Expression and Bioinformatics Analysis of Nipah Virus N Protein in Insect Cells

doi:10.7668/hbnxb.20194252

Abstract

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

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.

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Figures/Tables 18

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

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

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.

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.

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.

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.

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.

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.

Fig.8 DAB staining after membrane transfer

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

Fig.9 N protein affinity/hydrophobicity prediction result

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

Fig.11 The prediction results of N protein signal peptides

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

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

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

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

Fig.14 3D model prediction of N protein

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