| The 42kDa Carboxyl-Terminal Processing Fragment of Plasmodium falciparum Merozoite Surface Protein-1 (PfMSP-142) is one of the anti-malarial vaccine candidate antigens. In the present study, the nucleotide sequence of PfMSP-142 was modified at the 5 ' position and subeloned into transfer vector pBM030. Recombinant Bombyx mori Nuclear Polyhedrosis Viruses (BmNPVs) carrying PfMSP-1 42 were constructed by cotransfecting wild-type BmNPV genomic DNA and PfMSP-142-inserted recombinant pBM030 transfer vector into BMN cells. A total of three recombinant BmNPVs, each carrying a modified form of PfMSP-142, were generated. The recombinant BmNPVs were separately used to infect BmN cell cultures (in vitro) and silkworms (in vivo) to express the corresponding recombinant PfMSP-1 42 proteins. One of the constructs, with a honeybee melittin signal peptide sequence, gave the highest level of expression as determined by a sandwich ELISA. The average expression level of this construct achieved in silkworms was 378.86mug/mL hemolymph, which was 100-fold higher than that in BmN cell culture (3.52mug/mL culture medium). Northern blot analysis showed no significant difference in transcript level among the three constructs, suggesting that the expression of recombinant PfMSP-142 protein was regulated translationally. The recombinant PfMSP-142 protein could be detected in Western blots using a monoclonal antibody specific to the carboxyl terminal of the native PfMSP-1 protein. Immunoaffinity chromatography was employed to purify the recombinant PfMSP-142 protein to high degree of purity. On the other hand, conventional chromatography in ion-exchange media could only partially purify the protein. The recombinant PfMSP-1 42 protein was reduction-sensitive, and it was shown to approximate the native conformation in competitive inhibition ELISA. The recombinant PfMSP-1 42 protein was highly immunogenic. An extremely high antibody titer could be produced in rabbits immunized with the protein in Freund's adjuvant. Specificity test showed that the antibodies recognize conserved and common epitopes on the carboxyl terminal of native PfMSP-1 protein. Besides, the antibodies were capable of inhibiting parasitic growth in vitro. The present findings demonstrate the feasibility of high-level expression of biologically and immunologically active recombinant PfMSP-142 protein in BmNPV-silkworm expression system. The use of this expression system in preparation of recombinant PfMSP-142 protein for human clinical trials appears to be an attractive and economical choice. |
