鸭圆环病毒FJ0601株Rep蛋白的原核表达及其抗血清制备

通过PCR方法扩增鸭圆环病毒(Duck circorirus,DuCV)FJ0601株Rep基因的完整ORF,按正确的阅读框架将其克隆入原核表达载体pGEX-6P-1中,重组质粒转化BL21宿主菌后,经IPTG诱导表达。诱导菌体裂解物经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)后,将表达产物从凝胶中回收,免疫4周龄小鼠,3次免疫后,采血分离血清。所得抗GST-Rep多克隆抗血清分别与经PCR检验为阳性的病鸭脾脏、胸腺、法氏囊组织的冰冻切片进行间接免疫荧光试验(IFA),结果在感染DuCV的发病鸭3种免疫器官中均可检测到由阳性细胞组成的局部病灶,表明大肠杆菌表达的Rep融合蛋白至少保留了部分天然Rep蛋白的抗原性。

Prokaryotic expression and antiserum preparation of replication protein of duck circoviru F J0601 strain

Rep gene was amplified from genomic DNA of Duck circovirus (DuCV) FJ0601 strain by polymerase chain reaction(PCR)and then cloned into pGEX-6P-1 according to the right open reading frame(ORF). The recombinant plasmid was transformed into E. coli BL21 strain for expression with the induction of IPTG. After SDS-PAGE, the expressed specific band was harvested and injected into four-week-old mice 3 times,then the antiserum was collected for IFA with the positive froze histological section of spleen,thymus and bursa tested by PCR. The focuses of infec-tion composed of positive staining cells were found in the three immune organs infected by DuCV,showing that the expressed fusion protein of Rep in vitro has some epitopes of natural Rep.