中国莱姆病螺旋体PD91外膜蛋白A肽段的克隆表达及其免疫保护性的初步研究

目的:克隆并表达中国莱姆病螺旋体基因型代表菌株伽氏疏螺旋体( Borrelia garinii, B. garinii)PD91外膜蛋白A(OspA)的126~274 aa肽段,并对其免疫保护性进行初步研究。 方法:采用聚合酶链反应(PCR)扩增 B. garinii PD91的126~274 aa OspA肽段基因,克隆至原核表达载体pET-30a上,构建pET-30a-OspA-pep重组质粒,转入大肠埃希菌感受态细胞BL21(DE3)中,利用IPTG诱导表达,表达产物用Ni-IDA树脂层析纯化,采用Western blot分析其免疫原性。将不同剂量的重组OspA-pep(rOspA-pep)蛋白(20、30、40、50、60、80、100 μg)免疫新西兰家兔,采用间接免疫荧光法(IFA)检测免疫前后的抗体滴度,选取产生抗体滴度最高的剂量组为最佳剂量组。用最佳剂量组的免疫兔血清进行体外中和试验以检测rOspA-pep蛋白免疫后血清抗体的体外杀菌能力,同时用最佳剂量的rOspA-pep蛋白免疫新西兰家兔,观察其抗体滴度变化。 结果:重组质粒pET-30a-OspA-pep构建成功并在宿主菌体内高效表达。Western blot表明rOspA-pep蛋白与 B. garinii PD91的多抗有明显的免疫应答。IFA检测结果表明rOspA-pep蛋白免疫后的兔血清IgG抗体滴度明显升高(最高可达1∶2 480),40 μg为rOspA-pep的最佳免疫剂量。体外中和试验结果表明该剂量rOspA-pep蛋白免疫家兔后产生的抗体对10 6个/ml的 B. garinii和阿弗西尼疏螺旋体( Borrelia afzelii, B. afzelii)型代表菌株PD91和FP1的中和率达100%,对10 7个/ml的FP1的中和率为100%,对10 7个/ml的PD91的中和率为60%。用40 μg rOspA-pep在1 d和30 d免疫新西兰家兔2次后,其抗体达到高峰,持续时间为3~4个月,之后抗体滴度逐渐下降。 结论:中国莱姆病螺旋体基因型代表菌株 B. garinii PD91的126~274 aa OspA肽段具有较好的免疫原性,其诱导的抗体有较好的体外中和能力,可作为中国二代亚单位疫苗的候选成分。

Molecular cloning and expression of OspA peptide from a Chinese Borrelia garinii strain PD91 and preliminary study on its immunoprotectivity

Objective To clone and express the 126-274 aa OspA peptide(OspA-pep)of Chinese Borrelia garinii(B.garinii)strain PD91 and to preliminarily study its immune protectivity.Methods The gene encoding the 126-274 aa OspA-pep of B.garinii PD91 was amplified by polymerase chain reaction(PCR)and then cloned into the prokaryotic expression vector pET-30a to construct the recombinant plasmid pET-30a-OspA-pep.Escherichia coli BL21(DE3)competent cells transfected with the recombinant plasmid were induced by IPTG to express the target protein.The recombinant OspA-pep(rOspA-pep)was purified with Ni-IDA resin chromatography and its immunogenicity was analyzed by Western blot.New Zealand white rabbits were immunized with different doses of rOspA-pep(20,30,40,50,60,80 and 100μg).The titers of specific IgG antibodies in rabbit serum samples before and after immunization were detected by indirect immunofluorescence assay(IFA).The optimal immune dose was determined according to the antibody titer after immunization.In vitro neutralization test was performed to detect the immune protection of rOspA-pep using serum samples of the optimal immunization group.The optimal dose of rOspA-pep was used to immunize New Zealand white rabbits to observe the changes in antibody titer.Results The recombinant plasmid pET-30a-OspA-pep was successfully constructed and highly expressed in host bacteria.Western blot showed that rOspA-pep had obvious antigen-antibody reaction with polyclonal antibody against B.garinii PD91 strain.IFA results showed the titers of IgG antibody in serum samples of rabbits immunized with rOspA-pep increased significantly(up to 1∶2480)and 40μg was the optimal dose.The neutralization rates of antibodies induced by 40μg of rOspA-pep were 100%against 106 strain/ml of representative B.garinii PD91 and Borrelia afzelii(B.afzelii)FP1 strains,100%against 107 strain/ml of FP1 strain,and 60%against 107 strain/ml of PD91 strain.After immunization with 40μg rOspA-pep on 1 d and 30 d,the titers of specific IgG antibody in rabbit serum samples reached the peak within two months,and maintained at that level for about 3-4 months before a gradual decline.Conclusions The 126-274 aa OspA peptide fragment of Chinese B.garinii PD91 strain possessed good immunogenicity and induced antibodies with better in vitro neutralizing activity,which suggested that it could be used as a candidate component of the second generation subunit vaccine in China.