羊痘病毒P32蛋白编码基因的克隆及表达

为获得山羊痘病毒膜蛋白重组P32抗原，根据其基因序列设计合成了1对特异性引物，对CaPVP32基因进行了PCR扩增，产物大小约为969bp；产物回收纯化后，将其克隆至pBAD／Thio—TOPO载体中，转化TOP10大肠埃希氏菌感受态细胞，与已报道的多株CaPVP32基因序列的同源性高达99％；相应地，氨基酸序列同源性为98％。经测序筛选出阳性克隆，该重组菌株经L—arabinose诱导，可稳定、高效地表达CaPVP32抗原。表达蛋白为融合蛋白，分子质量约51．53ku．

Cloning and expression of capripoxvirus P32 protein gene

The P32 gene was amplified by PCR with a pair of primers designed according to the reported capripoxvirus P32 gene sequence. The amplified product of 969 bp in size was inserted into the pBAD/ Thio-TOPO vector. The recombinant plasmid was identified by PCR amplification and sequencing to confirm the correct sequences and the correct junctional orientations of the inserted P32 gene. The gene was expressed in the Escherichia coli TOP10 as a fusion protein with a N-terminal HP-thioredoxin and a C-terminal polyhistidine tag after induction by L-arabinose. SDS-PAGE and Western blotting analysis showed that the recombinant P32 protein had been expressed stably and reliably at a high level. The expressed protein was approximately 51.53 ku in molecular weight.