本试验将人CD14信号肽序列与卵清蛋白(ovalbumin,OVA)基因融合,旨在提高OVA在非输卵管上皮细胞中的表达。提取鸡输卵管上皮细胞总RNA,经反转录合成的cDNA,再利用修饰的特异性引物,通过PCR从cDNA中分别扩增出5'端与人CD14信号肽序列融合和非融合的OVA基因,并将它们分别插入到真核表达载体pcDNA3.1A中,构建成表达载体pcDNA-CD14sp-OVA和pcDNA-OVA。经磷酸钙介导,将质粒转染至HEK293T细胞,使其进行表达,用鼠抗OVA单克隆抗体通过免疫印迹(Western blot)法检测OVA表达水平。结果表明:本试验扩增的OVA基因序列与GenBank中的序列(登录号NM_205152)一致,构建的人CD14信号肽序列融合OVA基因的表达载体结构正确。经过在HEK293T细胞的表达,融合人CD14信号肽序列的重组蛋白OVA的表达水平明显得到提高。结果提示,融合人CD14信号肽序列后的重组蛋白OVA在非输卵管上皮细胞中的表达水平明显提高,为OVA基因作为DNA疫苗模式抗原的应用提供了必要的条件。
In order to increase the expression of ovalbumin (OVA) in non-oviduct epithelial cells, the OVA gene was fused with the sequence of human CD14 signal peptide in this study. Total RNA was extracted from chicken oviduct epithelial cells, and the cDNA was synthesized by reverse transcription. The OVA genes fused with or without human CD14 signal peptitde sequence at 5' terminal were amplified by PCR using modified specific primers, and both of them were inserted into eukaryotic expression vector pcDNA3.1A to construct their expression vectors (pcDNA-CD14sp-OVA and pcDNA-OVA). Then, pcDNA-CD14sp-OVA and pcDNA-OVA plasmids were transfected into HEK293T cells using calcium phosphate. The expressed recombinant OVA was detected by Western blot using mouse anti-OVA monoclonal antibody. The results showed that OVA gene sequence amplified in the study was in consistent with that in GenBank (accession number: NM_205152), and the expression vector of OVA gene fused with human CD14 signal peptide sequence was correct. The expression level of CD14 signal peptide sequence-fused OVA gene was significantly increased. The results indicate that after fused with human CD14 signal peptide sequence can effectively increase the expression of recombinant OVA in non-oviduct epithelial cells. This study provides necessary conditions for further investigation of OVA gene as a classical antigen in DNA vaccination.
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