| Ricin (RIC) and abrin (ABR) are two very toxic proteins isolated from the seeds of Ricinus communist and Abrus precatorius, the two toxins are very similar in molecular structures and the mechanisms of action. Ricin and abrin belong to the type II family of ribosome inactivating proteins, and are comprised of a galactose binding B chain coupled to a toxic A chain by a single disulfide linkage, with relative mass between 62～67 kDa, the A chains (RICA or ABRA) catalytically inactivates the 60S ribosomal subunit by removing an adenine from position 4324 of 28S rRNA, thereby inhibiting protein synthesis. Because of their extraordinary toxicities and easy to be obtained, the two toxins are considered as important biological warfare agents and highly hazardous bioterrorism agents. There are few vaccines that protect against ricin or abrin exposure. Therefore, it is urgent and important to avoid intoxation of ricin and abrin, more biodefense research efforts should be directed towards the development of safe vaccines against ricin or abrin.Objective: To construct the mutant ricin A chain (mRICA), mutant abrin A chain (mABRA), and chimeric protein mRICA/mABRA, express them in the cytoplasm of Escherichia coli, and evaluate their antigenicity. After vaccination on Balb/c mice with these purified proteins, evaluate the potential application of them as effective vaccine candidates for humans, to protect against a high-dose of native ricin or abrin.Methods: The mutant abrin A chain genes mABRAE164AR167L and mABRAE164AR167LN200P were created by using site-directed mutagenesis, and then the recombinant expression vectors, pETHis-mABRA1 and pETHis-mABRA2 were prepared by ligating the 753-bp DNA fragments (mABRAE164AR167L or mABRAE164AR167LN200P) into the pET-His vectors. The expression vectors pETHis-mABRA1 and pETHis-mABRA2 were then transformed into the E. coli strains BL21(DE3)pLysS and BL21(DE3), respectively. After the protein expression was induced with 1.0 mM IPTG at 30°C, we chose BL21(DE3)pLysS/pETHis-mABRA1 and BL21(DE3)pLysS/pETHis-mABRA2 for the production of mABRA. We constructed the expression vectors pETHis-mRICA and pET28a-mRICA by using the mutant ricin A chain gene mRICAD75AV76MY80A prepared before, then the two expression vectors pETHis-mRICA and pET28a-mRICA were transformed into the E. coli strains BL21(DE3)pLysS and BL21(DE3), respectively. The protein expression was induced with 0.5 mM IPTG at 16°C, then we chose BL21(DE3)/pETHis-mRICA for the expression of mRICA. The chimeric gene mRICA/mABRA was produced by connecting the genes of mRICAD75AV76MY80A and mABRAE164AR167L via linker, and then the chimeric gene mRICA/mABRA was ligated into four expression vectors, pQE-80L, pQE-30, pTIG-Trx and pET-28a, respectively. The recombinant expression vectors pQE80L-mRICA/mABRA and pQE30-mRICA/mABRA were transformed into E. coli M15, the other two expression vectors pTIGTrx-mRICA/mABRA and pET28a-mRICA/mABRA were transformed into E. coli BL21(DE3), respectively. After comparing the proteins expressed under induction with 0.1 mM IPTG at 18℃, we chose M15/pQE80L-mRICA/mABRA for the generation of mRICA/mABRA. The proteins mRICA, mABRA1, mABRA2 and mRICA/mABRA were purified by Ni–NTA affinity chromatography resin column under native conditions, and their antigenicities were determined by Western Blot and ELISA. Balb/C mice were vaccinated using the following five formations of immunogen(combined with alum), mRICA, mABRA1, mABRA2, mRICA/mABRA, mixture of mRICA and mABRA1, respectively. The mice were immunized via intramuscular or subcutaneous injection. Blood were sampled from the tail vein of every individual mouse 7 days after each vaccination to determine the titers of anti-mRICA or anti-mABRA antibodies. Then the vaccinated mice were challenged with i.p. injection of native RIC or ABR, or mixture of two toxins, respectively, and survival was evaluated. The sera samples, which contained anti-mRICA or anti-mABRA antibodies, were collected and tested for their passive protection against