犬TfR胞外区密码子的优化、真核表达及与犬细小病毒VP2蛋白结合

为制备大量具有天然活性的犬胞外区可溶性转铁蛋白受体(sTfR),本试验通过密码子优化提高sTfR在真核细胞中的表达水平.利用RT-PCR方法从犬肝脏中扩增sTfR编码基因,依据该基因编码的氨基酸序列,参照人偏爱的密码子,对该基因进行密码子优化并由公司合成.利用peDNA3.1-CD5质粒分别构建野生型和密码子优化的sTfR基因真核表达载体,经磷酸钙介导使其在HEK293T细胞中进行表达,利用Western-blotting鉴定表达产物,通过ELISA检测重组犬sTfR蛋白与犬细小病毒VP2蛋白的结合活性.结果显示本试验扩增的犬sTfR基因与GenBank该基因序列的同源性为100%;通过在HEK 293T细胞中进行瞬时表达,结果显示密码子优化可以明显提高sTfR基因在HEK 293T细胞中的表达水平,提高了75%.同时表达的sTfR蛋白能够与犬细小病毒VP2蛋白进行特异结合,表明表达的重组sTfR蛋白具有天然活性.

Study on codon optimization and expression of canine sTfR gene in eukaryotic cells and interaction with canine parvovirus VP2 protein

In order to prepare large-scale of the natural active soluble canine transferrin receptor(sTfR),the sTfR gene codons were optimized to increase sTfR expression level in eukaryotic cells.The gene encoding sTfR was amplified from canine liver tissues by RT-PCR.According to amino acid sequence,sTfR codons were optimized based on human main codons and synthesized.The eukaryotic expression vectors of wild-type and codon-optimized sTfR genes were eonstrueed by using pcDNA3.1-CD5 vector.The vectors were transfected into human embryonic kidney cells HEK293T cells mediated by calcium phosphate.The expressed sTfR proteins were identified by Western-blot.Binding activity of sTfR to canine parvovirus VP2 protein was analyzed by ELISA.The results showed that the amplified sTfR gene sequence was 100% similarity to the published in GenBank;The expression level of codon-optimized sTfR gene showed an increase of 75% compared with wild-type in 293T cells;sTfR protein was proved to have the specific binding activity for canine parvovirus VP2 protein.