禽腺联病毒Rep78和VP3蛋白的原核表达及抗血清制备

Prokaryotic expression of the Rep78 and VP3 proteins of avian adeno-associated virus and preparation of specific antisera

分别将禽腺联病毒(Avian adeno-associated virus,AAAV)的Rep78基因和VP3基因克隆入pET-47b原核表达载体并转化BL21(DE3)大肠杆菌,在IPTG的诱导下2种目的蛋白均成功得到了表达。SDS-PAGE显示,Rep78蛋白的相对分子质量约为85 000,而VP3蛋白相对分子质量约为60 000。Western-blot分析显示,表达产物均能与抗AAAV的阳性血清反应。将目的蛋白切胶免疫BALB/c小鼠分别制备了针对2种蛋白的多克隆血清。间接免疫荧光试验显示制备的抗血清能够与AAAV抗原特异反应。不与鸡胚致死孤儿病毒(CELO)抗原反应。结果表明,制备的抗Rep78和VP3蛋白的血清可以用于检测重组AAAV载体制备过程中Rep和Cap基因的表达水平。

Special antisera against Rep78 and VP3 proteins of avian adeno-associated virus（AAAV） were prepared to establish the serological methods for AAAV antigen detection. The Rep78 and VP3 gene were separately cloned into the pET-47b prokaryotic expression vectors and the recombinant plasmids were transformed into BL21 （DE3） E. coll. Thereafter,the proteins were successfully expressed following IPTG induction. The approximate molecular weight of Rep78 and VP3 protein analyzed by SDS-PAGE was 85 000 and 60 000, respectively. Western blotting assay revealed that the desired proteins could be recognized by the positive serum against AAAV. The antisera against Rep78 and VP3 proteins were produced by immunized BALB/c mouse. The prepared antisera reacted specifically with AAAV antigen, but not with CELO virus antigen in indirect immunofluorescence assay （IFA）. The result indicates that the antisera can be used for detection of Rep and Cap gene expression level in course of recombinant AAAV production.