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Recombinant fusion protein of cholera toxin B subunit (CTB) and poly-valent protective epitopes of plasmodium falciparum was given to i.m. to C57BL/6j mice and rhesus monkeys three times. In rhesus monkeys, high level of antibodies for CTB (1:6400) and malaria epitopes (1:3200) amtobpdoes were elicited as well as the specific CTL activity for P. plasmodium. After the mice were challenged with sporozoites of P. yeolli, about 50% of them were protected from the patent infection. A blood-stage challenge with 10(8) of P. cynomolgi parasite were given to rhesus monkeys, which showed that two animals in control group were patent infection for at least 30 days, in contrast, the two animals immunized were recovered respectively at the day of 11 and 15 after challenges. The results suggested that cholera toxin acts as an effective adjuvent in the development of malaria vaccine.

The expression level of the B subunit gene of cholera toxin (ctx) and E. coli heat labile toxin (ltx) is five to seven times more than that of A subunit gene. In these studies, a 80 basepair translation regulation element was found located in the structure gene of A gene of both toxin operon which consists of three translation initiation region. Site-directed mutation of the initiation codon of TIR3 resulted in the 9 time decrease of the expression of the downstream cistron which was translational coupled with A gene. The results indicated that translation from the internal of A gene and translation coupling are responsible for the differential expression level of the A and B gene of AB5 enterotoxin.

To demonstrate that there existed translation coupling between cholera toxin A subunit gene and B subunit gene, and give the answer why the expression level of B gene is five times more than that of A gene, alpha report system for the investigation of translation coupling was constructed by using lacZ gene as reporter. Frame-shift mutation was introduced near the C terminal of ctxA gene, and the ribosome would read through its normal stop codon. The report plasmid was constructed and it was found that the expression level of lacZ gene decreased five times after the frame-shift mutation. The translation of cholera toxin B subunit gene was translational coupled with A subunit gene, and was responsible for the differential expression level of the two genes.

A unique EcoRI site was introduced at the 3' end of cholera toxin B subunit (CTB) gene by site-directed mutagenesis, polynucleotides encoding 120-145aa epitope of HBV PreS2 were chemically synthesized and fused to the 3' end of cholera toxin B subunit gene. The fused gene was over-expressed (about 30 micrograms ml-1) in E. coli, and more than 95% of the fusion protein was secreted into the medium. The fusion protein expressed was purified by affinity chromatography. The chimera protein obtained bound to ganglioside GM1, and had the antigenicity of both cholera toxin B subunit and HBV PreS2 as confirmed by ELISA. After mice were immunized intramuscularly with the fusion protein, anti-CTB antibody and anti-PreS2 antibody were produced. These results indicated that the fusion protein retained not only the biological function of CTB but also the antigenicity and the immunogenicity of cholera toxin B subunit and HBV PreS2 epitope. This work provided a sound basis for further studies on the construction of engineered peptide vaccine.