边缘无浆体MSP5蛋白基因的克隆及原核表达

Cloning and prokaryotic expression of MSP5 protein-encoding gene of Anaplasma marginale

参照已发表的边缘无浆体主要表面蛋白5(MSP5)基因的核苷酸序列,设计了1对特异性引物,以边缘无浆体基因组DNA为模板,采用PCR技术扩增获得了MSP5蛋白基因。将其克隆到pGEM-T Easy载体,并进行测序分析。结果表明,克隆的MSP5基因与GenBank上登录的Florida株MSP5蛋白基因的序列同源性达98.6%,编码氨基酸的同源性为99.0%,并且该序列包含有完整的开放阅读框,大小为633 bp。将该基因亚克隆入原核表达载体pGEX-4T-1,构建了重组原核表达载体。将其转化到DH5α宿主菌中,用IPTG进行诱导表达,实现了融合表达,表达产物的分子质量为45 ku。Western-blot分析表明,此表达产物能够被抗边缘无浆体阳性血清所识别。

A pair of specific primers were designed according to the nucleotide sequence of the published MSP5 gene of Anaplasma marginale. The MSP5 gene was amplified by PCR based on the genomic DNA template of A. marginale,and the amplicon was cloned into pGEM-T Easy vector and identified by sequencing. In consequence,the identity at a nucleotide level was 98.6% with MSP5 gene from A. marginale Florida strain and the amino acid similarity was 99.0% correspondingly. There was an entire ORF 633 bp in size in the sequence of the cloned MSP5 gene. The MSP5 gene was subcloned into pGEX-4T-1 vector to construct a prokaryotic expression vector pGEX-4T-1/MSP5 and transformed into DHSα. The recombinant plasmid pGEX-4T-1/MSP5 was induced in Escherichia coli using IPTG. Western-blotting showed that the expressed recombinant fusion protein 45ku in size was able to be recognized by the rabbit anti-A. marginale positive serum.