Construction Of Oral Attenuated Recombinant Salmonella Gallinarum SG9R Vaccine Candidate Expressing Outer Membrane Protein Of E.coli And Related Immunoprotection Study

Chicken colibacillosis is caused by avian pathogenic Escherichia coli （APEC）, which is one of the most common pathogen in poultry, and cause local or systemic infection and leads to serious economic losses worldwide. With the improvement and the intensification of the poultry industry, Colibacillosis caused by avian pathogenic E.coli and/or its co-infection with other pathogens have been emerged as a major problem. At present, the prevention and treatment of this disease is given priority to drugs and vaccines.The effective way to treat this disease is to add antibiotics to the feed, but it is controversial and averse because of the problem of the bacteria with resistance to antibiotics. The most effective way to prevent APEC is vaccination,combination of application of antibiotics legitimately and preventive vaccine were recognized as the most useful measure in preventing APEC. However, there is still no genetic engineering vaccine which has high immunity specificity and wild spectrum. In this study, the chromosome-plasmid lethal system was used for expressing outer membrane protein of E.coli in attenuated & gallinarum SG9R Aasd strain, which lost the nutritious asd gene to investigate the protection opotential of this oral administered recombinant vaccine in preventing avian colibacillosis.1. Cloning and prokaryotic expression of E. coli outer membrane protein gene S632According to the sequence of the major encoding gene S632 for E.coli outer membrane protein published in GenBank, we designed a pair of primers of S632 by sequence analysis with software DNAStar.42 suspected E.coli strains of clinical cases from Jiangsu province were used as template and the S632 fragment was amplified by PCR. The PCR production was cloned into pMD19-T simple vector and then transformed into E. coli DH5a. Recombinant plasmid pMD19-T-S632 was obtained through subsequent screening and identification.28 isolates of all 42 suspected E.coli strains were identified to be E. coli. Sequence analysis revealed that S632 is ubiquity in E. coli and the conservatism of S632 is high. After induction culture of the BL21（DE3） carrying the recombinant plasmid pET-42a-,S<552, the target protein of a molecular weight of about 63 KDa could be detected at 4 hours induction of IPTG with a final concentration of 1.0 mM. Identified as in inclusion body forms by SDS-PAGE analysis, this fusion protein was renaturalized through urea denaturing and nickel column affinitive dialytic purification successively. The purified fusion protein with a final concentration of 0.964 mg/ml was obtained and named as pS632.This protein was used as envelope antigen in ELISA.2. Construction of attenuated Salmonella Gullinarum 9R strain expressing common outer membrane protein of E.coli and related immunoprotection studyThe S632 gene was amplified by PCR, then inserted into expressing vector pYA3342 which is containing asd gene. The recombinant plasmid was transformed into SG9RAasd mutant and obtained recombinant attenuated S.gallinarum vaccine SG9R （pYA3342-S’6’32）. All chickens could survive for 3 weeks after orally inoculated with 1 x 109cfu of SG9R （pYA3342-S6"32）, which confirmed the safety of the recombinant vaccine. Immune group were orally inoculated with recombinant vaccine strain SG9R （pYA3342-S632）, empty vector control group were inoculated with recombinant strain SG9R （pYA3342） and control group were fed with PBS buffer. In contrast to control groups, the SG9R （pYA3342-S632） could induce specific antibody responses and the level of antibody was significantly increased（P<0.05） at the end of the two weeks after primary immune. At the end of two weeks after secondary immune, the SG9R （pYA3342-S632） group could induce specific antibody responses in duodenum. The percent of CD3+ and CD8+ T lymphocytes in periphery blood were detected by flow cytometer after immune. The percent of CD3+and CD8+T lymphocytes of 3 groups all had increasing trend. Meanwhile, the percent of CD3+and CD 8+T lymphocytes of immunized groups were higher than that of the control groups. All chickens were challenged with 1 x 109cfu of APEC challenge strain QD2 by the airsac route on day 29 after primary immune. The mortality rate of chickens vaccinated with SG9R （pYA3342-S632）, SG9R （pYA3342） and PBS were 20%,70.0% and 80%, respectively. At the same time, all chickens were challenged with 1×109cfu of Salmonella challenge strain 50336 in the same way. The mortality rate of chickens vaccinated with PBS was up to 90%, the mortality rate of chickens vaccinated with SG9R （pYA3342-So-32） and SG9R （pYA3342） were 60% and 70%. To sum up, the recombinant vaccine strain can express the outer membrane protein ETEC₂479 stably and is a promising candidate vaccine.