[Construction, expression and identification of recombinant bacillus Calmette-Guérin vaccine secreting human interferon alpha-2a].
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.


CONCLUSIONS
The constructed recombinant BCG strain produces and secretes human IFNalpha-2a and it will be used in the treatment of superficial bladder cancer.
Liu
Hai-tao
HT
Department of Urology, The First People's Hospital of Shanghai, Jiaotong University, Shanghai, China.
Sun
Xiao-wen
XW
Zhang
Yi-nan
YN
Zhuo
Jian
J
Fan
Jie
J
Wang
Feng
F
Xia
Shu-jie
SJ
D001500:BCG Vaccine / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins; D014169:Transformation, Bacterial
D001500:BCG Vaccine; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins