Cloning, Expression Of Tetanus Toxin Fragment C Gene And The Immunogenicity

Tetanus is a severe spastic disease caused by tetanus toxin, which can be prevented by immunization with tetanus toxoid. There are some disadvantages in traditional tetanus vaccine based on chemical inactivated tetanus toxin by formaldehyde such as side effects, dangerous brought by the spore, pollution made by formaldehyde and the reversion of virulence. So current tetanus vaccine should be improved, especially needed to develop genetic engineering vaccines. In this study tetanus toxin fragment C was highly expressed in E.coli expression system and a recombinant attenuated Salmonella typhimurium strain expressing tetanus toxin fragment C was constructed, and the immunobiological properties of the expressed protein was evaluated, all of these provide some foundation for further studying of tetanus vaccine.1. Cloning, expression of tetanus toxin fragment C gene in E.coliThe gene of tetanus toxin fragment C (1356bps) was amplified from Clostridium tetani strain 64008 by PCR. The PCR product was inserted into the high-expression vector pET-30a(+) for sequencing and expressed in Exoli Rosetta(DE3). It was shown that sequence has 99.2% homogeneity to that in GenBank. The expressed protein was purified by His Bind Resin Kit. The result of SDS-PAGE verified that a desired recombinant protein with molecular weight of 50kD was expressed and accounted for33.5% of the total bacterial protein. Western-blot analysis further indicated this expressed product had the immunogenicity of tetanus toxin fragment C. The expressed protein initiated enough antibodies in immunized mice that were able to protect mice against a challenge with tetanus toxin.2. Construction and identification of the attenuated Salmonellatyphimurium strain expressing tetanus toxin fragment CTo construct a recombinant attenuated Salmonella typhimurium strain which expresses tetanus toxin fragment C, the gene fragment encoding for tetanus toxin fragment C has been cloned into the expression vector pYA3333 containing asd gene, generated a new recombinant plasmid pYA3333-TetC, which was transformed into Exoli X6212 with asd gene deletion. The recombinant plasmid pYA3333-TetC was further transformed into the host cells by electroporation, yielding the live vaccine strain X4550(pYA3333-TetC), which is a balanced lethal recombinant. Stability and growth curve of the recombinant strain was determined. The results showed that recombinant X4550(pYA3333-TetC) maintained good stability after 100 passages in vitro and the growth status of X4550(pYA3333-TetC) as same as that of X4550(pYA3333). The recombinant strain X4550(pYA3333-TetC) was safe and could provide significant protection against a challenge with tetanus toxin in immunized mice. This study lays the preliminary foundation for the construction of a novel tetanus vaccine.