CAJ | 학술논문

目的：克隆并表达中国莱姆病螺旋体( Borrelia afzelli)基因型代表菌株FP1的外膜蛋白C( OspC),并对其免疫保护性进行初步研究。方法聚合酶链反应( PCR)扩增莱姆病螺旋体FP1的ospC基因,克隆至原核表达载体pET-30a上,构建pET-30a-OspC重组质粒,转入大肠杆菌感受态细胞BL21(DE3)中,利用IPTG诱导表达。表达产物用Ni-IDA树脂层析纯化,SDS-PAGE电泳、Western blot分析。将rOspC免疫新西兰白兔,间接免疫荧光方法( IFA)检测免疫前、后兔血清中特异性抗体IgG的滴度,进行体外中和试验,从而对OspC的免疫保护性有初步的了解。结果重组质粒pET-30a-OspC构建成功并在宿主菌内高效表达；Western blot表明rOspC与莱姆病螺旋体FP1的多抗有明显的抗原抗体反应；rOspC免疫兔血清IgG效价显著升高(1∶3200或1∶6400)；体外中和试验表明实验组的免疫兔血清均能中和106个/ml的莱姆病螺旋体。结论莱姆病螺旋体的rOspC蛋白具有一定的免疫保护性,可作为研制莱姆病多价亚单位疫苗的一个成分。
목적：극륭병표체중국래모병라선체( Borrelia afzelli)기인형대표균주FP1적외막단백C( OspC),병대기면역보호성진행초보연구。방법취합매련반응( PCR)확증래모병라선체FP1적ospC기인,극륭지원핵표체재체pET-30a상,구건pET-30a-OspC중조질립,전입대장간균감수태세포BL21(DE3)중,이용IPTG유도표체。표체산물용Ni-IDA수지층석순화,SDS-PAGE전영、Western blot분석。장rOspC면역신서란백토,간접면역형광방법( IFA)검측면역전、후토혈청중특이성항체IgG적적도,진행체외중화시험,종이대OspC적면역보호성유초보적료해。결과중조질립pET-30a-OspC구건성공병재숙주균내고효표체；Western blot표명rOspC여래모병라선체FP1적다항유명현적항원항체반응；rOspC면역토혈청IgG효개현저승고(1∶3200혹1∶6400)；체외중화시험표명실험조적면역토혈청균능중화106개/ml적래모병라선체。결론래모병라선체적rOspC단백구유일정적면역보호성,가작위연제래모병다개아단위역묘적일개성분。
Objective To clone and express the outer surface protein C ( OspC) from a Chinese Borrelia afzelli FP1 strain and to evaluate the immune protectivity of the recombinant OspC protein ( rOspC) . Methods The gene encoding OspC protein of Borrelia afzelli FP1 strain was amplified by polymerase chain reaction (PCR) and then inserted into pET-30a plasmid to construct the recombinant expression plasmid pET-30a-OspC. The transformed E. coli BL21 strains carrying pET-30a-OspC plasmid were induced by IPTG to express OspC protein. The expressed proteins were purified by Ni-IDA resin chromatography and analyzed by SDS-PAGE and Western blot assay. Indirect immunofluorescence assay ( IFA) was performed to detect anti-rOspC protein antibodies in serum samples from rabbits immunized with rOspC protein. In vitro neutral-ization test was performed for evaluation the immune protectivity of rOspC protein. Results The recombi-nant expression plasmid pET-30a-OspC was successfully constructed and highly expressed in E. coli BL21. A strong antigen-antibody reaction between the rOspC protein and polyclonal antibody against Borrelia afzelli FP1 strain was detected by Western blot assay. The titers of IgG in serum samples from rabbits immunized with rOspC protein were significantly elevated. The in vitro neutralization test indicated that 106/ml of Borre-lia afzelli FP1 strains were neutralized by every anti-OspC protein serum sample from the experiment group. Conclusion The rOspC protein showed a strong immune protectivity against Borrelia afzelli, which could be used in the development of polyvalent subunit vaccine against lyme disease.