PCV2ORF2基因在大肠杆菌中的表达及ELISA检测方法的建立

Expression of ORF2 gene of porcine circovirus type 2 in Escherichia coli and establishment of an ELISA

采用PCR扩增了猪圆环病毒Ⅱ型广东分离株的ORF2 3′端基因,将其连接到pET32a(+)质粒上并转化DH5α细胞,重组质粒经PCR和酶切鉴定并测序后,转化BL21菌,用1 mmol/L IPTG诱导表达重组菌。经SDS-PAGE和Western-blot分析,表明ORF2基因已在大肠杆菌中正确表达,表达的融合蛋白的分子质量约为33 ku;表达产物经超声波处理后,用Ni-NTA柱纯化,复性,用此纯化产物包被酶标板,建立了ELISA诊断方法,经对临床516份血清样品进行检测,结果表明仔猪的血清阳性率为35.2%,育肥猪的血清阳性率为60.6%,种猪的血清阳性率为69.8%。

Based on the gene sequence of ORF2 of porcine circovirus type 2（PCV2）,a pair of primers were designed, and the carboxyl terminal fragment of 393 bp in size was amplified by PCR from PCV2 Guangdong isolate. The cap gene fragment was inserted into the expression plasmid pET32a（ ＋）, yielding the recombinant plasmid pET32a-ORF2. The recombinant plasmid pET32a-ORF2 was transformed into Escherichia coli BI.21 and induced with 1 mmol/L IPTG. SDS-PAGE analysis showed that the fusion protein pET32a-ORF2 was approximately 33 ku in molecular mass. The fusion protein was treated with purified resin 50 % Ni-NTA under denaturing conditions, resulting in obtaining the fusion protein with relati- vely high purity. With the purified fusion protein pET32a-ORF2 as coating antigen,an ELISA for detection of PCV2 was established. 516 serum samples were examined with this ELISA. The positive incidence in piglets,hogs and sows was 35.2% ,60.6% and 69.8% ,respectively.