禽腺联病毒Rep78、Rep52基因的原核表达及多抗制备

Prokaryotic expression of the Rep78 and Rep52 genes of avian adeno-associated virus and preparation of specific antiserum

为制备禽腺联病毒Rep蛋白多抗血清,根据已发表的禽腺联病毒基因组序列设计2对引物,扩增出Rep78、Rep52基因,分别将其克隆入原核表达载体pET-30a,并转化大肠杆菌BL21(DE3),在IPTG的诱导下,2种蛋白均获得了高效表达,SDS-PAGE显示2种重组蛋白分子量均正确。将重组蛋白切胶免疫ICR小鼠,分别制备了针对2种蛋白的多抗血清,Western blot实验显示制备的抗血清能够与Rep78和Rep52抗原发生特异反应。以上结果说明,Rep78和Rep52蛋白在大肠杆菌中获得成功表达,且制备的多抗血清可用于Rep基因的表达检测。

In order to prepare the antiserum of avian adeno associated virus Rep protein, according to the published genome sequences of AAAV, two pairs of specific primers were designed to amplify Rep78 and Rep52 gene by PCR. The amplified fragments were cloned into prokaryotic expression vector pET 30a, and the recombinant plasmids were transformed into BL21 （DE3） E. coli. Then the proteins were successfully expressed following IPTG induction, and SDS- PAGE confirmed the correct molecular weight of recombinant expression proteins. The antiserum against Rep78 and Rep52 proteins were produced by immunized ICR mouse. Western blotting results showed that the antiserum could react with Rep78and Rep52 proteins specificaily. These results showed that Rep78 and Rep52 proteins were successfully expressed in E. coli, and the antiserum could be used for detection of Rep gene expression.