| Objective: Coxsackievirus group B type 3 (CVB3) infections are common causes of acute and chronic myocarditis in human. It's an urgent task for the study of DNA vaccine providing of CVB3 infections. The VP1 gene is a major capsid protein of CVB3, which include many epitopes of B and T. It can induce the production of effective neutralizing antibody. Although the vaccine constructed with VP1 alone could stimulate the production of antibody, the titers of antibody produced were usually too low to protect the host from lethal CVB3 challenge. It has been proved cytokine as a molecular adjuvant can enhance the immunological effect of CVB3 vaccine. So the recombinant plasmid encoding MDC and CVB3VP1 had been constructed. It enhanced the effect of CVB3 vaccine. But the recombinant vaccine is imperfect. Maybe one or two domains of the fusion protein expressed in the animals after inoculation can't maintain native structure, which affect binding to antigen-presenting cells (APCs), or the immunological potency of VP1. So it need to be resolved how to construct recombined nucleic acid vaccines that can express active functional fusion protein and ensure protein stability. Fusion proteins are composed of two or more distinct modules that are joined into a single macromolecule by stretches of amino acids referred to as linkers. The selection of the linker sequence is particularly important for the construction of functional fusion proteins. The two functional domains can fold correctly because of the peptides linkers. The interdomain linkers allow the two modules to perform independent function. Linker length and composition should be concerned mainly. Several studies suggested the flexible and hydrophilic linker was chosen firstly for the construction of functional fusion proteins. The most widely used linker designs have sequences consisting primarily of stretches glycine (G) and serine (S) residues. The molecular weight of G is the smallest. Because of the absence ofβ-carbon , G provide the necessary flexibility. S is the best of all the hydrophilic amino acids. The use of long linker may result in low yield of active fusion proteins since unprotected and flexible regions are often susceptible to proteolytic cleavage during recombinant protein production. Use of a shorter linker might overcome problems associated with protease. On the other hand, there is a risk that a short linker brings the modules too close to each other, resulting in a loss of function. Previous studies indicated linkers of 10-22 residues were advisable candidate for the linker of functional fusion proteins. Three recombined eukaryotic expression plasmids pcDNA3/MDC-L-VP1 were constructed to engineer proteins with linker peptides of 10,15 and 19 aa residuces. Methods: (1) The DNA fragments of MDC-L15 and MDC-L19 were amplified by PCR from the plasmid of pcDNA3/MDC. Similarly, the DNA fragments of L15-VP1 and L19-VP1 were amplified by PCR from the plasmid of pcDNA3/VP1. (2) The PCR products of the four DNA fragments were linked to plasmid pGEM-T, E.coli DH5αwas transformed and cultured in medium containing Ampicilin (100 ug/ml) 2YT. Then plasmids were extracted, recombinant clones were selected and identified by endonuclease cutting and sequencing. (3) The constructed plasmids were cut by proper endonucleases and the target gene fragments were linked to pcDNA3 which was cut by proper endonucleases to construct the eukaryotic expressing plasmids of pcDNA3/MDC-L15-VP1 and pcDNA3/MDC-L19-VP1. (4) Large preparation were performed to obtain the plasmids of pcDNA3, pcDNA3/VP1, pcDNA3/MDC-L10-VP1, pcDNA3/MDC-L15-VP1 and pcDNA3/MDC-L19-VP1 from transformed E.coli DH5α, and purified by PEG. (5) BALB/c mice aged 6 weeks were divided into 5 groups at random: pcDNA3 control group, pcDNA3/VP1 group, pcDNA3/MDC-L10-VP1 group, pcDNA3/MDC-L15- VP1 group, and pcDNA3/MDC-L19-VP1 group. Every group consisted of 14 mice. The plasmids were inoculated to mice in a dose of 100ug of DNA per mouse, intramuscularly (i.m.) in quadriceps muscle at 4-week intervals. Fourteen days after every injection, sera were collected and CVB3-specific neutralizing antibodies were titrated. Three weeks after the third immunization, 3 mice from each group were sacrificed. Splenocyctes were isolated for detecting antigen-specific lymphoproliferative response and specific CTL response by CCK-8 assay. 