Expression of pseudorabies virus gD protein in rice cells and optimization of purification condition

doi:10.3969/j.issn.1004-1524.20250283

Acta Agriculturae Zhejiangensis ›› 2026, Vol. 38 ›› Issue (5): 867-875.DOI: 10.3969/j.issn.1004-1524.20250283

• Animal Science • Previous Articles Next Articles

Expression of pseudorabies virus gD protein in rice cells and optimization of purification condition

KOU Shaokang¹(), CHEN Hengyao¹, PAN Shiyuan¹, ZHANG Jiangnan¹, CHENG Wenbo¹, CHU Hongyan¹, ZHANG Lei¹, ZHANG Erqin¹^,²

¹ College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China
² Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China

Received:2025-04-09 Online:2026-05-25 Published:2026-06-02

Abstract

Abstract:

To explore the feasibility of expressing the pseudorabies virus gD protein in rice cells and to lay a foundation for developing a pseudorabies virus subunit vaccine, this study optimized and artificially synthesized the pseudorabies virus gD gene based on rice codon usage bias, constructing the recombinant plant expression vector pCAMBIA1300-gD. This vector was introduced into rice callus via Agrobacterium-mediated transformation, and resistant calluses were obtained after co-cultivation and dark culture screening. Callus genomic DNA was extracted using the CTAB method, and positive calluses were identified by PCR. Positive calluses were expanded in culture, ground, and added to liquid medium for shake-flask cultivation. Western blot was used to detect the expression of the gD protein. Based on the physicochemical properties of the target protein, purification conditions including pH value, cation exchange resin, and hydrophobic resin were screened and optimized. The results showed that 110 calluses were obtained after hygromycin screening, with 92 identified as positive by PCR, yielding a positive rate of 83.6%. Western blot analysis of 35 cell lines revealed successful gD protein expression in 15 lines. Purification via two-step chromatography using cation exchange resin 650F and hydrophobic resin GE Butyl 4FF yielded gD protein of certain purity. This study established a foundation for using rice as a bioreactor to produce pseudorabies virus subunit vaccines.

Key words: rice, callus, pseudorabies virus, plant expression vector, gD protein, codon optimization, protein purification

CLC Number:

S855.3

KOU Shaokang, CHEN Hengyao, PAN Shiyuan, ZHANG Jiangnan, CHENG Wenbo, CHU Hongyan, ZHANG Lei, ZHANG Erqin. Expression of pseudorabies virus gD protein in rice cells and optimization of purification condition[J]. Acta Agriculturae Zhejiangensis, 2026, 38(5): 867-875.

Figures/Tables 11

Fig.1 Double restriction enzyme digestion of plasmid pUC57-gD M,DNA marker; 1, Blank; 2-4, Verification of pUC57-gD plasmid through restriction analysis; N, Negative control.

Fig.2 Identification of the recombinant plasmid pCAMBIA1300-gD by double enzyme digestion M,DNA marker;1, Restriction enzyme analysis of pCAMBIA1300-gD; 2, PCR identification of the gD gene; N, Negative control.

Fig.3 Agrobacterium PCR verification of pCAMBIA1300-gD M,DNA marker;1, gD gene verification by PCR; N, Negative control.

Fig.4 Rice cell genetic transformation and transgene expression A, Seed-induced expression; B, Agrobacterium-plant co-culture; C, Hygromycin resistance screening; D, Plant cell culture; E, Cell suspension culture.

Fig.5 Identification of positive calli M, DNA marker; 1-23, Rice genomic DNA; N, Negative control.

Fig.6 Identification of gD protein expression in rice cells M, Protein marker; 1-9, gD protein test results.

Fig.7 Stability of gD protein under different pH values M, Protein marker; 1-3, The pH value of the solution is 6, 7 and 8, respectively.

