| Aeromonas Veronii is a kind of Human-animal-fish zoonotic pathogen in recent years, which widely exists in the natural environment, and causes massive death of aquatic animal diseases. Some countries have classified A.veronii as quarantine items. Although studies on A.veronii are gradually conducted in many countries, the progress remain slow. A.veronii contains a variety of virulence factors, including OmpAI, which the adhesin of A.veronii helping bacterium to adhere on the surface of skin cells. Maltoporin, encoded by LamB gene, can transfer maltodextrin and play a critical role in the survival of bacteria. There are currently no commercial vaccines against these important pathogens. Therefore, using the A.veronii strain JLL-1 isolated from carp in Jilin Province as materials. We prepared purified proteins by prokaryotic expression and observed immune protective effects, which with provide theoretical basis for the development of commercial vaccines against A.veronii.At first we extracted the genome of A.veronii strain JLL-1 and cloned OmpAI and LamB gene by PCR, pMD-18 T cloning vectors were connected respectively and transformed to the Escherichia coli DH5α. Plasmid was extracted from the right DNA identified by PCR and restrictive enzyme reaction.Then we extracted OmpAI and LamB gene which were connected to pET30 a and transformed to E.coli BL21(DE3). The right strains were preserved by glycerol method at-20℃. In this study, we successfully cloned the 1021 bp OmpAI gene and 1314 bp LamB gene respectively, and constructed the prokaryotic expression vector, which lays a foundation for the next step of the study.We cultivated the pET30a-Omp AI and pET30a-LamB strains, optimized the conditions of expression and collected target proteins purified by HisPur prepacked column. The concentration of purified proteins was determined by BCA and stored at-80℃. Under the optimal conditions, the two strains expressed about 43 kDa protein OmpAI and 55 kDa protein maltoporin with 6×His respectively.We used 160 carp koi to study the effect of two proteins on immune protection, which were randomly assigned to four groups, each group with 40. Group1 were intraperitoneally injected with sterile PBS as the control group; Group2 with purified recombination OmpAI; Group3 with purified recombination OmpAI combined with freund’s adjuvant; Group4 with inactivated A.veronii JLL-1 strain combined with freund’s adjuvant. Carp koi were immunized after one week in laboratory conditions. At the third week we continued seconary immunity and observed the changes of immune parameters. We collected the blood of 5 koi chosen randomly from each group every week and the koi were humanely sacrificed by anesthesia euthanasia. The ACP, AKP, SOD, and IgM in blood were detected. At the 4th week after first immunity, 20 koi from each group were intraperitoneally challenged with 0.2mL(2×105cfu/mL, 5×LD50) A.veronii strain JLL-1. We observed the RPS of each group and evaluated the immune protective effects.The immunization program of maltoporin followed the above same method. The results showed that immunized recombinat OmpAI and maltoporin could protect koi against A.veronii. Furthermore, the effects of immunized recombinat protiens combined with adjuvant were better than that of others. The RPS of recombinat OmpAI and maltoporin were 77.78% and 84.21% respectively. Immune parameters also showed that these two recombinat proteins could generate immune response. Compared to the inactivated A.veronii strain JLL-1 combined with freund’s adjuvant, the effect of immune protection generated by these two proteins was slightly weak, which conversely avoided the disadvantages of inactivated whole cell. Finally we found that the immune effects of recombinat protein maltoporin were stronger than that of recombinat protein OmpAI. |
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