Gene Expression And Immunogenicity Study Ofouter Membrane Proteins Of Vibrio Vulnificus And Edwardsiella Tarda

In this paper the immunogenicity of two recombinant outer membrane proteins of Edwardsiella tarda and Vibrio vulnificus obtained by gene engineering technology were studied to prepare for further research of OMP-based subunit vaccine of two bacteria pathogens.The main methods and results are as follows:?1?The gene fragments encoding the outer regions and showing rich epitopes of two outer membrane proteinswere amplified by PCR,after two full length genes of OmpA and OmpU selected from Edwardsiella tarda and Vibrio vulnificus respectively.Then two recombinant expression vectors?pGEX-2T-OMP-Edwa-his and pGEX-2T-OMP-Vibrio-his?were constructed and transformed into E.coli BL21.In order to express two recombinant OMPs efficiently under optimum conditiond,two recombinant express vectors were induced under different conditions.Results showed that two recombinant vectors can be highly expressed under the conditions of inducing 5-9 h by 0.25 mmol/L IPTG at 16?in a suitable initial bacteria concentraton(OD_600nm=0.3-0.8).Two expressed proteins were eluted and purified by nicked column,and then renaturated by dialysis with gradient of urea.?2?One hundred eighty healthy Japanese eels?Anguilla japonica?were divided equally into3 groups and intraperitoneally injected with bovine serum albumin?BSA group?,formalin killed-whole-cell of Edwardsiella tarda?FKC group?or the expressed OmpA of Edwardsiella tarda?Omp group?respectively.On 14,21,28 and 42 d post-vaccination,the whole blood cells were collected to evaluate the stimulation index and bactericidal activity,and the sera were also collected to assess the titers of specific antibody,lysozyme activity,complement activity and bactericidal activity.Stimutenously,the lysozyme activities in suspension of kidney,skin mucus and liver were also ascertained.Results showed that the stimulation index in Omp group was significantly?P<0.05?enhanced compared to BSA group on 28 d post vaccination;the titers of specific antibody in Omp group were significantly?P<0.05 or P<0.01?increased compared with FKC or BSA group on 28 d and 42 d;lysozyme activity of serum in Omp group was significantly?P<0.05?higher than BSA group,and it showed significantly?P<0.05?difference in kidney on 42 d;complement activity in Omp group was significantly?P<0.05?higher than BSA group.Omp group showed strong bactericidal capacity on 21 d and 28 d.The relative percent survival?RPS?of Omp vs BSA group after challenged by Edwardsiella tarda was 77.7%.These results showed that the expressed OmpA of Edwardsiella tarda could significantly improve the specific and non-specific immune function of Anguilla japonica and the resistance of eels to the infection of Edwardsiella tarda.?3?One hundred eighty healthy Japanese eels were divided equally into 3 groups and injected intraperitoneally with bovine serum albumin?BSA group?,formalin killed-whole-cell of Vibrio vulnificus?FKC group?or the expressed OmpU of Vibrio vulnificus?Omp group?respectively.On 14,21,28 and 42 d post-vaccination,the whole blood cells were collected to evaluate the stimulation index and bactericidal activity,and the sera were collected to assess the titers of specific antibody,lysozyme activity,complement activity and bactericidal activity.The lysozyme activities in suspension of kidney,skin mucus and liver were also ascertained.Results showed that the stimulation index in Omp group was significantly?P<0.05 or P<0.01?enhanced compared with FKC or BSA group on 28 d;the titers of specific antibody in Omp group were significantly?P<0.05 or P<0.01?increased compared with FKC or BSA group on28 d and 42 d;lysozyme activity of kidney and skin mucus in Omp group was significantly?P<0.05 and P<0.01?higher than BSA group on 42 and 14 d respectively;the result of complement activity showed no significant differences?P>0.05?between Omp and BSA group.Omp group showed strong bactericidal capacity on 21 d and 28 d.The relative percent survival?RPS?of Onp vs BSA group after challenged by Vibrio vulnificus on 28 d post vaccination was 80%.The results showed that the expressed OmpU of Vibrio vulnificus could significantly improve the specific and non-specific immune function of Anguilla japonica and the resistance of eels to the infection of Vibrio vulnificus.In short,the Edwardsiella tarda OmpA and Vibrio vulnificus OmpU improved stimulation index,specific antibody titers and bactericidal capacity of Japanese eels in this study.Therefore,the RPS of Japanese eels to the infection of Edwardsiella tarda and Vibrio vulnificus was increased significantly.