8 mice from each group were subjected to intraperitoneal (i.p.) challenge with 5 LD50 of CVB3 and the number of surviving animals was monitored up to 3 weeks post infection. The rest mice of each group challenge with 3 LD50 CVB3 and were sacrificed at 7th day, the titers of blood viruses were evaluated. Then the hearts fixed with 10% formalin, dehydrated with grading ethanol, embedded in paraffin, and cut into 5μm sections. Some sections were stained with hemotoxylin and eosin to observe the changes of the myocardium.Results: (1) The gene fragments of MDC-L15, MDC-L19, L15-VP1 and L19-VP1 were amplified by PCR. The length of the fragments was same as expected. (2) The prokaryotic clone vectors pGEM-T/MDC-L15, pGEM-T/MDC-L19, pGEM-T/ L15-VP1 and pGEM-T/L19-VP1 were successfully constructed, DNA sequencing showed that the sequences of our results are identical with that recorded in Genbank. (3) After these gene fragments were inserted to the pcDNA3, the recombined plasmid was identified by enzyme cutting and the results were identical as expected and suggested that the eukaryotic expressing plasmid of pcDNA3/MDC-L15-VP1 and pcDNA3/ MDC-L19-VP1 had been constructed successfully. (4) The mean titers of neutralizing antibody after every immunization in pcDNA3/VP1 group were 1:7.07, 1:10.59, 1:25.20, and that in pcDNA3/MDC-L10-VP1 group were 1:7.93, 1:22.45, 1:31.74, and that in pcDNA3/MDC-L15-VP1 group were 1:8.91, 1:23.78, 1:39.99, and that in pcDNA3/MDC-L19-VP1 group were 1:10.00, 1:25.20, 1:50.39, respectively. The mean neutralizing antibody titers in pcDNA3 group were lower than 1:5. The statistics analysis showed that the diffrences of neutralizing antibody titers of every group produced after three times of inoculation were significant. After the third inoculation, except the difference between pcDNA3/VP1 group and pcDNA3/ MDC-L10-VP1 group was not significant, all the others were significant (P<0.01). (5) Compared with mice injected with other plasmids, the antigen-specific lymphoproliferative response and specific CTL activity of mice immunized pcDNA3/MDC-L19-VP1 significantly enhanced (P<0.01). (6) Up to the 21th day after CVB3 challenge, the survival rates of pcDNA3/MDC-L19-VP1 group, pcDNA3/MDC-L15-VP1 group, pcDNA3/MDC-L10 -VP1 group and pcDNA3/VP1 were 37.5%, 37.5%, 25.0% and 12.5%, respectively. The mice of pcDNA3 group were dead within 11 days. (7) The virus titers of mice immunized with pcDNA3/MDC-L19-VP1 were lower than that of mice immunized with other plasmids (P<0.01). (8) The histopathology changes were less serious as the order pcDNA3/VP1, pcDNA3/MDC-L10-VP1, pcDNA3/MDC-L15- VP1 and pcDNA3/MDC-L19-VP1, but no significant difference were found between them. Conclusion: (1) The gene fragments of MDC-L15, MDC-L19, L15-VP1 and L19-VP1 were succesfully cloned by PCR. (2) The eukaryotic expressing plasmids pcDNA3/MDC- L15-VP1 and pcDNA3/MDC-L19-VP1 were constructed successfully. (3) The survival rates were raised as the linker length increased. So the recombinant DNA vaccine pcDNA3/MDC-L19-VP1 can help BALB/c mice against lethal CVB3 challenge in some extent. (4) After administration of pcDNA3/MDC-L19-VP1, pcDNA3/MDC-L-15-VP1, pcDNA3/ MDC-L10-VP1 and pcDNA3/VP1 to BALB/c mice, the neutralizing antibody could be induced, and the titers of antibody were increased along with the inoculation times. After the third immunization, the titers of mice immunized with pcDNA3/MDC-L19-VP1 were higher than that of mice immunized with other plasmids (P<0.01). (5) Compared with mice injected with other plasmids, the antigen-specific lymphoproliferative response and specific CTL activity of mice immunized pcDNA3/MDC-L19-VP1 significantly enhanced (P <0.01). (6) The results of neutralizing antibody titers, specific lymphocytic proliferative activity, specific CTL cytotoxic activity, virus titers and histopathology were all consistent, which indicated pcDNA3/MDC-L19-VP1 might be a promising vaccine. |
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