[Construction of recombinant shuttle plasmid pIMP1-eHER2/neu and screening and identification of its stable Clostridium sporogenes transformants].

AIM

To construct recombinant clostridium sporogenes modified with the extracellular domain of human oncogene HER2/neu, to lay a foundation for further study of its antitumor effect.

METHODS

The extracellular domain (ECD) of HER2/neu gene was attached to the downstream of promoter and signal sequence of clostridia endo-1, 4-glucanase (eglAp) by SOE-PCR to construct fusion gene eglAp-HER2/neu, which was then inserted into E.coli-clostridia shuttle plasmid pIMP1 to construct recombinant plasmid pIMP1-eHER2/neu. The recombinant plasmid was firstly transformed into E.coli DH5α.Then the correct construct was identified and introduced into C. sporogenes by electroporation. Positive clones were selected by erythromycin resistance, bacteria PCR were used for verification.

RESULTS

Restriction map and sequencing result showed that the sequence and ORF of fusion gene eglAp-HER2/neu in recombinant plasmid pIMP1-eHER2/neu was correct. Bacteria PCR results indicated that the recombinant plasmid pIMP1-eHER2/neu was successfully transformed into C.sporogenes. After more than 20 passages under antibiotic pressure, C.sporogenes transformants could stably carry the recombinant plasmid pIMP1-eHER2/neu.

CONCLUSION

Stable C.sporogenes transformants with the recombinant plasmid pIMP1-eHER2/neu are successfully acquired, which laid a foundation for further anti-tumor study.