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Published on: January 31, 2018
[Construction and screen of recombinant BCG strain expressing and secreting human interleukin 12 protein]
1Department of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Objective:
To screen and construct the recombinant Bacillus Calmette-Guerin (rBCG) strain expressing and secreting the human interleukin 12 (rBCG-12).
Methods:
IL-12 complete gene including p40 and p35 subunits was amplified by PCR from a plasmid pORF-h IL-12 and cloned into E. coli-Mycobacteria shuttle vector pMV361. The recombinant vector was named as rpMV-IL-12. rpMV-IL-12 was confirmed by DNA sequencing, and then rpMV-IL-12 was used, by electroporation way, to transform the BCG for getting the recombinant rBCG-12 strain. The positive rBCG-12 clone was screened and identified by kanamycin resistance and IL-12 target gene PCR amplification. The IL-12 protein expression of rBCG-12 was induced with heat shock reaction. Then rBCG-12 culture supernatant and bacterial precipitation were collected respectively and analyzed with SDS-PAGE electrophoresis for their protein components.
Results:
The recombinant shuttle plasmid rpMV-IL-12 was constructed successfully. The sequence of amplified IL-12 gene was consistent with GenBank report. By SDS-PAGE electrophoresis, it was confirmed that the IL-12 protein could express and secrete into the supernatant of rBCG-12 strain culture.
Conclusion:
The recombinant BCG strain expressing human IL-12 protein, rBCG-12 strain, is constructed and screened successfully. It is the foundation for further research on rBCG-12 immune function.
