[Cloning, expression and immunologic identification of C31B8.8 gene of Caenorhabditis elegans].

OBJECTIVE

To clone and express C31B8.8 gene of wild-type Caenorhabditis elegans, and study the immunological characteristic of the recombinant protein.

METHODS

Total RNA was extracted from cultivated C. elegans and reversely transcribed into cDNA. C31B8.8 gene was amplified by PCR and cloned into pMD-18T vector for sequencing. The accurate sequence was subcloned into the expression vector pET-30a with (His) 6-tag. The recombinant plasmid was transformed into E. coli BL21 and followed by expression of the protein induced by IPTG. The recombinant protein was identified by using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) and Western blotting 10 BALB/c mice were randomly divided into C31B.8-immunized group and PBS + adjuvant group Mice in C31B8.8-immunized group were immunized with 40 microg of purified C31B8.8 antigen formulated in Freund's adjuvant Mice in PBS + adjuvant group received only adjuvant emulsified with PBS. All the mice received four immunizations every week with the same dose of antigen. Serum samples were collected at pre-immunization and certain time after immunization and the antibody titer was analyzed by ELISA. The recombinant C31B8.8 protein and soluble components of Angiostrongylus cantonensis fourth-stage larvae were identified by Western blotting.

RESULTS

The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. MALDI-TOF-MS and Western blotting analysis showed that the recombinant C31B8.8 protein was the target protein. Compared with PBS + adjuvant group, mice immunized with purified protein C31B8.8 produced higher level of IgG. The anti-C31B8.8 serum recognized recombinant C31B8.8 protein, and reacted with soluble antigens of A. cantonensis fourth-stage larvae.

CONCLUSION

C elegans C31B8 gene shows certain immunogenicity and immunoreactivity, and the soluble antigens of A. cantonensis fourth-stage larvae can react with anti-C31B8.8 serum.