猪繁殖与呼吸综合征病毒山东分离株N蛋白基因的原核表达及其单克隆抗体的制备与鉴定  

Prokaryotic Expression of Gene of PRRSV SD-TA Strain N Protein and Preparation and Characterization of Monoclonal Antibodies

作　　者：曹丙蕾[1] 张群[1] 凌宗帅[1] 邱洪凯 杨超然 

机构地区：[1]济南出入境检验检疫局,山东济南250014 [2]山东正邦养殖有限公司,山东济南250010 [3]山东省实验中学,山东济南250010

出　　处：《西南农业学报》2014年第4期1772-1776,共5页Southwest China Journal of Agricultural Sciences

基　　金：山东出入境检验检疫局科研项目(SK201401)

摘　　要：采集某发病猪场病料进行疑似PRRSV分离鉴定,经克隆测序获得一株美洲型的高致病性PRRSV SD-TA株分离株,并对其N蛋白(核衣壳蛋白)基因设计引物进行克隆转化,构建重组质粒pET-30a-ORF7,鉴定后经诱导表达纯化获得大小约为21KD(含6×His标签蛋白)的重组N蛋白。将纯化蛋白免疫BALB/c小鼠,经间接ELISA检测后取脾细胞与SP2/0骨髓瘤细胞进行细胞融合,并对融合细胞上清进行抗体检测,通过有限稀释法,获得了2株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为6D10和3H5,经鉴定后两者均为IgG1型,且识别于不同的抗原位点,经Western-blot和IFA检测,单抗6D10和3H5均能与N蛋白发生特异性反应,与无关蛋白无交叉反应,且均能与PRRSV经典株(VR株)和高致病性变异株(SD-TA株)感染的Marc-145细胞产生特异性反应。The swine samples of dead or sick pigs obtained from some breeds were collected, and the vires was isolated and identified for PRRSV. A highly pathogenic PRRSV SD-TA strain of American type PRRSV was obtained by cloning and sequencing. N （Nuclcocapsid） protein-encoding gene of PRRSV SD-TA strain was cloned by the primers designed and transformed to construct the recombinant plasmid pET-30a-ORF7. The recombinant plasmid pET-30a-ORF7 was identified and then induced to express and purify the recombinant proteins. The size of the recombinant proteins was 21KD, including 6 x His tag proteins. The BALB/c mice were inoculated by the purified proteins and the level of the antibodies was detected by indirect ELISA. The splenocytes were fused with SP2/0 myeloma cells and the supernatents of the fusion cells were detected by indirect ELISA. The positive hybridoma cells were sub-cloned twice by limited dilution methods to obtain the two （ Mabs 6D10 and 3 I＂I5 secreting anti-N protein antibodies stably （ IgG1 isotype） and both bound to the distinct epitopes. Western- blot and IFA showed that both Mabs 6D10 and 3I-L5 could react with the recombinant N proteins specifically but could not react with the unrelated proteins. Moreover, the two Mabs could react with the Marc-145 cells challenged by the classical PRRSV VR-2332 strain and HPPRRSV SD-TA strain specifically.

关 键 词：PRRSV 单克隆抗体 抗原位点 WESTERN-BLOT IFA 

分 类 号：S855.3[农业科学—临床兽医学]