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| Construction of Recombinant Lactobacillus casei Expressing Gallibacterium anatis flfA and Evaluation of Oral Immune Effect |
| WANG Kun-Peng, LIU Pan-Pan, XIA Yin-He, GAO Dong-Sheng, CHANG Hong-Tao, WANG Xin-Wei, LI Yong-Tao, ZHAO Jun, CHEN Lu, WANG Chuan-Qing, YANG Xia* |
| College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China |
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Abstract Salpingitis and oviduct cyst of laying hens (Gallus?gallus domesticus) are serious diseases harmful to poultry industry. Gallibacterium anatis is one of the common pathogens. At present, Lactic acid bacteria (LAB) are widely used in the development of live vector vaccine as food-grade expression vector. Therefore, Lactobacillus casei CECT5276 (L. casei 5276) was used as a live vector for presenting antigens in this study. With L. casei 5276 as the host bacterium and G. anatis flfA gene as a target, a shuttle expression vector pMG36e were used to obtain recombinant plasmid pMG36e-flfA. The recombinant plasmid was transferred into L. casei by electroporation, then the expression of flfA protein and the growth status, stability, retention and tolerance of recombinant bacteria were detected and analyzed, and the immune effect of recombinant bacteria was preliminarily evaluated. The analyses of SDS-PAGE and Western blot showed that the recombinant L. casei successfully expressed fusion protein with a molecular weight of about 20.3 kD, which could react with Rabbit anti-G. anatis positive serum and had reactogenicity. The analysis of growth status and stability showed that the target gene insertion had no effect on the growth of L. casei and could be stably inherited in the host bacteria; it had good tolerance in simulated intestinal juice, gastric juice and high salt environment. On the 0, 14, 28, 42 d after immunization, the specific IgG and SIgA antibody levels in serum and intestinal mucus were higher than those in empty vector group and PBS control group (P<0.05). The results of animal challenge test showed that all chickens in 3 groups did not die after intraperitoneal injection of G. anatis PDS-RZ-1-SLG strain, but the isolation rate of G. anatis from chicken organs immunized with recombinant bacteria was lower than that of empty vector group and PBS group, suggesting that recombinant bacteria had certain immune protective effect on chickens. This study could provide a reference for further study of oral vaccine against G. anatis.
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Received: 10 February 2020
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Corresponding Authors:
* , yangxia66@163.com
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