建構嚴重急性呼吸道症候群冠狀病毒套膜基因轉譯密碼最佳化之核酸疫苗

嚴重急性呼吸道症候群（Severe Acute Respiratory Syndrome, SARS）是一種具高度傳染性和致死率的新興急性呼吸道症候群，2002年至2003年間對全球的公共衛生和經濟上產生嚴重的威脅。SARS的致病原被認為是一種新的冠狀病毒－SARS coronavirus (SARS-CoV)，而發展SARS-CoV 疫苗被認為是一種有效控制SARS疾病的方法。在SARS疫苗發展，質體核酸（DNA）疫苗被認為是SARS疫苗發展一種具潛力的方法，除了DNA疫苗能引起產生保護性中和抗體和病毒特異性毒殺T細胞的免疫效果，亦具有疫苗本質穩定、製作簡單、施打安全等優點。但是DNA 疫苗的效率還是會因致病原基因在異種生物系統內因轉譯密碼使用表現不好而無法高效率轉譯出蛋白質而。轉譯密碼擬人優化（Codon optimization，humanized codon）或許為增加DNA疫苗效力的一個策略。套膜蛋白質質是SARS-CoV結構蛋白質的一種，也是引起SARS 病毒顆粒組裝和釋出過程中必要之蛋白質。本論文將SARS-CoV 套膜蛋白質基因擬人優化後，和原始的SARS-CoV 套膜蛋白質基因分別做成DNA疫苗（pcDNA3/SE , pcDNA/hSE）免疫BALB/c小鼠，比較兩者做為核酸疫苗之效能。結果發現在經質體轉染293A細胞中，擬人優化套膜蛋白基因的完整mRNA表現量多於原始的套膜蛋白基因的mRNA表現量，且能產生完整套模蛋白質。未經修飾的SARS套膜蛋白質基因mRNA半數以上不完整，幾乎整段被剪除，不能產生完整之套模蛋白質。在TW1 SARS-CoV感染的Vero E6細胞中同時發現病毒感染18小時後細胞內的SARS套膜蛋白基因mRNA絕大部分亦是類似發生於質體轉染293A細胞中的剪除現象，顯示SARS-CoV套膜基因在宿主細胞內表現時，似乎有RNA切割修飾之現象。本研究將pcDNA3/SE、pcDNA3/hSE及對照組pcDNA3等質體DNA分別以肌肉注射施打於7週大雌性BABL/c小鼠，並於兩週後追加第二劑DNA疫苗，並收取施打疫苗前、後之血清。以IPTG刺激pRSET-A/SE轉型大腸桿菌後溶出的上清液所表現之SARS-CoV套膜蛋白質為抗原，進行西方墨點法分析，未能測出SARS-CoV套膜蛋白質特異性抗體的反應。將進一步自IPTG刺激pRSET-A/SE轉型大腸桿菌純化出SARS-CoV套膜蛋白質，以酵素免疫分析法進一步分析測試血清。

關鍵字

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

並列摘要

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.

並列關鍵字

Severe acute respiratory syndrome coronavirus (SARS-CoV) ； DNA vaccines ； envelope（E） gene ； codon optimization

參考文獻

Reference List

1. Peiris,J.S., Yuen,K.Y., Osterhaus,A.D. & Stohr,K. The severe acute respiratory syndrome. N. Engl. J. Med. 349, 2431-2441 (2003).

2. De Groot,A.S. How the SARS vaccine effort can learn from HIV--speeding towards the future, learning from the past. Vaccine 21, 4095-4104 (2003).

3. Wang,J.T. & Chang,S.C. Severe acute respiratory syndrome. Curr. Opin. Infect. Dis. 17, 143-148 (2004).

4. Peiris,J.S.M. et al. Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. The Lancet 361, 1767-1772 (2003).

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建構嚴重急性呼吸道症候群冠狀病毒套膜基因轉譯密碼最佳化之核酸疫苗

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