C57BL/6 mice were immunized orally with recombinants. In some time,vaccinated mice were challenged with 0.2ml Hp SS1. Mice were sacrificedby spinal dislocation. The gastric antrum from each stomach was assessed for H. pylori colonization by the detection of urease activity and bacterial culture.3、Results1. hp0410 was about 750bp and has been amplified from Helicobacter pylori strain NCTC11639 genomic DNA,In comparison with the Hp standard strains 22695 and J99 in GeneBank, the DNA sequence homology were respectively 95% and 94%. The recombinant plasmid has been transformed into L.acidophilus,the extracted plasmid have been identified by double enzyme digestion and by PCR. 2. There is purpose protein about 34kD in the supernatant by the experiment of SDS-PAGE electrophoresis and silver nitrate staining. The results of Western blot detected purpose protein can respond to the sera of all Hp-positive patients. The stability of the recombinant plasmid is respectively 97% and 90% in the environment of containing and not containing erythromycin.3. ELISA results showed that recombinants can elicite mucosal SIgA antibodies and induce mucosal immune to protect from Hp infection. Recombinants can stay 3-4 days in mice’s gastric and intestinal tract.4. The vaccinated and non-vaccinated SPF grade female C57BL/6 mice were attacked by highly pathogenic H. pylori SSI strains,some time later,stomach biopsies from the vaccinated mice were negative by urease assay and Bacterial separation test, However stomach biopsies from the non-vaccinated mice is positive. All of the mice were positive by colony counts. However, there was a significant difference in protection between the groups of mice vaccinated with the control groups.4、Conclusion1. The recombinants with constitutive expression of Helicobacter pylori adhesin Hp0410 have been constructed successfully.2. Recombinant Lactobacillus acidophilus that can induced mucosal SIgA antibodies have a significant protective effect against Helicobacter pylori infection.