Ding Guo-Qing, Shen Zhou-Jun, Chen Shan-Wen, Zhou Xie-Lai, Liao Guo-Dong
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.
Zhonghua Wai Ke Za Zhi. 2008 Jul 1;46(13):1022-6.
To construct a recombinant bacillus Calmette-Guérin vaccine (rBCG) secreting human interferon-alpha 2b (IFN alpha-2b).
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).
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.
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.
构建分泌人α-2b干扰素（IFNα-2b）的重组卡介苗（rBCG）。
分别通过聚合酶链反应（PCR）从卡介苗基因组和人外周血基因组中扩增卡介苗Ag85B信号序列和IFNα-2b基因。将IFNα-2b基因克隆到大肠杆菌-卡介苗穿梭载体pMV261中，得到pMV261-IFNα-2b。通过将卡介苗Ag85B信号序列插入pMV261-Ag85B-IFNα-2b构建新的重组质粒pMV261-IFNα-2b。然后，通过电穿孔用该重组质粒转化卡介苗，并命名为rBCG-IFNα-2b。分别通过PCR和蛋白质免疫印迹法测定rBCG中IFNα-2b基因的DNA和蛋白质表达。还通过酶联免疫吸附测定（ELISA）测定rBCG在培养上清液中分泌的IFNα-2b蛋白的量。
通过部分核苷酸测序，人IFNα-2b和卡介苗Ag85B的DNA序列与基因库中的序列一致，并正确插入穿梭表达载体pMV261中以构建重组质粒pMV261-Ag85B-IFNα-2b。通过电穿孔用该重组质粒成功转化卡介苗，重组卡介苗（rBCG-IFNα-2b）能够合成并分泌细胞因子IFNα-2b。通过ELISA对培养上清液中IFNα-2b的浓度进行定量，计算得出约为301.45 pg/ml。
成功构建了分泌人IFNα-2b的重组卡介苗（rBCG-IFNα-2b），rBCG疫苗可高效稳定表达特异性IFNα-2b蛋白。
