Related Experiment Video
Updated: May 27, 2026
CRISPR-based Shuttle Cloning: A High-throughput Cloning Method
Published on: June 13, 2025
[Construction of recombinant shuttle plasmid pIMP1-eHER2/neu and screening and identification of its stable
Yan-li Zhang1, Wen-qing Zhang, Qiu-bo Wang
1Qingdao University Medical College, Qingdao, China. qdzhangyanli1974@163.com
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.
