Studies On Construction And Immunological Characteristics Of Recombinant BCG-TSOL18 Vaccine Of Taenia Solium

Objective: The aim of this study is to construct a recombinant Bacillus Calmette-Guérin(BCG)-TSOL18 vaccine of Taenia solium by gene engineering technology and study on the expression of the TSOL18 gene in BCG; and then, dynamically observing the level changes of serum specific Ig G and Ig G2 a, the level of spleen lymphocyte proliferation and the level of IL-2 and IL-4 in culture supernatant of spleen lymphocytes induced by the recombinant BCG-TSOL18 vaccine(r BCG-TSOL18) immunized Kunming mice. So as to lay the foundation for the further research of the vaccine.Methods: The recombinant plasmid p GEX-TSOL18 provided by our laboratory was digested by restriction endonucleases Bam H I and Eco RⅠ to obtain TSOL18 gene. The TSOL18 gene was cloned into Escherichia coli-Mycobacterium shuttle expression vector p MV261 to construct the recombinant plasmid p MV261-TSOL18. The recombinant plasmid p MV261-TSOL18 was electroporated into BCG to construct the recombinant BCG-TSOL18 vaccine of Taenia solium, then the recombinant BCG-TSOL18 vaccine was identified by PCR. The recombinant BCG-TSOL18 vaccine produced protein via thermally-induced at 45 ℃and the expressed protein was identified by SDS-PAGE and Western blot analysis. To study the immune response induced by the recombinant BCG-TSOL18 vaccine of Taenia solium immunized Kunming mice, 80 Kunming mice were randomly divided into four groups, 20 mice in each group. They were intraperitoneal injection group with vaccinating 5×106 colony forming units(CFU) of recombinant BCG-TSOL18 vaccine 100μl, intragastric administration group with vaccinating 4×108CFU of recombinant BCG-TSOL18 vaccine 100μl, BCG control group, and PBS control group, respectively. The serum was separated and spleen lymphocytes were collected at 0 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks after immunization, respectively. The levels of specific Ig G and Ig G2 a were detected by enzyme linked immunosorbent assay(ELISA). The levels of interleukin-2(IL-2) and IL-4 were detected by ELISA. The level of spleen lymphocyte proliferation was detected by CCK-8.Results: The TSOL18 gene(393bp) was successfully obtained by enzyme digestion. The recombinant plasmid p MV261-TSOL18 was successfully constructed and confirmed by enzyme digestion, PCR, and sequencing. The recombinant BCG-TSOL18 vaccine of Taenia solium was successfully constructed and confirmed by PCR. The recombinant plasmid p MV261-TSOL18 could successfully express the recombinant TSOL18 protein via thermally-induced in BCG, its relative molecular mass(Mr) was approximately 14.7k Da and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). Result of Western blot showed the TSOL18 protein had the specific antigenicity. Dynamic observations showed that the levels of specific Ig G and Ig G2 a in mice serum increased in 2~8 weeks and reached a higher level at 6 weeks after immunization in intraperitoneal injection group and intragastric administration group with recombinant BCG-TSOL18 vaccine of Taenia solium. The level of spleen lymphocyte proliferation increased in 2~8 weeks and reached a higher level at 6 weeks after immunization. The levels of IL-2 and IL-4 in spleen lymphocyte culture supernatant increased in 2~8 weeks and 4~6 weeks and reached a higher level at 6 weeks and 4 weeks after immunization, respectively.Conclusion: 1. The recombinant plasmid p MV261-TSOL18 was successfully constructed. 2. The recombinant BCG-TSOL18 vaccine of Taenia solium was successfully constructed. 3. The expression of TSOL18 gene of Taenia solium was successful in BCG and the expressed protein of the recombinant TSOL18 had the specific antigenicity. 4. The recombinant BCG-TSOL18 vaccine of Taenia solium could induce seffective humoral and cellular immune response in the mice.