伯氏疏螺旋体外膜蛋白OspC和鞭毛蛋白FlaB多克隆抗体的制备和免疫原性研究  

Preparation and immunogenicity of a polyclonal antibody against outer membrane protein OspC and flagellin FlaB of Borrelia burgdorferi

作　　者：张学潮[1] 朱函坪[2] 姚苹苹[2] 徐海君[3] 徐芳[2] 孙一晟[2] 卢杭景 张云[4] 岳明[5] 杨章女[2,4] ZHANG Xue-chao;ZHU Han-ping;YAO Ping-ping;XU Hai-jun;XU Fang;SUN Yi-sheng;LU Hang-jing;ZHANG Yun;YUE Ming;YANG Zhang-nyu(Hangzhou Center for Disease Control and Prevention,Hangzhou 310021,Zhejiang Province,China;Zhejiang Provincial Center for Disease Control and Prevention;Institute of Insect Sciences,Zhejiang University;Institute of Military Medicine,Eastern Theater of the Chinese People’s Liberation Army;The First Affiliated Hospital of Nanjing Medical University)

机构地区：[1]杭州市疾病预防控制中心免疫预防所,浙江杭州310021 [2]浙江省疾病预防控制中心,浙江省传染病疫苗与预防控制研究重点实验室,浙江杭州310051 [3]浙江大学昆虫科学研究所,浙江杭州310058 [4]中国人民解放军东部战区军事医学研究所,江苏南京210002 [5]南京医科大学第一附属医院,江苏南京210029

出　　处：《中国媒介生物学及控制杂志》2020年第5期531-535,共5页Chinese Journal of Vector Biology and Control

基　　金：浙江省公益研究计划(LGF20H260003);国家重点研发计划(2017YFC1601503);国家自然科学基金(81171609)。

摘　　要：目的对伯氏疏螺旋体(又称莱姆病螺旋体,Borrelia burgdorferi)外膜蛋白OspC和鞭毛蛋白FlaB原核表达后,制备多克隆抗体并进一步进行免疫原性检测。方法以伯氏疏螺旋体BgNMJW1基因组DNA为模板,PCR扩增出外膜蛋白OspC和鞭毛蛋白FlaB的基因片段,克隆入表达载体pGEX-6p-1,转入表达菌株Rosetta,经异丙基硫代半乳糖苷(IPTG)诱导产生融合蛋白,并经谷胱甘肽转移酶柱蛋白纯化或割胶纯化。纯化后的蛋白用于免疫新西兰大白兔得到多克隆抗血清。结果经聚丙烯酰氨凝胶电泳(SDS-PAGE)鉴定,经过诱导的携带目的基因载体,与对照相比,有明显的目的蛋白表达,大小分别约为49×103和63×103,诱导结果表明,在细菌培养至吸光度(A)值为0.4时,加入1 mmol/L的IPTG,在25℃下,诱导10 h,此时产生的蛋白表达量较大。将融合表达产生的蛋白纯化后免疫新西兰大白兔得到多克隆抗血清,应用抗血清对B.garinii和B.burgdorferi 2种基因型的伯氏疏螺旋体代表菌株(BgNMJW1和BbB31A3)的OspC和FlaB进行蛋白免疫印迹检测,可得到清晰检测条带。结论OspC和FlaB的联合应用可对莱姆病的诊断起到有效作用。Objective To prepare a polyclonal antibody against the outer membrane protein OspC and the flagellin FlaB of Borrelia burgdorferi(also called Lyme disease spirochete) after prokaryotic expression, and to test its immunogenicity.Methods The genomic DNA of B. garinii strain BgNMJW1 was used as a template to amplify the gene segments of OspC and FlaB using polymerase chain reaction(PCR);then the PCR products were subcloned into the expression vector pGEX-6p-1 and transformed into the expression strain Rosetta. The fusion proteins were expressed after induction with isopropyl thiogalactoside(IPTG) and purified using glutathione transferase(GST) column or via gel cutting. The purified proteins were then used to immunize New Zealand white rabbits to obtain polyclonal antiserum. Results The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) showed that compared with the control, the vector carrying the target genes had obvious expression of recombinant proteins after induction, and the sizes of the recombinant proteins were about 49×10~3 and 63×10~3, respectively. The induction result showed that the expression of the induced proteins reached its peak level if 1 mmol/L IPTG was added when the bacteria were cultured to an absorbance(A) of 0.4, followed by inducing at 25 ℃ for 10 hours. By immunizing New Zealand white rabbits with the purified fusion proteins, the polyclonal antiserum was obtained and used to detect OspC and Flab of B. garinii strain BgNMJW1 and B. burgdorferi strain BbB31A3 in Western blot, then the clear detection bands were obtained. Conclusion The combined application of OspC and FlaB can play an effective role in the diagnosis of Lyme disease.

关 键 词：伯氏疏螺旋体 原核表达 外膜蛋白 鞭毛蛋白 多克隆抗体 

分 类 号：R377[医药卫生—病原生物学]