Effect Comparative Study Of Recombinant Ricin A Chain With The Natural Ricin

This research systematically studies immune effect comparative study of recombinant ricin A chain with the natural ricin from two aspects.In the first experiment,the study was aimed to produce recombinant ricin A-chain(r-RTA) with highbiological activity and compare its toxicity with native ricin(n-RT). We synthesized the published RTA gene sequence on NCBI and cloned it into the p ET-28 a vector, and then prokaryoticly expressed by the E.coli. After purification with Ni2+-NTA resin column, the r-RTA were eluted by 500mmol/L of imidazole solutions. The purified protein was identified by SDS-PAGE and Western blotting, and the immunogenicity was determined by ELISA.Animal experiments and cell toxicity analysis were conducted to compare the toxicity between the r-RTA and n-RT.The rate of recombinant expression was 31.2%.About 20 mg fusion proteinswere obtained from1000 mL cultures with the protein purity of ≥90%.The results of ELISA showed that the immunogenicity of r-RTA were about 1.27 times of that of n-RT.The half inhibitory concentration(IC50) of n-RT to Raw 264.7cells was 0.01μg/mL and the median lethal dose(LD50)to mouse was 3.27±0.44μg/kg. We used the same dose of toxin to challenge the Raw 264.7cells or mouse, and found that the virulence of n-RT was 2700 times of that of the r-RTA.The mortality rate of n-RT was 40%, while r-RTA was all survive.In the second experiment,the purpose of this research is to compare the immune effect of recombinant ricin A chain(r-RTA), recombinant mutant ricin A chain(m-RTA) and natural ricin(n-RT). In cell test, the toxicity of m-RTA and r-RTA is 2700 times lower than n-RT. After Intraperitoneal injection immunization without adjuvant r-RTA, m-RTA and n-RT, all mice were losing weight during the immune prograss, compared to the group immunized with r-RTA and n-RT, mice in the group immunized with m-RTA have smallest weight change. After four times immunized,the serum antibody titers of mice could reach 10-4 above, using 6000μg/kg m-RTA immunized mice could provide full protection for 20 × LD50 dose of ricin infected. The results show that the r-RTA, m-RTA and n-RT has a certain toxicity in mice, the mutant m-RTA irritation can provide better protection to mice. which provided vital data and theoretical supports for the development of RTA-based vaccine.