RESUMO
OBJECTIVE: To construct and express Echinococcus granulosus recombinant bacille Calmette-Guerin (BCG) strain rBCG-EgG1Y162. METHODS: The encoding gene of the antigen EgG1Y162 of E. granulosus was recombined with E. coli-Mycobacterium shuttle expression plasmid vector pMV361 by genetic engineering technique, and transformed into E. coli for amplification. The recombinant plasmid rpMV-EgG1Y162 was identified by PCR, double digestion with restriction enzymes, and sequence analysis. The confirmed rpMV-EgG1Y162 was transformed into BCG strain via electroporation technique to construct the recombinant rBCG-EgG1Y162. After identification by PCR and double digestion with restriction enzymes, the recombinant strain was cultured for about 2 weeks. In order to induce the expression of target protein, the rBCG was placed in 45 degrees C for 30 min. SDS-PAGE and Western blotting were used to analyze the expressive protein. RESULTS: The product of recombinant plasmid rpMV-EgG1Y162 was approximately 360 bp by PCR amplification and double digestion with restriction enzymes, consistent with the expected fragment length. Sequencing results showed that the inserted sequence was correct. The rBCG-EgG1Y162 grew well and the identification of PCR and enzyme digestion revealed accuracy. The results of SDS-PAGE and Western blotting showed that the relative molecular weight (M(r)) of the protein was about 71 000. CONCLUSION: The E. granulosus rBCG-EgG1Y162 strain is constructed and expressed.
Assuntos
Vacina BCG/genética , Echinococcus granulosus/genética , Vacinas de DNA/genética , Animais , Antígenos de Helmintos , Vacina BCG/imunologia , Clonagem Molecular , Echinococcus granulosus/imunologia , Escherichia coli/genética , Feminino , Expressão Gênica , Mycobacterium bovis/genética , Coelhos , Vacinas de DNA/imunologiaRESUMO
A pair of primers (egG1Y162) were designed according to the nucleotide sequence of Echinococcus multilocularis emY162 antigen gene. Using genomic DNA and cDNA from protoscoleces and adult worms of E. granulosus as templates, PCR was performed with the primers to obtain fragments of egG1Y162 gene. PUCm-T/egG1Y162 recombinant plasmids and PUCm-T/egY162 cDNA recombinant plasmids were constructed and identified by PCR, digestion with restriction enzyme and sequencing. The egG1Y162 antigen gene was amplified in protoscoleces and adult worms of E. granulosus. The size of the egG1Y162 gene was 1 648 bp and cDNA was 459 bp, and GenBank accession numbers were AB458258 and AB458259, respectively.