| Streptococcus agalactiae was a common amphixenosis spreading between human being, mammals and fish. It occurred in the mainly tilapia farmed area in GuangDong, HaiNan, FuJian and GuangXi province of China and was a great threaten to the healthy development of tilapia industry chain in our country. So far, the therapeutic effect of tilapia disease infected by Streptococcus agalactiae is poor, and And there had no vaccines available. The present research mainly studied the C antigen alpha protein of group B Streptococcus agalactiae. The subunit vaccine and nucleic acid vaccine were established according to the N-terminal conserved sequence region of bca gene which was analyzed by bioinformatics. The effects of vaccines were also evaluated. The following was the main work of this research:1. The cloning, expressing and bioinformatics analysis of alpha protein of Streptococcus agalactiae.The bca gene of Streptococcus agalactiae HN0101 strain isolated from tilapia was cloned. The sequences were analyzed by bioinformatics software. The results showed that ORF of bca gene was composed of 1341bp and coded a polypeptide including 446 amino acids. A conserved YSIRK signal peptide and an alpha C N super family structure domain were included in N-terminal. However, C-terminal sequences included a conserved Gram_pos_anchor super family structure domain and two repeated Rib structural domain. Comparing the two alpha protein structures of different source of isolates, the main difference was the repeat number of Rib structure. The phylogenetic tree analyzing found alpha protein of our strain went to the same branch of A909 isolated from human being and GD201008-001 isolated from fish and the homology reached 100%. It also predicted that alpha protein was a hydrophilic protein, and had a transmembrane domain,37 potential phosphorylation site and one potential N-glycosylation site. Secondary structure analysis found it included lots of random coil.12 amino acid areas might be B cell epitomes. When Escherichia coli were expression host, the solubility of the recombinant protein could reach 100%. Subcellular localization showed it located in the cell wall.2. The recombinant expression vector establishing and immunogenic analyzing of N-terminal bca gene of Streptococcus agalactiae.According to the bioinformatics analyzing results of bca gene, primers were designed on the basis of 51-401 amino acids which were remained by getting rid of transmembrane region and Gram_pos_anchor region. Amplified fragments pbca was cloned to pMD19-T vector after gel extracting. It was cut by using BamHl and Xho I after identified and linked to expressing vector pET-32a (+), so the recombinant expression vector pET-32a (+)-pbca was established. The recombinant vector transferred to BL21(DE3) after identified correctly and the inducing temperature, IPTG concentration and inducing time were optimized. The recombinant protein were expressed under the optimal condition and injected to rabbit after purified to preparing rabbit anti-bacteria serum. The immuneoreactivity between antibody and recombinant protein was detected by western-blot. The results showed that the express host which had recombinant vector would express a 57.9 KDa alpha protein (pBCA) when cultured in 37℃ and induced with 0.1 mmol/L IPTG for 4h. Furthermore, the antibody titer of rabbit anti pBCA serum reached 1:32 checking by agarose diffusion test. Western-blot analysis showed the serum would specific bound with the recombinant protein.3. The immune effect of subunit vaccine established from pbca gene of Streptococcus agalactiae on tilapia.Recombinant protein BCA (pBCA) was made following the method of chapter three. Healthy tilapias were immunized by pBCA, pBCA+adjuvant and PBS control group respectively. Fish serums were collected on 7,14,21,28,35, and 42d right after immunizing. Their antibody was checked by ELISA and the results showed that pBCA and pBCA+adjuvant groups can induce fish producing antibody. The antibody titer of pBCA and pBCA+adjuvant groups reach the peak on the fourth week and the fifth week respectively. After the second week, the antibody titer of pBCA and pBCA+adjuvant groups was significantly higher (P<0.01) than that of PBS control group. From the second week to sixth week, lysozyme activity of pBCA group and pBCA+adjuvant group kept on high level, and reach the highest on week four of pBCA group, also on week five of pBCA+adjuvant group. The transcription level of IL-1β and TNF-α gene analynizing showed that the both immune gene transcription level significantly up regulated in spleen and kidney and also found the transcription level in spleen was higher than that in kidney. Challenge test showed that the protection rates of pBCA+adjuvant group and pBCA group were 66.67% and 52.38% respectively. The above results showed that the recombinant protein pBCA had a good immune protection, and might be as a candidate vaccine for preventing Streptococcus agalactiae infection.4. DNA vaccine establishing of Streptococcus agalactiae pbca genepbca gene was cloned to eukaryotic expression vector pcDNA3.1(+) that constructed recombinant plasmid pcDNA3.1(+)-pbca. It was identified by PCR to bacteria colony and double enzyme cutting. The positive colony including pcDNA3.1(+)-pbca vector was cultured in LB solution medium. Vector was extracted again and identified by agarose gel electrophoresis. The results showed it recombinant vector was 7200bp which was the same with predicted and the concentration was 1.4mg/L, OD 268/280>1.8 that demonstrated the purification of the recombinant vector was high and could be used for making DNA vaccine.5. The immune effect of DNA vaccine established from pbca gene of Streptococcus agalactiae on tilapiaThe tilapias were immunized with recombinant vector by muscle injection. Three groups, DNA vaccine group, blank vector group and PBS control group, were included. Samples from muscle were collected on 7d,14d, and 21d after injection and used RT-PCR checking the target gene. The results showed that the target gene were checked positive on all the three time but negative in blank vector group and PBS control group. It demonstrated that the recombinant vector expressed successfully in fish body and the constructed vaccine was right and successful.Fish serums were collected on 7,14,21,28,35, and 42d right after immunizing. Their antibody was checked by ELISA and the results showed that the recombinant vector could induce fish producing specific antibody. The antibody titer reached the top on the fourth week and it also kept on high level on fifth and sixth week. Lysozyme activity analysis showed that the lysozyme activity in DNA vaccine group was significantly higher (p<0.01) than that of PBS control group from week one to week six. And the activity level kept on high level during week three to week six and it reached the highest valued when week five. The transcription level of IL-1 β and TNF-α gene analyzing showed that the both immune gene transcription level significantly up regulated in spleen and kidney and also found the transcription level in spleen was higher than that in kidney. Challenge test showed that the protection rates of DNA vaccine was 47.62%. All the results above showed the DNA vaccine constructed by pbca gene had a good immune protection, and could be as a candidate vaccine for preventing Streptococcus agalactiae infection. |
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