尼帕病毒N蛋白在昆虫细胞上的表达及蛋白生物信息学分析

doi:10.7668/hbnxb.20194252

摘要/Abstract

摘要：

为获得具有和天然蛋白质类似功能与活性的尼帕病毒核衣壳蛋白(N蛋白),通过PCR扩增NiV N蛋白开放阅读框基因,克隆pFastBac^TMHTB载体,转化DH10Bac感受态细胞,转染sf9昆虫细胞,蛋白免疫印迹鉴定,生物信息学分析研究N蛋白特性。结果表明,经PCR和双酶切鉴定,获得重组质粒pFastBac^TMHTB-NiV-N;经SDS-PAGE和Western Blotting鉴定,获得具有免疫活性的重组蛋白Bacmid-NiV-N;经分子生物信息学软件分析预测,重组蛋白含有532个氨基酸,分子大小为58.34 ku,为不稳定、亲水性蛋白,无跨膜区,蛋白二级结构主要以无规则卷曲为主,有5个T细胞抗原表位和18个B细胞抗原表位。上述研究为深入进行NiV N蛋白的功能和建立相应抗原检测方法提供物质基础。

关键词: 尼帕病毒, N蛋白, 昆虫细胞, 生物信息学分析

Abstract:

In order to obtain Nipah virus nucleocapsid protein(N protein)with similar function and activity to natural proteins,the NiV N protein open reading frame gene was amplified by PCR,cloned into pFastBac^TMHTB vector,transformed into DH10Bac receptive cells,and transfected into sf9 insect cells.The protein was identified by Western Blotting and bioinformatics analysis was used to study the characteristics of N protein.The results showed that the recombinant plasmid pFastBac^TMHTB-NiV-N was obtained through PCR and double enzyme digestion identification.Recombinant protein Bacmid-NiV-N with immune activity was identified by SDS-PAGE and Western Blotting.According to molecular bioinformatics software analysis and prediction,the recombinant protein contained 532 amino acids with a molecular size of 58.34 ku.It was an unstable and hydrophilic protein without a transmembrane region.The secondary structure of the protein was mainly irregular curls,with 5 T cell epitopes and 18 B cell epitopes.The above research provides a material basis for further understanding the function of NiV N protein and establishing corresponding antigen detection methods.

Key words: Nipah virus, N protein, Insect cell, Bioinformatics analysis

王腾芬, 叶玲玲, 李书宁, 张伟, 胡世享, 卓娜, 艾军, 陈培富. 尼帕病毒N蛋白在昆虫细胞上的表达及蛋白生物信息学分析[J]. 华北农学报, 2024, 39(S1): 259-266. doi: 10.7668/hbnxb.20194252.

WANG Tengfen, YE Lingling, LI Shuning, ZHANG Wei, HU Shixiang, ZHUO Na, AI Jun, CHEN Peifu. Expression and Bioinformatics Analysis of Nipah Virus N Protein in Insect Cells[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(S1): 259-266. doi: 10.7668/hbnxb.20194252.

http://www.hbnxb.net/CN/Y2024/V39/IS1/259

图/表 18

表1 试验所用引物

Tab.1 Primers used in the experiment

引物名称 Primer name	引物序列(5'— 3') Primer sequence(5'— 3')	片段大小/bp Fragment size	退火温度/℃ Annealing temperature
Niv-N-F	GGGCGCCATGGGATCATGAGTGATATTTTTGAAGAGGCGG	1 596	56
Niv-N-R	TTCTCGACAAGCTTGTCACACATCAGCTCTGACAAAGTC
M13-F	GTTTTCCCAGTCACGAC		55
M13-R	CAGGAAACAGCTATGAC

图1 N基因PCR扩增产物 M.DL2000 DNA Marker;1.N基因PCR扩增产物;2.阴性对照。

Fig.1 PCR amplification products of N gene M.DL2000 DNA Marker;1.PCR products of N gene;2.Negtive control.

图2 重组质粒pFastBac^TMHTB-Niv-N菌液 PCR扩增产物 M.DL5000 DNA Marker;1—20.重组质粒pFastBac^TMHTB-Niv-N菌液PCR扩增产物;21—22.阴性对照。

Fig.2 PCR amplification products of recombinant plasmid pFastBac^TMHTB-Niv-N in bacterial solution M.DL5000 DNA Marker;1—20.Recombinant plasmid pFastBac^TMHTB-Niv-N bacterial solution PCR amplification product;21—22.Negative control.

图3 重组质粒pFastBac^TMHTB-Niv-N双酶切鉴定 M.DL5000 DNA Marker;1—2.重组质粒pFastBac^TMHTB-Niv-N双酶切产物。

Fig.3 Recombinant plasmid pFastBac^TMHTB-Niv-N double restriction enzyme digestion identification M.DL5000 DNA Marker;1—2.Recombinant plasmid pFastBac^TMHTB-Niv-N double enzyme cleavage product.

图4 M13引物PCR扩增重组杆粒产物 M.DL5000 DNA Marker;1—20.M13引物PCR扩增重组杆粒产物;21.阴性对照。

Fig.4 M13 primer PCR amplification of recombinant rod-shaped products M.DL5000 DNA Marker;1—20.Amplification of recombinant bacmid by M13 primer PCR product;21.Negative control.

