Construction of SARS-CoV envelope DNA vaccines using codon-optimization strategy
Date Issued
2005
Date
2005
Author(s)
Lin, Chia-Jung
DOI
zh-TW
Abstract
Severe acute respiratory syndrome (SARS) is a newly emerged disease with high infectivity and morbidity. SARS global pandemic occurred in 2002 and 2003 caused a serious threat to public health and the economy. The SARS coronavirus (SARS-CoV) has been identified as the etiological agent for SARS. Thus, vaccination against SARS-CoV may represent an effective approach to control SARS pandemics.
DNA vaccination regimen reveals attractive approaches for SARS vaccine development, as they can elicits both humoral and cellular immune responses and offer many advantages over conventional vaccines, including stability, simplicity, and safety. However, efficiency of DNA vaccine is restricted by the limited performance of genes directly cloned from the pathogenic organisms may not be able to translated efficiently in a heterologous host expression system as a consequence of codon usage bias. Therefore, genes subjected to optimization by changing into humanized codons represent a novel strategy to increase the efficacy of DNA vaccination.
SARS-CoV envelope（E）protein is a structural protein and is required for the initiation of virus assembly and budding in SARS-CoV replication cycle. To substitute codons that are preferentially used in the Homo sapienes system, we synthetically assembled humanized SARS-CoV E genes （hSE）, constructing pcDNA3/hSE plasmid and compared them with the wild-type counterparts, for their efficiency as DNA vaccine in BALB/c mice. The complete E gene mRNA and E protein expression can only be detected in pcDNA3/hSE transfected 293A cells. The E mRNA showed extended trimming-off and E protein was not detected in pcDNA3/SE transfected-293A cells. We also found SARS-CoV E mRNA underwent similar trimming in SARS-CoV infected Vero E6 cells（18 hours post infection）. These finding hinted the native SARS-CoV E gene might undergo a modulating process similar to RNA splicing in host cells.
We set up DNA vaccination protocol by immunizing 7-week-old female BABL/c with pcDNA3/SE, pcDNA3/hSE, and pcDNA3 intra-muscularly and boost after 2 weeks. Mouse sera were collected before the first and the second shot, two and four weeks after the second shot. The sera were analyzed by western blotting on IPTG stimulated pRSET-A/SE bacterial lysate supernatant for E-specific antibody. We could not detect SARS-CoV E-specific antibodies in the test sera. Further protein purification and settling up enzyme-linked immunosorbent assay（ELISA）might be necessary for detecting the effect of DNA vaccine.
Subjects
嚴重急性呼吸道症候群
套膜基因核酸疫苗
轉譯密碼擬人優化
Severe acute respiratory syndrome coronavirus (SARS-CoV)
DNA vaccines
envelope（E） gene
codon optimization
SDGs
Type
other
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