动物营养学报2012，Vol.24Issue(11)：2165-2171,7.DOI:10.3969/j.issn.1006-267x.2012.11.014
人CD14信号肽介导卵清蛋白在HEK293T细胞的高效表达
Highly Expression of Ovalbumin in HEK293T Cells Mediated by Human CD14 Signal Peptide
摘要
Abstract
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. IA to construct their expression vectors (pcDNA-CD14.sp-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. [ Chinese Journal of Animal Nutrition, 2012, 24 (11) : 2165-2171关键词
卵清蛋白/人CD14信号肽/重组蛋白表达/HEK293T细胞Key words
ovalbumin/ human CD14 signal peptide/ expression of recombinant/ HEK293T cells分类
农业科技引用本文复制引用
能昌爱,李秀锦,仲飞,李振,张峰,陈慧慧,张考,韩冬梅..人CD14信号肽介导卵清蛋白在HEK293T细胞的高效表达[J].动物营养学报,2012,24(11):2165-2171,7.基金项目
国家自然科学基金(30771586) （30771586）
河北省自然科学基金(C2008000244) （C2008000244）
