伪狂犬病毒gD蛋白在水稻细胞中的表达及其纯化条件优化

doi:10.3969/j.issn.1004-1524.20250283

浙江农业学报 ›› 2026, Vol. 38 ›› Issue (5): 867-875.DOI: 10.3969/j.issn.1004-1524.20250283

伪狂犬病毒gD蛋白在水稻细胞中的表达及其纯化条件优化

寇少康¹(), 陈恒耀¹, 潘世源¹, 张江楠¹, 程文博¹, 楚红燕¹, 张磊¹, 张二芹¹^,²

¹ 河南农业大学动物医学院, 河南郑州 450046
² 河南省农业科学院动物免疫学重点实验室, 河南郑州 450002

收稿日期:2025-04-09 出版日期:2026-05-25 发布日期:2026-06-02
作者简介:寇少康,主要从事免疫学研究。E-mail:kshaokang@163.com
基金资助:
河南省重大科技专项项目(221100110600);河南省科技攻关项目(222102110210)

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 Published:2026-05-25 Online:2026-06-02

摘要/Abstract

摘要：

为探索利用水稻细胞表达猪伪狂犬病毒gD蛋白的可行性,并为开发伪狂犬病毒亚单位疫苗奠定基础,本研究根据水稻密码子偏好性,对猪伪狂犬病毒gD基因进行优化与人工合成,构建重组植物表达载体pCAMBIA1300-gD。通过农杆菌转化法将该载体导入水稻愈伤组织,经共培养和暗培养筛选获得抗性愈伤组织。采用CTAB法提取愈伤组织基因组DNA,通过PCR鉴定阳性愈伤组织。将阳性愈伤组织扩大培养后研磨,加入液体培养基,于摇床中振荡培养;利用Western blot检测gD蛋白的表达情况。根据目标蛋白质的理化性质,对纯化条件中的pH值、阳离子填料和疏水填料进行筛选优化。结果表明,经潮霉素筛选获得110个愈伤组织,PCR鉴定出92个阳性愈伤组织,阳性率为83.6%。Western blot检测35个细胞株,其中15个成功表达gD蛋白。通过阳离子填料650F和疏水填料GE Butyl 4FF两步层析纯化,可获得一定纯度的gD蛋白。本研究为以水稻为生物反应器制备伪狂犬病毒亚单位疫苗奠定了基础。

关键词: 水稻, 愈伤组织, 伪狂犬病毒, 植物表达载体, gD蛋白, 密码子优化, 蛋白质纯化

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

中图分类号:

S855.3

寇少康, 陈恒耀, 潘世源, 张江楠, 程文博, 楚红燕, 张磊, 张二芹. 伪狂犬病毒gD蛋白在水稻细胞中的表达及其纯化条件优化[J]. 浙江农业学报, 2026, 38(5): 867-875.

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.

图/表 11

图1 pUC57-gD质粒的双酶切鉴定 M,DNA marker;1,空白;2~4,酶切鉴定pUC57-gD;N,阴性对照。

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.

图2 重组质粒pCAMBIA1300-gD的双酶切鉴定 M,DNA marker;1,pCAMBIA1300-gD酶切鉴定;2,gD基因PCR鉴定;N,阴性对照。

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.

图3 pCAMBIA1300-gD的农杆菌PCR验证 M,DNA marker;1,gD基因PCR鉴定;N,阴性对照。

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

图4 水稻细胞遗传转化表达 A,种子诱导;B,农杆菌和愈伤组织共培养;C,潮霉素抗性筛选;D,植物细胞培养;E,细胞悬浮培养。

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.

图5 阳性愈伤组织鉴定 M,DNA marker;1~23,水稻基因组DNA;N,阴性对照。

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

图6 gD蛋白在水稻细胞中的表达鉴定 M,蛋白质相对分子质量标准物;1~9,gD蛋白检测结果。

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

图7 不同pH值条件下的gD蛋白的稳定性 M,蛋白质相对分子质量标准物;1~3,溶液pH值分别为6、7和8。

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.

表1 阳离子填料的筛选

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	+	+

表2 疏水填料的筛选

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	+	+

图8 两步法纯化gD蛋白的SDS-PAGE分析 M,蛋白质相对分子质量标准物;1,650F流穿液;2,650F 10%洗脱液;3,650F 40%洗脱液;4,650F 100%洗脱液;5,GE Butyl 4FF流穿液;6,GE Butyl 4FF 100%洗脱液。

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.

图9 两步法纯化gD蛋白的Western blot分析 M,蛋白质相对分子质量标准物;1,纯化的gD蛋白。

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

参考文献 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.

伪狂犬病毒gD蛋白在水稻细胞中的表达及其纯化条件优化

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

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