| Cysticercosis, as a serious zoonosis caused by the larvae Taenia solium, is among the most 17 neglected tropical diseases prioritized by the World Health Organization. This globally distributed disease can cause huge harms to the farming industry as well as human health. Although the broad-spectrum anthelmintics praziquantel and albendazole have therapeutic effects against cysticercosis, the serious side effects and drug resistance can occur with their long-term therapies. Therefore, researches on effective vaccine is of particular importance for the disease control. As a major vaccine candidate, the recombinant protein of Taenia oncosphere antigen TSOL18 can improve immune protection. However, the immune effect could be affected because its protection is unstable and its molecular weight is too small(only 16ku).As a highly conserved protein instructure and function, Heat shock Protein70(HSP70) plays an important role in biological processes, e.g., chaperone, antigen presentation, anti-infection immunity and inhibition of apoptosis. HSP70 has been widely used as an adjuvant or a vector to enhance the protective immunity of a vaccine antigen. In order to improve the stability and protective immunity of the recombinant protein TSOL18, we studied the potential of TsHSP70-4 as an immunological adjuvant for it. This study can provide new insights on the adjuvant for the engineered vaccine TSOL18 against cysticercosis. The results of this study are described as follows:1. TsHSP70-4 was amplified by RT-PCR and it was 1953 bp in length, using primers designed based on the gene models from Gene DB database. The ORF was cloned into the vector pPIC9 K after codon optimization based on the codon bias in Pichia pastoris. The recombinant protein with a molecular weight about 95 kD was obtained in P. pastoris expression system. The Western-blot and mass spectrometry analysis verified the expression and amino acid sequence of the expressed TsHSP70-4.2. TsHSP70-4 was linked to the N-teminus of the recombinant protein TSOL18 by a flexible peptide linker. The recombinant protein was successfully expressed in P. pastoris and its molecular weight was about 115 kD.3. As an adjuvant, a mixture of TsHSP70-4 and TSOL18 could significantly extend the immunization duration for TSOL18 in mice. In the first 18 days of immunization, the significant differences of IL-4 and IFN-γ were observed in the serum of immuned mice(P<0.05) of the mixture group, compared with those of the TSOL18 group. On the 21 th day, significant difference of the concentrations of IL-10 between the mixed group and the TSOL18 group was observed(P<0.01). In addition, on the 21 th and 28 th days, the percentages of CD3+CD4+ were significantly increased(P<0.05) in the analysis of whole blood T lymphocyte subtypes. The result reveals that the residence of TSOL18 in mice can be extended when TSOL18 was immuned together with TsHSP70-4. Therefore, TsHSP70-4 can enhance immune response of the T. solium oncosphere TSOL18 recombinant proteins, especially in humoral immunity.4. The immune effect of TsHSP70-4-TSOL18 fusion protein group was significantly less than those of the groups TsHSP70-4+TSOL18 mixture and TSOL18.Consequently, TsHSP70-4 can be used as a potential immunological adjuvant of the recombinant protein TSOL18. The pattern and orientation may lead to less effect of immunization of fusion protein. However, the specific reason for this phenomenon remains to be illustrated. |
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