RESUMO
OBJECTIVE: To construct recombinant Mycobacterium smegmatis vaccine expressing Cysticercus cellulosae cC1 antigen. METHODS: The recombinant pET28a-cC1 plasmid was extracted and double digested by Xho I and BamH I restriction enzymes, and shuttle plasmid pMV261 was extracted and double digested by Hind III and BamH I restriction enzymes. Both fragments were modified by Klenow fragment to form blunt end, then the large fragments of cC1 and pMV261 plasmid were purified and ligated by T4 ligase enzyme. The recombinant pMV261-cC1 plasmid was constructed and sequenced. Then the pMV261-cC1 plasmid was transformed into Mycobacterium smegmatis by the electrotransformation method. The recombinant cC1-Mycobacterium smegmatis was induced by heat and identified by the Western blotting method with the sera of cysticercosis patients. In addition, the growth states of the Mycobacterium smegmatis and the recombinant cC1-Mycobacterium smegmatis were compared and the growth curves were drawn. RESULTS: The restriction enzyme and sequencing results showed that the recombinant pMV261-cC1 plasmid was successfully constructed. After heat induction, a 40 kD band was showed by PAGE analysis of cC1-Mycobacterium smegmatis. The Western blotting results showed that the sera of cysticercosis patients could recognize the 40 kDa band, which suggested that cC1 protein was expressed in Mycobacterium smegmatis. Compared with the Mycobacterium smegmatis, the recombi- nant cC1-Mycobacterium smegmatis showed no significant difference in proliferation characteristics. CONCLUSION: The recombinant cC1-Mycobacterium smegmatis vaccine has been successfully constructed.
Assuntos
Antígenos de Helmintos/genética , Cysticercus/imunologia , Engenharia Genética/métodos , Mycobacterium smegmatis/genética , Animais , Antígenos de Helmintos/imunologia , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA/metabolismo , DNA Recombinante/genética , Expressão Gênica , Dados de Sequência Molecular , Mycobacterium smegmatis/crescimento & desenvolvimento , Plasmídeos/genética , Transformação Genética , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
OBJECTIVE: To clone the coding gene of the stage-specific antigen cC1 from Cysticercus cellulosae and express high levels of soluble cC1 in E.coli. METHODS: The cC1 gene was amplified from Cysticercus cellulosae by RT-PCR and cloned into pMD18-T vector, followed by subcloning into the prokaryotic expression plasmid pET28a. The recombinant plasmid was transformed into E.coli BL21(DE3) and the expression conditions were optimized. The expressed product was purified by Ni(+)-affinity chromatography, analyzed by high-performance liquid chromatography (HPLC), and identified with SDS-PAGE and Western blotting. RESULTS: The fragment length of the amplification product by RT-PCR was 1056 bp. Comparison of the amplified gene sequence with the cC1 gene in Genbank identified a samesense point mutation at 423 position in the gene cloned into the expression plasmids. After a 6-h induction with 0.05 mmol/L IPTG at 37 degrees celsius;, the expression of the 40 kd soluble fusion protein exceeded 60% of the total bacterial protein, and the fusion protein was recognized by Cysticercus-infected human sera. The purity of the fusion protein was about 94% after purification by affinity chromatography. CONCLUSION: The stage-specific antigen cC1 from Cysticercus cellulosae has been successfully cloned and the soluble protein efficiently expressed in E.coli, which provides the basis for its further study and application.