| Objective: Group A streptococcus(Group A streptococcus, GAS) is a common Gram-positive bacteria, can cause a variety of serious invasive infections and a lot of autoimmune diseases. In most developing countries, rheumatic fever, rheumatic heart disease and glomerulonephritis, caused by GAS infection, are still a major factor to threat the quality of life. Currently, GAS infection is mostly treated by penicillin, macrolides, or intravenous immunoglobulin, but the rate of recurrence of GAS infection is high due to antimicrobial resistance and/or the short period of protective effect. Thus, The development of high effective vaccine of GAS become the focus of research in GAS prevention and therapy.In our previews research, the recombinant plasmid p ET28a/F7M5 has constructed successfully, which contains seven Fba A epitopes and some epitopes of M protein of M1, M3, M6, M18 serotypes and J gene, the common sequence at C-terminal of M protein[1]. However, the recent epidemiological survey shows that M12 GAS is the major strain of the prevailing GAS strains in most area of China. Therefore, in this study some epitopes of the M12 gene were inserted into the F7M5 sequence of pet28a/F7M5 which named F7M6. Additionally, F7M6 served as the template and M6 gene segment was amplified by PCR(Hereafter M6), which cloned to pet28 a. Subsequently, the purified F7M6, M6 protein and the full length of M1 protein(Hereafter M protein) preserved in our lab were immunized mice. ELISA, ELISPOT and other experimental techniques were used to detected the cellular immunity and humoral immune effect, and the survival rate of each group was calculated after mice were challenged by M1 GAS to determine the protective effect of the vaccine.Methods:1 GAS M12 gene sequence was obtained from the United States Centers for Disease Control and Prevention(CDC) database by the bioinformatic methods, and the specific sequence of M12, no cross-reaction with human tissue genes were selected through nucleotide blast online.At last, F7M6 sequence was got through inserting the special gene fragment of M12 into F7M5 by overlap PCR.2 Epitopes of F7M6 were predicted online.3 The conformation and the hydrophily of F7M6 sequence were predicted online.4 The gene of F7M6 as template, the gene sequence contained the serotype with M Protein of M1, M3, M6, M18 serotypes and J gene were amplified, and named M6.5 Recombinant plasmid p ET28a/F7M6 and p ET28a/M6 were successfully constructed and transformed to Escherichia coli BL21 to express recombinant protein F7M6 and M6. The two proteins were identified by SDS-PAGE and Western blot, and purified by affinity chromatography method.6 12 mice of each group were immunized at 0, 21, 42 and 63 days with 20 μg per dose of F7M6, M6 or M protein. At the same time, mice were immunized with PBS as control group. All immunizations were performed via subcutaneous route. Serum samples were collected on day 10 after each immunization.7 Half of the mice in each group were sacrificed on 10 days following the last immunization, and humoral and cell-mediated immune responses, including the level of Ig G, Ig G1, Ig G2 a, IL-4 and IFN-γ, were detected by ELISA and ELISPOT.8 The specific binding ability between the sera from ASO-positive patients and the healthy children with F7M6 protein were detected by ELISA.9 The specific binding ability of different GAS strains from clinic with the mouse sera of F7M6 group were detected by ELISA.10 The immunized animals were intraperitoneally challenged with a lethal dose of M1 GAS, and monitored on a daily basis to determine the protective effect of the vaccine.Results:1 Recombinant plasmids of p ET28a/F7M6 and p ET28a/M6 were constructed and expressed successfully. Western blot showed that F7M6 and M6 can significantly bind F7M5 antibody.2 The levels of sera Ig G of each experimental group were gradually increased after mice were immunized several times. The titer of F7M6, M6 and M protein group were 1:669309, 1:58158, and 1:32980, respectively. The Ig G level of F7M6 group was signicantly higher than that of the other groups(P<0.001), while there was no significant difference between M6 group and M protein group.3 The level of sera Ig G1 and Ig G2 a of each group was significant higher than PBS group(P<0.001), but the level of Ig G1 of F7M6 was no significant difference from M6 and M protein group. In contrast, the level of Ig G2 a of F7M6 was higher than M6 groups(P=0.009), while there was no significant difference of Ig G2 a between F7M6 group and M protein group, as well as M6 group and M protein group.4 The levels of IL-4 and IFN-γ were detected by ELISPOT, and the results showed that F7M6, M6 and M protein groups were all higher than the PBS group(P<0.001). F7M6-immunized mice exhibited the highest level expression of IL-4 and IFN-γ, while the levels of IL-4 and IFN-γ from M protein groups were significantly higher than that of M6 group.5 The purified F7M6 was used as antigen to detect antibodies in the sera from ASO-positive patients and healthy children sera as negative control. We found the positive rate of ASO-positive patient sera was 95%,while that of healthy children was just 47.3%,the difference between of them had the significance in statistics(P<0.005).6 The binding ability of four clinical GAS strains was detected by ELISA, and the results showed that all of them couldbindwell with antiF7M6 sera.7 The four groups were challenged with type M1 GAS bacterial suspension, and the survival rates of each group were respectively that F7M6 was 66.7%, M6 was 3.3%, M protein was 66.7%, and no mice in PBS groups survived challenge. Statistical analysis showed that those groups were higher than PBS group.Conclusions:1 Recombinant plasmids of p ET28a/F7M6 and p ET28a/M6 were constructed and expressed successfully.2 F7M6, M6 and M protein could elicit well humoral and cell-mediated immune response, and induce a protective effect in immunized miceagainst GAS infection. The F7M6 protein elicited the best effect among all the groups, and the multivalent epitopes of F7M6 cover the most of GAS serotypes at present. |
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