COVID-19病毒N基因在昆虫细胞内的表达

doi:10.7668/hbnxb.20192941

摘要/Abstract

摘要：

为获得具有和天然蛋白质类似功能与活性的COVID-19核衣壳蛋白并应用于实际检测中,首先根据Bac-to-bac昆虫表达系统和人工合成的COVID-19核衣壳蛋白(N蛋白)序列,将pFastBac^TMHTB载体上的酶切位点BamH Ⅰ和Xba Ⅰ分别加入上下游引物,利用PCR技术对N基因进行扩增,先后连接T-Vector pMD19(simple)载体与pFastBac^TMHTB载体,并获得重组质粒pMD19-T(simple)-COV19-N和pFastBac^TMHTB-COV19-N,最终在DH10Bac细胞中构建重组杆粒DH10Bac-pFastBac^TMHTB-COV19-N并使其在昆虫细胞Sf9内进行表达,获得重组蛋白后进行SDS-PAGE和WB分析。通过PCR方法成功扩增N基因,构建的重组质粒pMD19-T(simple)-COV19-N经PCR和双酶切鉴定均证明正确,在DH10Bac细胞内构建的重组杆粒DH10Bac-pFastBac^TMHTB-COV19-N通过PCR鉴定得到预期2条带,大小分别为2 430,3 690 bp,证明成功得到重组杆粒,用该重组杆粒转染Sf9昆虫细胞,同时设立重组GFP蛋白对照组,转染120 h后分别收集重组N蛋白与重组GFP蛋白并制样;分别进行SDS-PAGE和WB分析,使用HRP-His标记抗体验证转染成功且重组N蛋白与重组GFP蛋白均在Sf9细胞内顺利表达,试验结果与预期相符,重组N蛋白条带大小约为46 ku。该试验成功构建了呼吸道冠状病毒N基因真核表达载体,并在昆虫细胞内成功表达,为建立ELISA检测方法等相关研究提供试验基础。

关键词: COVID-19病毒, N蛋白, 昆虫表达系统, 重组杆粒, 蛋白鉴定

Abstract:

In order to obtain COVID-19 nucleocapsid protein with similar function and activity to natural protein and apply it to practical detection. Firstly,according to Bac-to-bac insect expression system and synthetic COVID-19 nucleocapsid protein(N protein)sequence,BamH Ⅰ and Xba Ⅰ on pFastBac^TMHTB vector were added to upstream and downstream primers respectively. The N gene was amplified by PCR technology,and T-Vector pMD19(simple)vector and pFastBac^TMHTB vector were connected successively and recombinant plasmids pMD19-T(simple)-COV19-N and pFastBac^TMHTB-COV19-N,and finally construct recombinant bacmid DH10Bac-pFastBac^TMHTB-COV19-N in DH10Bac cells was expressed in insect cell Sf9. The recombinant protein was obtained and analyzed by SDS-PAGE and WB. The recombinant plasmid pMD19-T(simple)-COV19-N was identified by PCR and double enzyme digestion. The recombinant bacmid DH10Bac-pFastBac^TMHTB-COV19-N was constructed in DH10Bac cells was identified by PCR and the expected two bands were 2 430,3 690 bp,respectively,which proved that the recombinant bacmid was successfully obtained. The recombinant bacmid was transfected into Sf9 insect cells. At the same time,the recombinant GFP protein control group was established. After 120 h of transfection,the recombinant N protein and recombinant GFP protein were collected and samples were prepared;SDS-PAGE and WB analysis were carried out respectively. HRP-His labeled antibody was used to verify that the transfection was successful,and both recombinant N protein and recombinant GFP protein were successfully expressed in Sf9 cells. The experimental results were consistent with the expectation,and the size of recombinant N protein band was about 46 ku. The eukaryotic expression vector of respiratory coronavirus N gene was successfully constructed and successfully expressed in insect cells,which provides an experimental basis for the establishment of ELISA detection methods and other related research.

