Synthsis And Inducible Expression Of A Gene Encoding Pre-erythrocytic Chimeric Protein Of Plasmodium Falciparum In Attenuated Salmonella Typhi And Detection Of Its Immunogenicity In Mice

Thrombospondin-related anonymous protein(TRAP) and circumsporozote protein(CSP) ofPlasmodiumfalciparum have been considered important candidates for pre-erythrocytic malaria vaccine. The sequences of ectodomain of TRAP (aa: 26 ?30) and (NANP)19 repeat region and entire carboxy terminus of CSP were fused to generate an Chimeric Protein 4 ofPlasmodiumfalciparum(designated as PtCP-4) via a hinge consisting of Gly-Pro-Gly. The sequence of pfcp-4 was redesigned using yeast codon usage and synthesized by PCR-based method. To synthesize the gene, the sequence of 1577 bp pfcp-4 gene was divided into two fragments( designated as pfcp-4a and pfcp-4b, respectively). The pfcp-4a fragment was assembled by eight oligonucleitides with the length of 120, 116, 126, 125, 113, 115, 118 and 105 bases, respectively using Asymmetric PCR method while the pfcp-4b fragment by another eight oligonucleitides with length of 117, 105, 132, 138, 115, 119, 118 and 96 bases, respectively. The two fragments were inserted into pBluescript plasmid to DNA sequence analysis. Any errors including substitutions and deletions were corrected. After confirming the error-free, the two fragments were combined using restriction site Pst Ito generate the entire pfcp-4 synthetic gene. To examine the expression of the gene, the synthetic gene was inserted into pQE vector where expression of foreign gene was controlled by IPTG. The resulting plasmid was transformed into E. ccli SG13009 for inducible expression. Our data showed that PIrP-4 recombinant protein was produced under IPTG induction whereas no product was detected in the cell without induction. The Molecule Weight of the protein is 57 kDa which is identical to the expected size and the product was recognized by polyclonal antibodies against CSP protein. Tetracycline-controlled expression of interest gene in attenuated Salmonella typhi CVD9O8 strain was established via insertion of tet repressor(tetR) cassette into defined locus aroC. Inducible expression of foreign gene in this system was achieved in vitro and in vivo by tetracycline. Moreover, via inducible expression of foreign gene in vivo the stability of plasmid in vivo was improved. Additional objectives of this investigation is to establish tetracycline-controlled expression of pfcp-4 in CVD9O8 vaccine strain and to induce PfCP-4 specific immune response in mice via immunization of mice with the salmonella expressing the protein. Therefore, the synthetic pfcp-4 gene was inserted into pZE1 1 vector and introduced into CVD9O8aroC::tetR strain for inducible expression. Western blot analysis of PFCP-4 demonstrated the expression of the gene was completely shut off in absence of tetracycline whereas full expression of the gene was achieved in presence of the inducer. The CVD9O8aroC: :tetR strain expressing PfCP-4 was utilized for immunization of mice intraperitonelly with three times at one week interval. Tetracycline was injected into mice in the induction group via tail vein. Sera was collected from immunized mice for detecting the specific antibodies using ELISA. Our result showed that the specific antibodies were detected in the mice injected of tetracycline whereas no PICP-4 specific antibodies were detected in the uninduction group and negative control group. This data suggest that PtCP-4 was produced in vivo under tetracycline induction and presented to host immune system leading to the immune response. In summary, a chimeric protein consisting of TRAP and CSP of Plasm odium falciparum...