堆型艾美耳球虫乳酸脱氢酶基因的克隆与表达

Cloning and expressing of lactate dehydrogenase gene of Eimeria acervulina

克隆了堆型艾美耳球虫(Eimeria acervulina)乳酸脱氢酶(LDH)基因(EaLDH),并在大肠杆菌中成功表达出重组蛋白。根据GenBank中收录的EaLDH核苷酸序列设计特异性引物,以其第一代裂殖体总RNA为模板,采用RT-PCR方法扩增获得EaLDH基因。将EaLDH基因片段与原核表达载体pET28a(+)连接,构建重组表达质粒pET28a(+)-LDH,并在大肠杆菌BL21(DE3)中诱导表达。结果表明,扩增出的EaLDH基因含993bp的开放阅读框,与GenBank中收录的EaLDH基因序列相似性为98%。诱导后,重组蛋白能够在大肠杆菌中高效表达,融合蛋白相对分子质量约为4.1×104。

The lactate dehydrogenase （LDH） gene of Eimeria acervulina （EaLDH） was cloned and successfully expressed in Escherichia coli.According to the neucleotide sequence of EaLDH gene available in GenBank,the specific primers were designed and the EaLDH gene was amplified by RT-PCR using total RNA extracted from the first generation schizonts of E.acervulina as templete.The cloned EaLDH gene was then ligated with prokaryotic expression vector pET28a（＋） and transformed into BL21 （DE3） for expression.The results showed that the open reading frame （ORF） of EaLDH gene was 993 bp in length,which shared nucleotide sequence similarity of 98% with the data in GenBank.The recombinant protein was successfully expressed in E.coli with the relative molecular weight of 4.1 × 104.