猪痘病毒P35基因的原核表达及间接ELISA抗体检测方法的建立  

Establishment of an indirect ELISA for detection of antibodies against swinepox virus with the recombinant P35 as the coating antigen

作　　者：邓舜洲[1] 蒋新华[1] 冷闯[1] 朱芝秀[1] 何后军[1] 张文波[1] 

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045

出　　处：《中国兽医学报》2013年第10期1488-1492,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31160498)

摘　　要：为建立猪痘病毒(SWPV)血清学检测方法,根据SWPV的P35基因设计1对引物,从SWPV江西分离株(JX01)的细胞培养物中扩增到975bp的P35基因。将P35基因克隆到原核表达载体pGEX-4T-1,将其进行原核表达,经SDS-PAGE分析,表达P35重组蛋白约64 000。采用MagneGST蛋白纯化试剂盒纯化P35重组蛋白,以纯化的重组蛋白为检测抗原,建立了SWPV抗体间接ELISA检测方法,间接ELISA条件为:P35重组蛋白包被质量浓度为10mg/L,被检猪血清的稀释倍数为1∶80。以建立的间接ELISA方法对江西省47个猪场的471份猪血清进行了SWPV抗体的检测,结果猪血清抗体阳性率为35.7%,表明江西省部分猪场存在较普遍的SWPV感染。In order to develop ELISA method for detection of antibodies against swinepox virus （SWPV） ,a pair of specific primers was designed and synthesized according to the published P35 gene sequence of SWPV in GenBank （AF410153） ,and the P35 gene （975 bp） of SWPV was ampli- fied by polymerase chain reaction （PCR）. The amplified P35 gene was cloned into pGEX-4T-1 pro karyotic expression vector,and transformed into Escherichia coli BL21 competent cell. The recom bination was induced with 1 mmol/L IPTG. SDS-PAGE analysis revealed that a 64 000 protein was expressed as expected. The P35 recombinant protein was expressed in inclusion bodies and the re combinant protein was purified by MagneGST protein purification kit. To detect the antibody a gainst SWPV,an indirect ELISA method was established,according to the following protocol：the optimal concentration of coated antigen with P35 recombinant protein was 10 mg/L and optimal dilution of pig＇s sera was 1 ： 80. A total of 471 pig serum samples collected from 47 pig farms in Jiangxi province were detected by the established indirect ELISA,35.7% sera samples were SW- PV antibodies positive. The results indicated that SWPV infection was prevalent in pigs of Jiangxi province.

关 键 词：猪痘病毒 P35基因 原核表达 间接ELISA 

分 类 号：S852.65[农业科学—基础兽医学]