图5 Bacmid-GFP第3代在sf9昆虫细胞上的表达 A.转染Bacmid-GFP 第3代48 h的sf9细胞;B.转染Bacmid-GFP 第3代72 h的sf9细胞;C.转染Bacmid-GFP 第3代96 h的sf9细胞;D.转染Bacmid-GFP第3代120 h的sf9细胞。

Fig.5 Expression of Bacmid GFP third-generation on sf9 insect cells A.Transfection of sf9 cells from Bacmid-GFP 48 h in the third generation;B.Transfection of sf9 cells from Bacmid-GFP 72 h in the third generation;C.Transfection of sf9 cells from Bacmid-GFP 96 h in the third generation;D.Transfection of sf9 cells from Bacmid-GFP 120 h in the third generationv.

图6 转染Bacmid-NiV-N 第3代的sf9细胞病变 A.正常 sf9 细胞;B.转染 Bacmid-NiV-N第3代48 h的 sf9 细胞;C.转染 Bacmid-NiV-N第3代72 h的 sf9细胞;D.转染Bacmid-NiV-N第3代120 h的sf9细胞。

Fig.6 sf9 cell lesion in the third generation transfected with Bacmid-NiV-N A.Normal sf9 cells;B.sf9 cells transfected with Bacmid-NiV-N for 48 h in the third generation;C.sf9 cells transfected with Bacmid-NiV-N third-generation for 72 h;D.sf9 cells transfected with Bacmid-NiV-N for 120 h in the third generation.

图7 SDS-PAGE电泳考马斯亮蓝染色 M.蛋白分子质量标准;1.N蛋白第3代重组产物;2.N蛋白第2代重组产物;3.sf9细胞阴性对照。图8同。

Fig.7 SDS-PAGE electrophoresis Coomassie Brilliant Blue staining M.Protein molecular mass standard;1.The third-generation recombinant protein product of N;2.The second-generation recombinant protein product of N;3.sf9 cell negative control.The same as Fig.8.

图8 转膜后DAB染色

Fig.8 DAB staining after membrane transfer

表2 N蛋白氨基酸组成

Tab.2 The amino acid composition of N protein

氨基酸 Amino acid	数量 Quantity	占比/% Percentage	氨基酸 Amino acid	数量 Quantity	占比/% Percentage
Ala (A)	58	10.9	Lys(K)	25	4.7
Ser (S)	52	9.8	Gln (Q)	22	4.1
Leu (L)	47	8.8	Asn (N)	21	3.9
Glu (E)	40	7.5	Phe (F)	19	3.6
Arg (R)	38	7.1	Pro (P)	19	3.6
Gly (G)	35	6.6	Met (M)	18	3.4
Ile (I)	33	6.2	Tyr (Y)	11	2.1
Thr (T)	31	5.8	Trp (W)	5	0.9
Val (V)	30	5.6	His (H)	2	0.4
Asp (D)	26	4.9

图9 N蛋白亲/疏水性预测结果

Fig.9 N protein affinity/hydrophobicity prediction result

图10 N蛋白跨膜区预测结果

Fig.10 Prediction results of the transmembrane region of N protein

图11 N蛋白信号肽预测结果

Fig.11 The prediction results of N protein signal peptides

表3 N蛋白T细胞抗原表位预测肽段

Tab.3 Predicted peptides of N protein T cell epitope

序号 No.	肽段 Peptide	长度 Length	分值 Score
1	MVEILIEVKKGGSAKGRAVEIISDIGNYVEETGMAGFFATIRFGLETRYPALALNEFQSDLNTIKSLMLLYREIGPRAPY	80	1.219 68
2	YGLRITDMSTLVSAVITIEAQIWILIAKAVTAPDTAEESETRRWAKYVQQKRVNPFFALTQQWLTEMRNLLSQSLSVRKF	80	0.469 42
3	MVLLEESIQTKFAPGGYPLLWSFAMGVATTIDRSMGALNINRGYLEPMYFRLGQKSARHHAGGIDQNMANRLGLSSNQVA	80	-0.203 39
4	ELAAAVQETSAGRQESNVQAREAKFAAGGVLIGGSDQDVDEEEEPIEQSGRQSVTFKREMSISSLADSVPSSSVSTSGGT	80	-0.825 06
5	RLTNSLLNLRSRLAAKAAKEAASSNATDDPAISNKDQRESEKKNNQDLKPTQNDLDFVRADV	62	-1.350 91

表4 N蛋白B细胞抗原表位部分预测肽段

Tab.4 Partial Predicted peptides of N protein B cell epitope

序号 No.	位置 Position	肽段 Peptide	长度 Length
1	6—30	EEAASFRSYQSKLGRDGRASAATAT	25
2	109—127	NGIPVMERRGDKAQEEMEG	19
3	136—158	RDSSKGKTPFVDSRAYGLRITDM	23
4	185—216	TAEESETRRWAKYVQQKRVNPFFALTQQWLTE	32
5	344—354	SMGALNINRGY	11

图12 N蛋白Bepipred线性表位预测结果

Fig.12 The prediction results of the linear epitope of N protein Bepipred

图13 N蛋白二级结构预测 H.α-螺旋;e.延伸链;c.无规则卷曲;t.β-转角。

Fig.13 Prediction of the secondary structure of N protein H.Alpha helix;e.Extened chain;c.Random coil;t.Beta turn.

图14 N蛋白的三维模型预测

Fig.14 3D model prediction of N protein

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