中国强毒钩端螺旋体膜蛋白基因的真核表达及基因免疫

Eukaryotic expression and gene immunization of outer envelope protein from Chinese strong virulent Leptospira

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 expressions of two recombinant plasmids pBC-OmpL1 and pcDNA3-OmpL1 in mammalian cell, respectively(the OmpL1 gene is from virulent Leptospira serovar Lai), and to study the humoral immune responses in mice immunized with these two plasmids. Methods The two recombinant plasmids were transfected into COS7 cell with liposome respectively. The cells with plasmids stably transfected were selected by its G418 resistance provided by the plasmid. The expression of plasmids was detected with RT-PCR and Western blot. Then the plasmid was injected intramuscularly into BALB/c for three times at intervals of one week. At last, the antibody titer was measured by ELISA. Results The result of RT-PCR showed that the cell transfected with recombinant plasmid revealed a specific amplified band of about 1kb, but the cell transfected with blank plasmid vector had no band at all. Western blot showed that there was a specific 33.5kD band in the group transfected with recombiant plasmids, but the group transfected with blank plasmids had no band. Two weeks after second immunization with 100μg pBC-OmpL1 or pcDNA3-OmpL1 DNA, the mice produced antibody with titer of 1/19 683, and the mice injected with 50μg pcDNA3-OmpL1 together with 50μg pcDNA3 had lower level antibody with a titer of 1/6 561. However, two weeks later from the third immunization, the antibodies in all mice decreased. Conclusion Both of these two recombinant plasmids (OmpL1 gene of Leptospira Lai) can be expressed in mammalian cells. When the plasmids are inoculated into mice, they can elicit high titer antibodies similar to that induced by inactived whole Leptospira cell vaccine.