| Abrin and ricin are highly toxic plant proteins which are very similar in structure and function. The holotoxin consists of two chains(A chain and B chain) joined by a disulfide bond, and the A chain is the toxic chain which can enter into the eukaryotic cells to inhibit the protein synthesis and cause the cell death finially. The B chain is non-toxic and can bind to galactose residues on the surface of cells. The median lethal dose of the two toxins are nanogram level in mouse model. Although the plant proteins are non-infectious, the plants are widly distributed in the world as a economic crop and the manufacturing process of the two toxins are simple. Therefore, they have been listed as the category B bio-agents. Furthermore, some terrorist incidents caused by ricin had been happened in the USA and other European countries, and this threat also should be attach more importance in our country. Study on the toxin detection have been greatly developed, and the detective equipment have also been applied in some important events, but there is no vaccine available against the two toxins currently. Therefore, the studies on the defensive medical vaccine against these toxins are significance to both military and society.Because the A chain is more toxic and immunogenic than the B chain in the A/B toxins, so the aim of this study is to obtain a candidate vaccine which is of non-toxic and highly immunogenic by modifying the gene of the A chain(such as: truncation and site mutations). Then the modified genes were expressed as recombinant proteins with the help of E. coli strain, and the proteins were purified for the following experiments, such as toxicity detection, immunological assay, and so on. These proteins were screening step by step. Finally, the optimizing candidates were screened out to lay the fundation for the further clinical trials.Study on the vaccine against abrin is based on the A chain, we designed five different pairs of primers to amplify the five overlapped fragments. Then the five fragments were expressed with the help of the E. coli strain. Only two in five fragments(t ATA1 and t ATA4) were expressed in soluble form after a series of attempts, therefore, the two soluble protein were used in the next experiments. The recombinant proteins were purified with the help of Ni Sepharose High Performance column, and the purified proteins were used to detect its antigenicity in the Western blotting experiment. Then the toxicity of the recombinant proteins were detected both in vitro and in vivo. After that, the t ATA1 and t ATA4 were used to immunize mice mixed with distinct adjuvants. The vaccination scheme was three times injections at an interval of two weeks and the sera from the mice’s tail vein were used to detect the level of the antibody titers one week after each immunization. Two challenge routes were carried out one week after the final immunization and the status of the mice were recorded every 24 h until 10 days. Study on vaccine against ricin was similar to that on abrin. The first step was to introduce some key site mutations, and then truncated the C-terminal residues from 199-267. The modified gene was constucted and the recombinant protein was expressed and purified in the same way. The purified protein was called mt RTA, which was then used in the next series of experiments, such as western blotting, toxicity detection, mice immunization and protection. Study on the bivalent vaccine against ricin and abrin was carried out based on the two vaccine candidates. There were two forms of the bivalent vaccine, one is the chimera of the two vaccine candidates, called ―mt RTA-t ATA4‖, and the other is the physical mixture of the two vaccine candidates, called ―mt RTA+t ATA4‖. The two forms of the bivalent vaccine had also been done in a series of experiments. Data from all these experiments laied the foundation for the further clinical trials.All the constructed expression vectors were the same as the anticipation after the PCR, double digestion and sequencing confirmation. The sequence of the t ATA4 contains 564 bp bases, which coding 188 amino acid and its relative molecular mass is about 22 k Da. The sequence of the mt RTA contains 594 bp bases, which coding 198 amino acid and its relative molecular mass is also about 22 k Da. The two recombinant proteins were expressed in both soluble and insoluble forms with the help of E. coli strain, and the soluble proteins were used to confirm its antigenicity in the western blotting analysis after the process of purification. The toxicity of the recombinant proteins were not detected both in vitro and in vivo. The recombinant proteins were used to immunize mice mixed with alum or SPO1 adjuvant, and the Ig G antibody were detected as high as 1:106 in the serum from mice’s tail vein after the third immunization. There were two distinct challenge routes, which were i.p. injection and intratracheal aerosol spraying. When the t ATA4 immunized mice were challenged with different doses of abrin in the two routes, the highest whole protection doses were 40 or 30 × LD50 respectively, and the highest passive protection dose was 20 × LD50. When the mt RTA immunized mice were challenged with different doses of ricin in the two routes, the highest whole protection doses were 40 or 20 × LD50 respectively, and the highest passive protection dose was 25 × LD50. The bivalent vaccine experiment confirmed that the ―mt RTA+t ATA4‖ was better than the chimera ―mt RTA-t ATA4‖, the ―mt RTA+t ATA4‖ immunized mice can resist 10 × LD50 of ricin or 20 × LD50 of abrin, when the two toxin challenged together the highest resistence dose was 10 × LD50.The recombinant proteins t ATA4 and mt RTA were successfully designed, constructed, expressed and screened out. The soluble expression was realized in the E. coli strain, and the recombinant proteins were identified with good antigenicity and low toxicity in mouse model. Moreover, the two proteins were confirmed with good immune stimulation and protection. All these results had laied great foundations to the further clincial trails. |
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