Table 1 Screening of cationic resin

阳离子填料 Cationic resin	流穿液 Flow-through	100%洗脱液 100% elution
Boglon SP HP	-	+
Chutian TH SP	+	+
650F	-	+
GE SP HP	+	+
Boglon Diamond	-	+
BioRad Nuvia S	+	-
BioRad High S	+	+
Komyx SP HP	+	+

Table 2 Screening of hydrophobic resin

疏水填料 Hydrophobic resin	流穿液 Flow-through	100%洗脱液 100% elution
GE MMC	+	+
Boglon MMC	+	+
GE Butyl HP	+	+
GE Butyl 4FF	+	-
GE Hi Trap FF	+	+
Boglon Butyl 4FF	+	+

Fig.8 SDS-PAGE analysis of gD protein purified by the two-step method M, Protein marker; 1, 650F flow-through; 2, 650F 10% elution; 3, 650F 40% elution; 4, 650F 100% elution; 5, GE Butyl 4FF flow-through; 6, GE Butyl 4FF 100% elution.

Fig.9 Western blot analysis of gD protein purified by the two-step method M, Protein marker; 1, Purified gD protein.

References 28

[1]	FAN S Y, YUAN H X, LIU L, et al. Pseudorabies virus encephalitis in humans: a case series study[J]. Journal of NeuroVirology, 2020, 26(4): 556-564.
[2]	LEE J Y, WILSON M R. A review of pseudorabies (Aujeszky’s disease) in pigs[J]. The Canadian Veterinary Journal, 1979, 20(3): 65-69.
[3]	POMERANZ L E, REYNOLDS A E, HENGARTNER C J. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine[J]. Microbiology and Molecular Biology Reviews, 2005, 69(3): 462-500.
[4]	HOU Y, WANG Y M, ZHANG Y, et al. Human encephalitis caused by pseudorabies virus in China: a case report and systematic review[J]. Vector-Borne and Zoonotic Diseases, 2022, 22(7): 391-396.
[5]	HE W T, AUCLERT L Z, ZHAI X F, et al. Interspecies transmission, genetic diversity, and evolutionary dynamics of pseudorabies virus[J]. The Journal of Infectious Diseases, 2019, 219(11): 1705-1715.
[6]	HU F, WANG J W, PENG X Y. Bilateral necrotizing retinitis following encephalitis caused by the pseudorabies virus confirmed by next-generation sequencing[J]. Ocular Immunology and Inflammation, 2021, 29(5): 922-925.
[7]	SKINNER G R B, AHMAD A, DAVIES J A. The infrequency of transmission of herpesviruses between humans and animals; postulation of an unrecognised protective host mechanism[J]. Comparative Immunology, Microbiology and Infectious Diseases, 2001, 24(4): 255-269.
[8]	FREULING C M, MÜLLER T F, METTENLEITER T C. Vaccines against pseudorabies virus (PrV)[J]. Veterinary Microbiology, 2017, 206: 3-9.
[9]	ZOUHAROVA D, LIPENSKA I, FOJTIKOVA M, et al. Antiviral activities of 2, 6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: in vitro study results with pseudorabies virus (PrV, SuHV-1)[J]. Veterinary Microbiology, 2016, 184: 84-93.
[10]	PETRINA M, MARTIN J, BASTA S. Granulocyte macrophage colony-stimulating factor has come of age: from a vaccine adjuvant to antiviral immunotherapy[J]. Cytokine & Growth Factor Reviews, 2021, 59: 101-110.
[11]	GRANZOW H, KLUPP B G, FUCHS W, et al. Egress of alphaherpesviruses: comparative ultrastructural study[J]. Journal of Virology, 2001, 75(8): 3675-3684.
[12]	SUN Y, LUO Y Z, WANG C H, et al. Control of swine pseudorabies in China: opportunities and limitations[J]. Veterinary Microbiology, 2016, 183: 119-124.
[13]	CHEN H Y, CHEN Z D, BAI N, et al. Construction of a eukaryotic expression system with stable and secretory expression of Mycobacterium tuberculosis 38 kDa protein[J]. World Journal of Microbiology and Biotechnology, 2021, 37(10): 175.
