Zhonghua wai ke za zhi [Chinese journal of surgery] · 2008
[Construction of recombinant bacillus Calmette-Guérin vaccine secreting human interferon-alpha 2b].
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Ding Guo-Qing, Shen Zhou-Jun, Chen Shan-Wen, Zhou Xie-Lai, Liao Guo-Dong
Аннотация
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.
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.