toxin challenge.Results: Both PCR and sequencing analysis proved that mutant genes mABRAE164AR167L, mABRAE164AR167LN200P, mRICAD75AV76MY80A and mRICA/mABRA had a 100% homology with the designed sequences. The gene of mABRAE164AR167L or mABRAE164AR167LN200P was 753bp, encoding 251 amino acid residues; the length of mRICAD75AV76MY80A was 801bp, encoding 267 amino acid residues; while chimeric gene mRICA/mABRA was 1572 bp, which could encode the chimeric protein mRICA/mABRA with 524 amino acid residues. Successful ligation of recombinant expression vectors were confirmed by PCR amplification and digestion of endonuclease. The relative mass of mRICA, mABRA and mRICA/mABRA were approximately 32 kDa, 30 kDa and 62 kDa, respectively. The soluble 6×His-tagged proteins were one-step purified by Ni–NTA affinity chromatography resin column under native conditions, and the purities were greater than 98% as determined by analysis with TotalLab software. Western Blot and ELISA proved that mRICA could be recognized by the rabbit polyclonal antibody against native RIC, both mABRA1 and mABRA2 had high reactivities with the rabbit polyclonal antibody against native ABR. Furthermore, the chimeric protein mRICA/mABRA showed specific affinity with both polyclonal antibodies against native RIC and ABR. The toxicities of mRICA, mABRA1 and mABRA2 were tested by measuring the cytotoxicities in a human hepatoma cell line (SMMC-7721) and a myeloma cell line (Sp2/0). mRICA decreased toxicity to 1/2500-4100 and 1/8200-10000 that of rRICA or native ricin. mABRA1 had toxicity as low as 1/1350 or 1/8000 that of rABRA or native abrin. mABRA2 reduced toxicity to as low as 1/2700-2800 and 1/16000-17000 that of rABRA or native abrin, respectively.The anti-mRICA or anti-mABRA antibody was detected in the sera of mice vaccinated with mRICA, mABRA1 or mABRA2, respectively. Both anti-mRICA and anti-mABRA antibodies were detected in the sera of mice vaccinated with chimeric protein mRICA/mABRA or mixture of mRICA and mABRA1. After three continuous immunizations, the sera titers of immunized mice reached 1:106 or 1:107. Comparisons of ELISA titers between vaccinated mice and control mice indicated significant difference (p<0.05), while the titers have no significant difference among mice vaccinated via intramuscular or subcutaneous injection (p>0.05). When challenged with native ricin or abrin, a strong secondary response was induced in vaccinated mice, the antibody titers increased 10-fold one week after challenge. Challenge trials with native ricin confirmed that mRICA-immunized mice were able to survive against a dose of 10×LD50 of native ricin. Mice immunized with mABRA1 or mABRA2 were able to survive against a dose of 10×LD50 of native abrin. Furthermore, the immunized sera provided effective passive protection when an in vitro mixture of immunized sera and 10×LD50 of native ricin or abrin were co-injected into naive mice. Mice vaccinated with mixture of mRICA and mABRA1 were able to survive against a mixture of 5×LD50 of native ricin and abrin, and the survival rate decreased to 80% when challenged with 6×LD50 of native ricin and abrin, 20% when challenged with 8×LD50 of native ricin and abrin. While the mice vaccinated with chimeric protein mRICA/mABRA were able to survive against a mixture of 6×LD50 of native ricin and abrin, and the survival rate was 60% when 8×LD50 of native ricin and abrin mixture given.Conclusion: This study describes the generation of a substantial amount of mRICA, mABRA1, mABRA2 and their chimeric protein mRICA/mABRA from E.coli. The mutant or chimeric proteins are dramatically less able to inhibit protein synthesis than native ricin and abrin, but are immunogenic and safe to animals. Mice vaccinated with mRICA, mABRA1, mABRA2, chimeric protein mRICA/mABRA or mixture of mRICA and mABRA1 are completely protected against lethal doses of ricin and abrin challenge, as immunogens they could stimulate strong immune responses and are potential candidates for novel ricin and abrin vaccines. |
