摘要
表达P36基因的重组菌BL21(6P-1-P36)经诱导表达,超声波裂解后高速离心,分别收集上清和沉淀。可溶性表达产物亲和层析纯化后用于单抗筛选;同时,用GST-P36包涵体抗原腹腔注射免疫BALB/c小鼠(400μg/只)。利用淋巴细胞杂交瘤技术,获得了1株能分泌特异性抗体的杂交瘤细胞株5A7-2。生物学特性鉴定表明,腹水单抗的间接ELISA效价为1∶2.5×106;免疫球蛋白亚类为IgG2a。免疫印迹结果显示,腹水单抗能与GST-P36融合蛋白反应而不与谷胱苷肽转移酶(GST)反应,并且在猪肺炎支原体(Mycop lasma hyopneumoniae,Mhp)蛋白36000位置也有明显的条带,说明5A7-2所分泌的是针对MhpP36蛋白的单抗。P36蛋白是Mhp种特异性蛋白,其单克隆抗体的制备,为Mhp种特异性检测方法的建立奠定了基础。
The recombinant bacteria BL21 （6P-1-P36）, harboring expression plasmid of P36 gene fused with GST gene, were induced with IPTG and disrupted by sonication. After super-centrifugation, the soluble fusion protein GST-P36 was purified by affinity chromatography on Glutathione Sepharose 4B beads. The 8-week-old female BALB/c mice were injected intraperitoneally with 400 μg inclusion bodies containing GST-P36 fusion protein. By using the hybridoma technique,one hybridoma cell line, 5A7-2, secreting specific monoclonal antibody （MAb） was established. The subtype of 5A7-2 was IgG2a. and the titer of its ascitic fluid was about 1 ： 2.5×10^6 in indirect ELISA. The immunoblotting result indicated that MAb 5A7- 2 could react with 36 000 protein of Mycoplasma kyopneunmniae（Mhp） and GST-P36 fusion protein, but could＇t bind with GST protein. The results suggested that MAb 5A7-2 was specific to the P36 protein of Mhp. The P36 protein of Mhp carries the species-specific antigenic determinants and don＇t share common epitopes with the other mycoplasmas and bacteria. It suggested that MAb 5A7-2 would be very useful for the detection of Mhp infection specifically.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2006年第6期619-621,共3页
Chinese Journal of Veterinary Science
基金
教育部"高校青年教师奖"资助(175)
江苏省"六大人才高峰"计划(G-2002-026)
江苏省"三药"项目资助
