Construction Of The Orally Attenuated Recombinant Salmonella Typhimurium Vaccine Expressing Tsol18 And Research Of Its Immunology

Cysticercosis, which is known as a social-economic disease in the world, is an important zoonosis infecting human and pigs caused by Taenia solium larval Cysticercus cellulosae. It is a major public health problem and a potential threat to human security in most area of china, especially in minority regions. This disease must be prevented for its influence on international competition of the meat product and the great economic loss. Howerer, the choice and source of vaccinal antigen always puzzles veterinarians. Tsol18 is a specifically expressed antigen at T.solium oncosphere stage and secreted in intermediary host infected by oncosphere at early stage. The immune serum of Tsol18 can kill oncosphere in vitro and thus Tsol18 is one of the important potential antigens of cysticercosis vaccine. However, the Tsol18 recombinated protein was limited to utilization for that it is usually expressed as in cytoryctes pattern in E. coli system, which cause many difficulties such as renaturation, purification, lower activity, short conservation time and fast degradation.Salmonella typhimurium which one of the invasive bacterial species can be attenuated without loss of invasiveness by gene engineering techniques and used for delivering directly immunizing antigen into host cells and resulting in subsequent induction of humoral and cellular immune responses. Recent studies showed that the attenuated S.t is one of the focuses of vaccine development for its usage as vehicle of orally vaccine. The main purpose of this study is to optimize the potential Tsol18 antigen for cysticercosis vaccine, determine the immunologic responses of orally live recombinant vaccine and investigate the immunoprophylaxis effection.T.solium eggs were collected and total RNA was extracted from hatched and activated eggs in vitro. Tsol18, which was site-directed mutation of 4 bases and removed the encoding sequence of the signal peptide at N-terminal, was cloned by RT-PCR using specificity primers. The Tsol18 DNA fragment was ligated into the expression vector pGEX-4T-1 and transformed into competent E.coli BL21 to express Tsol18 recombinant protein. The Tsol18 recombinant protein was analyzed by SDS-PAGE, Western blot, stability test and used to prepare rabbit anti-Tsol18 serum. Tsol18 gene was inserted into the attenuated S.t expression vector pYA3341 containing asd gene. The reconstructed pYA3341 -Tsol18 was electrotransformed into a balanced-lethal recombinant attenuated S.t strain X4550 which is delta Cya, Crp and asd by twice. The immunogenicity, growth stability, orally safety and distribution in mice after oral administered of the recombinant X4550(pYA3341-Tsol18) were analyzed. The possible immune mechanism of X4550 (pYA3341-Tsol18) was investigated by detection of the antibody through ELISA and assaying the spleen cell subset through flow cytometry in immunized mice. The antibody of immunized pigs were detected by ELISA.The GST-Tsol18 recombinant protein was expressed mainly as soluble fusion pattern and count to 24 % of the total bacteria protein. Western blot indicated the GST-Tsol18 fusion protein could recognize cysticercosis positive serum and had a good stability for it only degradated 20.1% after conservation for 120 days at 4℃. All of the randomly selected recombinant vaccine stains were able to grow after 100 passages without selection pressure. This indicated the stability of the recombinant attenuated S.t oral vaccine 4550(pYA3341 -Tsol18). All BALB/c mice could survive well after orally taking X4550 (pYA3341-Tsol18) 2.0×1012 CFU for 30 days and verified the safety of the recombinant vaccine. The X4550 (pYA3341-Tsol18) could be detected in mice spleen 21 days post immuneization, indicating it can survive in vivo and stimulate host immune response for a long time. The X4550 (pYA3341-Tsol18) could induce a high titre of antibodies, the OD value of immuned mice could reach to 0.645 at four weeks and the secreted IgA could be detected in mouse liquor entericus at six weeks after secondary immunization. In contrast to controls, the immunized mice had an obviously augment of CD4+ and CD8+ T cells and the percentage of CD4+/ CD8+ T cells was significantly increased (P<0.01). These hinted that the recombinant oral vaccine could induce Th1 and Th2 immunity response at the same time. In pig immuniza- tion trial, the titre of antibody began to rise at 20 days after immunization and reach to 1.025 at 30 days after second-immunization. This suggested that the recombinant vaccine could induce a immunological reponse against cysticercosis.In this study a high performance expressive, satisfactory immunogenicity and stabile potential immune recombination GST-Tsol18 antigen of cysticercosis was obtained. A recombinant attenuated S.t. vaccine X4550(pYA3341-Tsol18) which expressed recombinant Tsol18 protein was constructed successfully and the immunology of recombinant vaccine was studied. All these layed the foundation for the development and application of genetically engineering vaccines against cysticercosis.