[14]	CAO Z, ZHANG K, ZHANG H, et al. Efficacy of a gB + gD-based subunit vaccine and the adjuvant granulocyte-macrophage colony stimulating factor for pseudorabies virus in rabbits[J]. Frontiers in Microbiology, 2022, 13: 965997.
[15]	NANDI S, KWONG A T, HOLTZ B R, et al. Techno-economic analysis of a transient plant-based platform for monoclonal antibody production[J]. mAbs, 2016, 8(8): 1456-1466.
[16]	BARONE P W, WIEBE M E, LEUNG J C, et al. Viral contamination in biologic manufacture and implications for emerging therapies[J]. Nature Biotechnology, 2020, 38(5): 563-572.
[17]	DESHPANDE P, JHAVERI A, PATTNI B, et al. Transferrin and octaarginine modified dual-functional liposomes with improved cancer cell targeting and enhanced intracellular delivery for the treatment of ovarian cancer[J]. Drug Delivery, 2018, 25(1): 517-532.
[18]	张绩, 周上铃, 何发, 等. 水稻α-淀粉酶基因的表达模式与颖花开放的关系[J]. 中国农业科学, 2023, 56(7): 1275-1282.
	ZHANG J, ZHOU S L, HE F, et al. Expression pattern of the rice α-amylase genes related with the process of floret opening[J]. Scientia Agricultura Sinica, 2023, 56(7): 1275-1282.
[19]	屈小天, 王雅楠, 许倩茹, 等. H5N1亚型禽流感病毒HA蛋白在水稻胚乳中的表达及其纯化[J]. 河南农业科学, 2024, 53(3): 125-132.
	QU X T, WANG Y N, XU Q R, et al. Expression and purification of H5N1 subtype avian influenza virus HA protein in rice endosperm[J]. Journal of Henan Agricultural Sciences, 2024, 53(3): 125-132.
[20]	陈焕春. 猪系统性疾病的流行现状与防控措施[J]. 饲料与畜牧, 2018(2): 45-50.
	CHEN H C. Epidemic situation and prevention and control measures of systemic diseases in pigs[J]. Animal Agriculture, 2018(2): 45-50.
[21]	TONG W, LIU F, ZHENG H, et al. Emergence of a pseudorabies virus variant with increased virulence to piglets[J]. Veterinary Microbiology, 2015, 181(3/4): 236-240.
[22]	CHEN F Z, ZHU Y X, WU M Z, et al. Comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus[J]. Viruses, 2015, 7(10): 5525-5538.
[23]	METTENLEITER T C. Aujeszky’s disease (pseudorabies) virus: the virus and molecular pathogenesis-state of the art, June 1999[J]. Veterinary Research, 2000, 31(1): 99-115.
[24]	KONDIBAEVA Z B, YESPEMBETOV B A, ABEUOV K B, et al. Inactivated vaccine against aujeszky’s disease[J]. Veterinary World, 2021: 2957-2963.
[25]	REDDY P S, IDAMAKANTI N, PYNE C, et al. The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle[J]. Veterinary Immunology and Immunopathology, 2000, 76(3/4): 257-268.
[26]	ZHANG T, LIU Y C, CHEN Y M, et al. A single dose glycoprotein D-based subunit vaccine against pseudorabies virus infection[J]. Vaccine, 2020, 38(39): 6153-6161.
[27]	MOON A, HUANG J S, SONG X, et al. Immune responses induced by a recombinant lactiplantibacillus plantarum surface-displaying the gD protein of pseudorabies virus[J]. Viruses, 2024, 16(8): 1189.
[28]	CORBIN J M, MCNULTY M J, MACHAROEN K, et al. Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures[J]. Biotechnology and Bioengineering, 2020, 117(10): 3053-3065.

Expression of pseudorabies virus gD protein in rice cells and optimization of purification condition

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