摘要
利用本室构建的质粒PGEX-ORF 2转化BL 21(DE3),经IPTG诱导表达,得到PCV 2-ORF 2的重组蛋白,经复性纯化后作为免疫原,采取常规免疫与体外脾脏免疫结合的方式免疫BALB/c小鼠,取其脾细胞与Sp2/0的骨髓瘤细胞融合,经间接酶联免疫吸附试验(EL ISA)筛选,获得了2株抗PCV 2-ORF 2蛋白的单克隆抗体的杂交瘤细胞,分别命名为3B 2F 4和9C 3D 2,其细胞培养上清效价分别为1∶1 024、1∶512,小鼠腹水效价分别为1∶204 800、1∶102 400。EL ISA结果显示,3B 2F 4和9C 3D 2仅与融合表达的PCV 2-ORF 2蛋白反应,而与PGEX-KG表达的蛋白、猪细小病毒(PPV)、猪繁殖与呼吸综合征病毒(PRRSV)不反应,说明此2株单抗特异性好。间接免疫荧光结果显示,3B 2F 4和9C 3D 2能与PCV 2发生反应,而不与PCV 1发生交叉反应,表明所获得的2株单抗是PCV 2型特异性的。这2株单抗的获得为进一步研究PCV 2-ORF 2基因的功能,及建立准确快速的鉴别PCV 1和PCV 2的诊断方法提供了强有力的手段。
The PCV2-ORF2 fragment was expressed as glutathione-s-tansferase fusion protein in Escherichia coli by lsoprophhhio-β-D-galactoside（IPTG） inducing after the recombinant plasmid PGEX-ORF2 was transformed into BL21. Two hybridoma cell strains against PCV2-ORF2 fusion protein named 3B2F4 and 9C3D2 respectively,were developed by indirect enzyme-linked immunosorbent assay method after fusion between Sp2/0 myeloma cells and spleen cells BALB/e mice immunized with redentatured and purified fusion protein. The ELISA titer of culture supernatant were l ： 1 024 and 1 ： 512,and ascites were 1 ： 204 800 and 1 ： 102 400. The results of ELISA indicated that the two monoclonal antibodies were only against PCV-ORF2 fusion protein but not reacted with PGEX-KG protein,PPV and PRRSV indicative of its high specificity. And the results of IFA indicated that the two monoclonal antibodies only can combine with PCV2 but not with PCV1 which proved them to be specific to PCV2. This research provided a powerful tools for the research of PCV2-ORF2 gene and established the method for discriminating and diagnosing the two sero-type of PCV.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2005年第6期567-569,共3页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(30170701)
