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.
NENG Chang
,
LI Xiujin
,
ZHONG Fei
,
LI Zhen
,
ZHANG Feng
,
CHEN Huihui
,
ZHANG Kao
,
HAN Dongmei
. Highly Expression of Ovalbumin in HEK293T Cells Mediated by Human CD14 Signal Peptide[J]. Chinese Journal of Animal Nutrition, 2012
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: 2165
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DOI: 10.3969/j.issn.1006-267x.2012.11.014
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