摘要
本研究参照已发表的PCV2基因组序列,设计合成1对特异性引物,以PCV2基因组DNA为模板,PCR扩增了长约480 bp的ORF2基因片段。将目的片段定向克隆到pGEX-6p-1原核表达载体,酶切及测序鉴定正确后,转化BL21(DE3)表达菌,经IPTG诱导得到了以包涵体形式表达的重组蛋白。采用亲和层析法在变性的条件下纯化重组蛋白,纯化的重组蛋白浓度为0.396 mg/mL。纯化蛋白经免疫印迹、间接ELISA检测证明具有良好的抗原性和特异性。
Designing and synthesizying one pair of primers according to the published PCV2 genome sequence,a fragment of about 480 bp long was amplified by RT-PCR technique with specific primers.Then the target fragment was directionally cloned into pGEX-6p-1 vector.After identifying with enzyme cut and sequencing,the recombinant plasmid was transformed into E.coli BL21（DE3）.The recombinant Cap protein was expressed in inclusion body form after induction with IPTG.After denaturation,renaturation of recombinant protein by affinity chromatography purification,purification of the recombinant protein concentration was 0.396 mg/mL.Western blotting and indirect ELISA testing showed that the purified recombinant protein retained better antigenicity and specificity.
出处
《中国畜牧兽医》
CAS
北大核心
2012年第8期91-94,共4页
China Animal Husbandry & Veterinary Medicine
关键词
猪圆环病毒2型
CAP蛋白
原核表达
活性检测
porcine circovirus type 2
Cap protein
prokaryotic expression
activity detection
