Construction And Immunoassay Of Oral Vaccine

Aims:Edwardsiella tarda is a kind of pathogenic bacteria that can put great threats on fish. It has caused huge economic loss on the fishery industry in China and other countries around the world. A vaccine against the bacteria has been developed elsewhere, however, it is inconvenient to vaccinate because injection is needed to carry out on small individual fish. The market is urgently calling for a new type of vaccine against the severe bacteria, especially a vaccine that is delivered by oral. This project aims to construct and develop such an oral fish vaccine and employ a cyanobacterium to express and protect this vaccine.Methods:At first, the genome DNA of Edwardsiella tarda was extracted using normal DNA extraction method. Then the genome DNA was used as a template and PCR was employed to amplify two genes in Edwardsiella tarda that have been demonstrated to possess strong immunogenicity against Edwardsiella tarda. After that, overlap extension PCR was carried out to fuse these two genes together. The fusion gene was inserted into the multiple cloning site of the expression vector pRL489. In the presence of a conjugal plasmid and a helper plasmid, this expression plasmid was transformed into Anabaena sp. PCC7120 using the method of triparental conjugative transfer. Then, the transgenic cyanobacterium was screened out using neomycin. The presence of the fusion gene was confirmed by PCR, sequencing and RT-PCR. The Western-blot was employed to detect the expression of the fusion gene.Another pair of primers was designed to amplify the fusion gene in order to insert it into the multiple cloning site of pET-30a. Escherichia coli strain BL21 (DE3) was used as a host to express the fusion gene. SDS-PAGE and Western-blot were used to detect the expression of the fusion protein. Ni-NTA spin columns affinity chromatography and Sephadex G-100 were used to purify the fusion protein. After its purification, the fusion protein was used as a protein molecular marker to determine the expression level of the fusion protein in Anabaena sp. PCC7120. This could be done since it possessed a His-tag and can be detected by anti-His antibody.After determining the expression level of the fusion protein, the transgenic cyanobacteria were cultivated in a 5 L bioreactor in order to harvest enough number of cyanobacterial cells. A batch of cyanobacterial culture was collected by centrifugation and freeze-dried by a vacuum freeze dryer. The weight of dried cyanobacterial cells was determined. Different quantities of these cells were used to feed three groups of zebrafish. At the same time, wide-type cyanobacteria and cyanobacteria bearing empty pRL-489 vector were also received the same treatment and were used to feed zebrafish as controls. There were also two groups of fish received normal food as controls. In total, seven groups of zebrafish with 25 in each group were received different treatments during the experiment. After the vaccination process, pathogenic Edwardsiella tarda strain EIB202 was used to infect the fish and PBS was used to mock infect the fish as a control. After the infection, the mortality of each group was recorded and the cumulative survival rate of each group was determined. This was regarded as a method to evaluate the immune protective effect of the potential vaccine.Results:A shuttle expression vector bearing the target fusion gene was successfully constructed and was designated as pRL-489-etal-L-gapdh. It was then successfully transformed into both Escherichia coli strain HB101 and cyanobacteria Anabaena sp. PCC7120. The expression of the fusion protein was detected in both systems. However, the results showed that the molecular weights of the detected fusion proteins in the two systems were both lower than the theoretical value of the fusion protein but were identical to each other. On the other hand, the fusion protein in the pET-30a system was expressed at a high level and the molecular weight was identical to its theoretical value. It was purified and served as a marker to successfully determine the expression level of the fusion protein in cyanobacteria. The expression level was very low, about 2.46%. Considering the protective effect of the transgenic cyanobacteria, it actually showed no protective effect on zebrafish.Conclusion and significance:Two expression vectors that express target gene in cyanobacteria and Escherichia coli strain BL21 (DE3) respectively were successfully constructed. Expression of the target gene was successfully detected in both systems. This project provided a new vision of developing oral vaccine for fish diseases treatment. Firstly, cyanobacteria were selected as a protective vehicle against the digestion effects of fish digestive tract. The cyanobacteria-based vaccine can be easily administrated by feeding fish since cyanobacterium is a kind of natural food source for fish. Secondly, the thought of combine separate genes that own strong protective effect together to create a fusion gene for the purpose of enhance the overall effect of the vaccine can be well employed in the future. In addition, the target fusion protein was expressed at a high level in the pET system, so the fusion protein expressed in this system can be easily separated and purified to develop into a potential vaccine.