以大腸桿菌表現系統製備SARS-CoV-2重組棘蛋白和核衣殼蛋白

Abstract

2019年12月，新型冠狀病毒 (SARS-CoV-2) 最先在中國武漢的華南海鮮市場出現感染案例。現今已經造成了全球大流行，超過兩千一百萬感染案例及七十萬死亡案例。目前並無有效的抗體或藥物能夠治療 SARS-CoV-2的感染，也尚未研發出疫苗，因此能夠快速並精準地檢測SARS-CoV-2，是目前能夠延緩疫情蔓延的重要任務。 本論文利用大腸桿菌表現SARS-CoV-2的重組棘蛋白N-terminal domain (NTD)、receptor binding domain (RBD)、S1、S2、S全長與核衣殼蛋白(nucleocapsid protein; NP)；除NP重組蛋白為可溶外，以大腸桿菌表現的NTD、RBD、S1、S2與S重組蛋白皆存在於inclusion body中，因此利用含8 M尿素的溶液使蛋白質溶解，再以HisTrap層析管柱進行純化，得到高純度的重組蛋白。接著利用劃線機將重組蛋白塗佈到PVDF膜上，再與血清樣本反應後，以西方墨點法偵測訊號。實驗結果顯示確診Coronavirus disease 2019 (COVID-19) 之患者的血清結合於NP重組蛋白之訊號為最強，但是正常人之血清樣本亦有些許的交叉反應；S2、S與NTD重組蛋白的訊號較弱，但是專一性較高，正常人之血清樣本並無交叉反應；使用RBD重組蛋白為抗原時並無偵測到任何訊號；而使用S1重組蛋白為抗原的專一性不佳，確診者與正常人之血清樣本皆可獲得訊號，無法正確診斷是否受到SARS-CoV-2的感染。本論文研究結果顯示，使用大腸桿菌表現之NP、NTD、S2與S重組蛋白所製成的試片可運用於COVID-19的疾病診斷。

At the end of December, 2019, the new coronavirus (SARS-CoV-2) appeared in the seafood market in Wuhan, China. Recently, more than 21 million infection cases and 7 hundred thousands of death cases have been reported. Currently, there is no effective antibody or drug that can treat SARS-CoV-2 infection, and no vaccine has yet been successfully developed. Therefore, the ability to detect SARS-CoV-2 quickly and accurately is an important task to prevent the spread of the epidemic. The research utilized E. coli to express the recombinant N-terminal domain (NTD), receptor binding domain (RBD) of Spike protein (S), S1, S2, S and nucleocapsid protein (NP) of SARS-CoV-2. Except NP, the NTD, RBD, S1, S2, S recombinant protein were expressed as inclusion body in E. coli. Therefore, a solution containing 8M urea was used to dissolve the protein, and the NTD, RBD, S1, S2, S and NP recombinant protein were purified with HisTrap chromatography column to obtain high purity of proteins. The recombinant proteins were coated on the PVDF membrane, and then incubated with the patient's serum for Western blotting analysis. The experimental results showed that the patient’s serum diagnosed as Coronavirus disease 2019 (COVID-19) positive has the strongest signal for binding to NP recombinant protein, but the normal patient’s serum also has a slight cross reaction with NP recombinant protein. The signals were weak by using S2, S and NTD recombinant protein as the antigens, but they were more specific for not causing any cross reaction. There was no signal while using RBD recombinant protein as the antigen. The specificity was not good while using S1 recombinant protein as the antigen since both of the COVID-19 positive and negative sera could obtain the signal. This research showed that the test strips coated with the E. coli-expressed NP, NTD, S2 and S recombinant protein can be applied for the diagnosis of COVID-19.