Analysis And Screening Of Secreted Proteins In Marine Bacteria And Its Application In Carrier Vaccine

According to the statistics, more than 5000 species of diseases have been found in aquaculture at present, of which the bacteriosis was one of the most common and serious diseases and often caused aquaculture huge economic losses. Thus, the immune control for the pathogens has practical significance. In the traditional solutions, the solution of feeding antibiotics and high-quality feedstuff played a positive role to a certain extent, but after a long-time frequently using of antibiotics and other chemicals, the environment has been caused some defects such as drug tolerance of the pathogenic, non-pathogenic microorganisms and drug residues in the aquatic animal's immune. At present, in the microbial vaccines research and development, live bacteria vaccine vector developement has become a focus mainly in food and medicine, but seldom-reported in the immune prevention of aquatic animals.Our research started with enhancing the protein expression from transforming recombinant protein expression vector, and constructed the visual expression vectors which can efficiently express the foreign protein. Then we screened out of the secreted proteins of aquatic bacterias and analyzed their signal sequences.This study includes two parts.PartⅠ: Study on the method of modifying recombinant vector for improving protein expression. In this part of the study, the commercial recombinant green fluorescent protein expression vector pGFPuv was modified to phGFPuv with inserting of a oligo nucleotide sequence coding more amino acid codon following the iniation codon by PCR method firstly. The difference of GFP expression level between the two vectors expressing in E. coli DH5αafter IPTG inducing was analyzed by SDS-PAGE. Both of the vectors can successfully express green fluorescent protein by IPTG inducing. However, the GFP proportion expressed by the modified vector reached more than 20% of the bacterial total protein, while the proportion expressed by the original vector was less than 9%. It shows that the oligo nucleotide sequence insert coding several amino acids with synonymous codons following the iniation codon can increase the expression level of exogenous proteins for 2-5 times.PartⅡ: Mass spectrometry identification of secreted proteins from aquatic bacterial isolates of animal origin and analysis of their secretive sequences. In this study, the secreted proteins of Vibrio anguillarum MN, V. anguillarum 3101, V. harveyi and Bacillus firmus which were isolated and conserved by our laboratory, were extracted. The components of secreted proteins were separated by SDS-PAGE. The 8 high expressed protein bands in the SDS-PAGE were identified by mass spectrometry MALDI-TOF/TOF as the metalloprotease of Vibrio anguillarum MN, the metalloprotease of Vibrio anguillarum 3101, the zinc metalloproteinase of Vibrio anguillarum 3101, the hypothetical protein VFMJ11₁094 and outer membrane protein of V. fischeri ES114, the putative chitinase, enterotoxin A and protein BCG9842 of Bacillus firmus. After downloaded the sequences of above proteins from NCBI database, PCR primers were designed and specific DNA bands were amplified , cloned and sequence analisised. 8 signal peptides were given by using of the online software SignaIP 3.0, namely angMN-35, ang3101-35, ang3101-25, vf-38, vf-23, bf-43, bf-37 and bf-16. The cellular localization of the secreted sequences were analyzed by PSORT. And we found that all the signal peptides located in the outer membrane, outside or periplasmic space of the cell. The results may be useful for construction of a secreted vector.