Key words: COVID-19 virus, N protein, Insect expression system, Recombinant bacmid, Protein identification

宿放, 韩佃刚, 叶玲玲, 张冲, 尹尚莲, 罗倩敏, 董仙兰, 李瑶瑶, 李凌枫, 艾军, 信吉阁. COVID-19病毒N基因在昆虫细胞内的表达[J]. 华北农学报, 2022, 37(5): 174-180. doi: 10.7668/hbnxb.20192941.

SU Fang, HAN Diangang, YE Lingling, ZHANG Chong, YIN Shanglian, LUO Qianmin, DONG Xianlan, LI Yaoyao, LI Lingfeng, AI Jun, XIN Jige. Expression of COVID-19 Virus N Gene in Insect Cells[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(5): 174-180. doi: 10.7668/hbnxb.20192941.

http://www.hbnxb.net/CN/Y2022/V37/I5/174

图/表 7

图1 连接T-Vector pMD19(simple)载体后进行PCR验证 M.DL5000 DNA Marker;1—4. pMD19-T(simple)-COV19-N的PCR产物。

Fig.1 PCR verification after connecting T-Vector pMD19(simple)vector M.DL5000 DNA Marker;1—4. PCR products of pMD19-T(simple)-COV19-N.

图2 连接T-Vector pMD19(simple)载体后进行双酶切验证 M.DL5000 DNA Marker;1—4.pMD19-T(simple)-COV19-N双酶切结果;5.pMD19-T(simple)-COV19-N的PCR产物对照;6.T-Vector pMD19(simple)空载体对照。

Fig.2 Double enzyme digestion verification after connecting T-Vector pMD19(simple)vector M.DL5000 DNA Marker;1—4.pMD19-T(simple)-COV19-N double enzyme digestion results;5.PCR product control of pMD19-T(simple)-COV19-N;6.T-Vector pMD19(simple)empty vector control.

图3 从菌液中提取的pFastBac^TMHTB空载体电泳 M.DL5000 DNA Marker;1—2.pFastBac^TMHTB空载体。

Fig.3 Electrophoresis of pFastBac^TMHTB empty carrier extracted from bacterial solution M.DL5000 DNA Marker;1—2.pFastBac^TMHTB empty vector.

图4 重组杆粒DH10Bac-pFastBac^TMHTB-COV19-N的PCR M.DL5000 DNA Marker;1.M13引物PCR扩增重组杆粒。

Fig.4 PCR of recombinant bacmid DH10Bac-pFastBac^TMHTB-COV19-N M.DL5000 DNA Marker;1.Amplification of recombinant bacmid by M13 primer PCR.

图5 昆虫细胞转染试验 A.DH10Bac-pFastBac^TMHTB-GFP对照组(明视野),约12 h,40×10;B. DH10Bac-pFastBac^TMHTB-GFP对照组(暗视野),约12 h,40×10;C.DH10Bac-pFastBac^TMHTB-GFP对照组,120 h,40×10;D.DH10Bac-pFastBac^TMHTB-COV19-N组,120 h,40×10。

Fig.5 Insect cell transfection experiment A.DH10Bac-pFastBac^TMHTB-GFP control group(bright field),about 12 h,40×10;B.DH10Bac-pFastBac^TMHTB-GFP control group(dark field),about 12 h,40×10;C.DH10Bac-pFastBac^TMHTB-GFP control group,120 h,40×10;D.DH10Bac-pFastBac^TMHTB-COV19-N group,120 h,40×10.

图6 DH10Bac-pFastBac^TMHTB-COV19-N重组蛋白电泳 N.PageRuler预染蛋白Marker;1.DH10Bac-pFastBac^TMHTB-GFP荧光蛋白对照;2.DH10Bac-pFastBac^TMHTB-COV19-N重组蛋白。图7同。

Fig.6 Electrophoresis of recombinant DH10Bac-pFastBac^TMHTB-COV19-N protein N.PageRuler pre stained protein Marker;1.DH10Bac-pFastBac^TMHTB-GFP fluorescent protein control;2.DH10Bac-pFastBac^TMHTB-COV19-N recombinant protein.The same as Fig.7.

图7 转膜后使用DAB染色法染色

Fig.7 DAB staining after membrane transfer

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