title: [Construction of recombinant bacillus Calmette-Guérin vaccine secreting human interferon-alpha 2b].
abstract: OBJECTIVE
  To construct a recombinant bacillus Calmette-Guérin vaccine (rBCG) secreting human interferon-alpha 2b (IFN alpha-2b).
  
  
  METHODS
  BCG Ag85B signal sequence and IFN alpha-2b gene were amplified from the genome of BCG and of human peripheral blood by polymerase chain reaction (PCR), respectively. IFN alpha-2b gene was cloned in E. coli-BCG shuttle-vector pMV261 to get pMV261-IFN alpha-2b. A new recombinant plasmid pMV261-IFN alpha-2b was constructed by inserting BCG Ag85B signal sequence into pMV261-Ag85B-IFN alpha-2b. Then, BCG was transformed with this recombinant plasmid by electroporation, and designated as rBCG-IFN alpha-2b. The DNA and protein expressions of IFN alpha-2b gene in rBCG were determined by PCR and Western blot respectively. Also the quantity of IFN alpha-2b protein secreted by rBCG in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA).
  
  
  RESULTS
  By partial nucleotide sequencing, the DNA sequences of human IFN alpha-2b and BCG Ag85B were consistent with that in the Gene Bank, and were correctly inserted into the shuttle expression vector pMV261 to construct recombinant plasmid pMV261-Ag85B-IFN alpha-2b. BCG was successfully transformed with this recombinant plasmid by electroporation and the recombinant BCG (rBCG-IFN alpha-2b) was capable of synthesizing and secreting cytokine IFN alpha-2b. The concentration of IFN alpha-2b in culture supernatants was quantified by ELISA and calculated to be approximately 301.45 pg/ml.
  
  
  CONCLUSIONS
  Recombinant BCG secreting human IFN alpha-2b (rBCG-IFN alpha-2b) was constructed successfully and the specific IFN alpha-2b protein can be expressed highly and steadily by rBCG vaccine.
authors.affiliation: Department of Urology, Sir Run Run Shaw Hospital, Affiliated with School of Medicine, Sir Run Run Shaw Institute of Clinical Medicine, Zhejiang University, Hangzhou 310016, China.
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Ding
authors.forename: Zhou-Jun
authors.initials: ZJ
authors.lastname: Shen
authors.forename: Shan-Wen
authors.initials: SW
authors.lastname: Chen
authors.forename: Xie-Lai
authors.initials: XL
authors.lastname: Zhou
authors.forename: Guo-Dong
authors.initials: GD
authors.lastname: Liao
mesh_terms: D001500:BCG Vaccine / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins; D014169:Transformation, Bacterial
chemical_list: D001500:BCG Vaccine; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins