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Published on: January 31, 2018
[Construction, expression and identification of recombinant bacillus Calmette-Guérin vaccine secreting human
Hai-tao Liu1, Xiao-wen Sun, Yi-nan Zhang
1Department of Urology, The First People's Hospital of Shanghai, Jiaotong University, Shanghai, China.
Objective:
To construct a recombinant bacillus Calmette-Guérin vaccine (BCG) secreting human interferon alpha-2a (IFNalpha-2a).
Methods:
BCG Ag85B signal sequence was amplified from the genome of BCG by using polymerase chain reaction (PCR) and cloned in E.coli-BCG shuttle-vector pMV261 to get pMS. The cDNA fragment encoding human IFNalpha-2a was amplified from the plasmid pBIFNalpha-2a by using PCR and inserted into the shuttle expression vector pMV261. The recombinant plamid pMSIFNalpha-2a was identified by restriction endonuclease digestion, PCR amplification and nucleotide sequencing. pMSIFNalpha-2a was electroporated into BCG to get rBCG. The DNA and protein expressions of IFNalpha-2a gene in rBCG were determined by PCR and Western blotting respectively. IFNalpha-2a in the culture supernatant of rBCG was detected by enzyme-linked immunosorbent assay (ELISA).
Results:
The recombinant plamid pMSIFNalpha-2a was constructed successfully and confirmed by restriction endonuclease analysis, PCR detection and nucleotide sequencing analysis. pMSIFNalpha-2a was successfully transformed into BCG by electroporation and were capable of synthesizing and secreting cytokine IFNalpha-2a. Western blotting revealed that the secretive proteins could specially combine with antibody against human IFNalpha-2a. the level of IFNalpha-2a (324.57 pg/ml) in the culture supernatant of rBCG was higher than control group by ELISA assay.
Conclusion:
The constructed recombinant BCG strain produces and secretes human IFNalpha-2a and it will be used in the treatment of superficial bladder cancer.
