Human CD14Signal Peptide Sequence Mediates Highly Secretory Expression Of Ovalbumin Gene In HEK293T Cells

Ovalbumin is a major nutritional component of eggs, which is a common classicalantigen in immunological study. Although the OVA gene can express much less innon-oviduct epithelial cells than in oviduct epithelial cells, it would impact its applicationas antigen of DNA vaccine. Apart from this, the OVA gene sequence was analyzed bySignalP3.0server, we got that OVA gene sequence did not comprise signal sequence.Whether it is the reason that OVA gene cannot express in non-oviduct epithelial cells, westill do not get it.In order to improve its secretary expression in non-oviduct epithelial cells, the humanCD14signal sequence was fused to OVA gene. The total RNA was extracted from chickenoviduct epithelial cells by Trizol Reagent, the first chain of cDNAs was synthesized byReverse Transcription. The OVA gene was amplified by PCR using primers OVA-F andOVA-R-s with the template of cDNAs; Meanwhile the CD14sp-OVA gene that is the OVAgene fused with CD14signal sequence was amplified by twice PCR using modifiedspecific upper primers CD14sp-OVA-F1, CD14sp-OVA-F2and down primer OVA-R-s.Both of OVA gene structures were inserted into eukaryotic expression vector pcDNA3.1Ato generate their reorganization eukaryotic expression vectors, pcDNA-CD14sp-OVA andnegative control pcDNA-OVA. Then, the pcDNA-CD14sp-OVA and pcDNA-OVAplasmids were transfected into HEK293T cells using calcium phosphate. After theplasmids was expressed in the expression medium about48hours, the protein wasconcentrated by TCA and separated by SDS-PAGE method, then protein was transferredto nitrocellulose filter. At last, the expressed recombinant OVA was detected by Westernblot using mouse anti-OVA monoclonal antibody.The results show that OVA gene sequences and OVA gene fused with CD14signalsequence amplified in this paper were in consistent with the OVA gene sequence inGenBank. Western blot analysis show that the expression level of CD14signalsequence-fused OVA gene was significantly higher than that of the non-fused OVA gene,which indicates that human CD14signal sequence can effectively promote OVA gene expression in non-oviduct epithelial cells. This research provides necessary conditions forfurther investigation of OVA gene as a classical antigen in DNA vaccination.