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目的 检测含有强毒赖型钩体OmpL1外膜蛋白基因的 2个重组质粒pBC OmpL1和pcDNA3 OmpL1在哺乳动物细胞中的表达 ,及其作为DNA疫苗诱导小鼠体液免疫应答的效果。方法 采用脂质体转染法 ,以重组质粒转染COS7细胞 ,通过抗性标志筛选稳定转染入质粒的细胞 ,RT PCR和Westernblot检测被转染细胞OmpL1基因的表达情况。然后 ,将重组质粒以肌注法免疫BALB c小鼠 ,每 2周 1次 ,共 3次 ,ELISA检测小鼠的体液免疫应答水平。结果 RT PCR结果显示 ,重组质粒转染组在约 1kb处出现特异的扩增带 ;Westernblot检测显示 ,重组质粒转染组总蛋白样品在相对分子质量 (Mr)为 33.5× 10 3 处出现特异的蛋白带 ,而空质粒载体转染组均没有此相应的DNA或蛋白质条带。两质粒第 2次免疫小鼠 2周后 ,10 0 μg重组质粒pBC OmpL1和pcDNA3 OmpL1DNA免疫组产生了效价为 1 196 83的高滴度抗体 ,注射 5 0 μgpcDNA3 OmpL1+5 0 μgpcDNA3的小鼠产生滴度为 1 6 5 6 1较高的抗体 ,第 3次免疫 2周后 ,抗体水平下降。结论 两重组质粒者均能在体外培养的哺乳动物细胞中表达钩体OmpL1蛋白质 ,以其免疫小鼠后 ,均诱导产生高滴度的特异性抗体 ,其效价与全钩体疫苗相当。
Objective To detect the expression of two recombinant plasmids pBC OmpL1 and pcDNA3 OmpL1 containing OmpL1 outer membrane protein genes in mammalian cells and their effects as humoral immune responses induced by DNA vaccine in mice. Methods The recombinant plasmids were transfected into COS7 cells by lipofection method and stably transfected into the plasmids by resistance markers. The expression of OmpL1 gene in transfected cells was detected by RT PCR and Western blot. Then, the recombinant plasmids were immunized BALB mice by intramuscular injection once every 2 weeks for 3 times. The level of humoral immune response in mice was detected by ELISA. Results RT-PCR results showed that a specific amplification band appeared at about 1kb in the recombinant plasmid transfection group. Western blot showed that the total protein sample in the recombinant plasmid transfection group had a specific molecular weight (Mr) of 33.5 × 10 3 Protein band, empty plasmid vector transfection group did not have the corresponding DNA or protein band. Two weeks after the two plasmids were immunized, the mice were immunized with 100 μg recombinant plasmid pBC OmpL1 and pcDNA3 OmpL1 DNA with high titer of 1 196 83 and 50 μg pcDNA3 OmpL1 + 50 μg pcDNA3 Antibodies produced higher titers of 1 6 5 6 1 and decreased antibody levels 2 weeks after the third immunization. CONCLUSION: Both recombinant plasmids can express OmpL1 protein in mammalian cells cultured in vitro. All the recombinant plasmids can induce high titers of specific antibodies after immunizing mice. The titer is comparable to that of whole-cell vaccine.