不同原核表达载体对非洲猪瘟病毒CD2v蛋白可溶性表达及免疫反应性比较

doi:10.3785/j.issn.1008-9209.2022.11.071

浙江大学学报（农业与生命科学版）

2023, Vol. 49

Issue (6): 873-880 DOI: 10.3785/j.issn.1008-9209.2022.11.071

动物科学与动物医学

不同原核表达载体对非洲猪瘟病毒CD2v蛋白可溶性表达及免疫反应性比较

冯梦珂^1,²(

),王星博^1,²,林璐璐^1,²,崔明仙^1,²,颜焰^1,²,周继勇^1,²(

)

^1.浙江大学动物医学中心，浙江杭州 310058
^2.农业农村部动物病毒学重点实验室，浙江杭州 310058

Comparisons on soluble expression and immunoreactivity of African swine fever virus CD2v protein expressed by different prokaryotic expression vectors

Mengke FENG^1,²(

),Xingbo WANG^1,²,Lulu LIN^1,²,Mingxian CUI^1,²,Yan YAN^1,²,Jiyong ZHOU^1,²(

)

^1.Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China
^2.Key Laboratory of Animal Virology of Ministry of Agriculture and Rural Affairs, Hangzhou 310058, Zhejiang, China

全文: PDF(2580 KB) HTML

摘要：

本研究旨在系统性地探究不同原核表达载体对非洲猪瘟病毒CD2v蛋白的可溶性表达差别，并利用临床非洲猪瘟病毒抗体阳性血清比较包涵体和可溶性CD2v蛋白的免疫反应性。利用5种原核表达载体pCold-TF、pET28a、pMAL-C6T、pGEX-4T-1、pET32a分别表达去除信号肽和跨膜区的非洲猪瘟病毒CD2v蛋白，利用镍-氨三乙酸（nickel-nitrilotriacetic acid, Ni-NTA）亲和层析法分别纯化源自pET28a和pCold-TF表达载体的包涵体和可溶性CD2v蛋白，并用间接酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）方法比较纯化蛋白的免疫反应性。结果显示，pCold-TF载体以表达可溶性蛋白为主，pMAL-C6T载体同时表达包涵体和可溶性蛋白，而其他载体主要表达包涵体蛋白。临床非洲猪瘟病毒抗体阳性血清检测数据显示，可溶性蛋白的免疫反应性显著优于包涵体蛋白（P＜0.05）。pCold-TF载体的触发因子（trigger factor, TF）标签可促进CD2v蛋白的可溶性表达，其表达蛋白的免疫反应性优于包涵体蛋白。本研究结果为CD2v蛋白的免疫原性研究奠定了基础，亦为其他重要抗原的可溶性表达提供了思路。

关键词： 非洲猪瘟病毒; CD2v蛋白; 原核表达; 可溶性表达; 免疫反应性

Abstract:

The aim of this study was to systematically investigate the differences in the soluble expression level of the African swine fever virus (ASFV) CD2v protein by different prokaryotic expression vectors, and the immunoreactivities of the inclusion body and soluble CD2v proteins were compared using clinical anti-ASFV antibody-positive sera. Five prokaryotic expression vectors, namely, pCold-TF, pET28a, pMAL-C6T, pGEX-4T-1 and pET32a, were utilized to express the CD2v protein without the signal peptide and transmembrane region, respectively. The inclusion body CD2v protein expressed by the pET28a vector and the soluble CD2v protein expressed by the pCold-TF vector were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography, and the immunoreactivity of the purified proteins was detected by indirect enzyme-linked immunosorbent assay (ELISA). The results showed that the CD2v protein expressed by the pCold-TF vector was soluble mainly, while the CD2v protein expressed by the pMAL-C6T vector was insoluble (inclusion body) and soluble, and the CD2v protein expressed by the other vectors was mainly as inclusion body. The indirect ELISA results for clinical anti-ASFV antibody-positive sera showed that the immunoreactivity of soluble protein was significantly better than that of the inclusion body protein (P＜0.05). The trigger factor (TF) tag of pCold-TF promoted the soluble expression of the CD2v protein, and the immunoreactivity of the expressed protein was greater than that of the inclusion body protein. This study lays the foundation for further immunogenicity research on the CD2v protein and provides a candidate strategy for the soluble expression of other important antigens.

Key words: African swine fever virus CD2v protein prokaryotic expression soluble expression immunoreactivity

收稿日期: 2022-11-07 出版日期: 2023-12-25

CLC:

S855.3

基金资助: 国家自然科学基金区域创新发展联合基金项目(U21A20256);浙江省重点研发计划项目(2020C02013)

通讯作者: 周继勇 E-mail: mengkefeng@zju.edu.cn;jyzhou@zju.edu.cn

作者简介: 冯梦珂（https://orcid.org/0000-0002-5338-8403），E-mail：mengkefeng@zju.edu.cn

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	冯梦珂
	王星博
	林璐璐
	崔明仙
	颜焰
	周继勇

引用本文:

冯梦珂,王星博,林璐璐,崔明仙,颜焰,周继勇. 不同原核表达载体对非洲猪瘟病毒CD2v蛋白可溶性表达及免疫反应性比较[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(6): 873-880.

Mengke FENG,Xingbo WANG,Lulu LIN,Mingxian CUI,Yan YAN,Jiyong ZHOU. Comparisons on soluble expression and immunoreactivity of African swine fever virus CD2v protein expressed by different prokaryotic expression vectors. Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(6): 873-880.

链接本文:

https://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2022.11.071 或 https://www.zjujournals.com/agr/CN/Y2023/V49/I6/873

表1 不同原核表达载体标签及重组蛋白分子量

图1 重组质粒的PCR鉴定结果M：DNA分子标志物；1～5：pET-28a-CD2vΔSP-TM、pET32a-CD2vΔSP-TM、pGEX-4T-1-CD2vΔSP-TM、pMAL-C6T-CD2vΔSP-TM和pCold-TF-CD2vΔSP-TM重组质粒的PCR扩增产物；6：阴性对照。

图2 5种不同原核表达载体对CD2v重组蛋白表达的SDS-PAGE分析A～E.分别为pET-28a-CD2vΔSP-TM、pET32a-CD2vΔSP-TM、pGEX-4T-1-CD2vΔSP-TM、pMAL-C6T-CD2vΔSP-TM、pCold-TF-CD2vΔSP-TM重组质粒。M：蛋白质相对分子质量；1：未诱导菌体；2：诱导后菌体；3：诱导后菌体超声裂解上清液；4：诱导后菌体超声裂解沉淀。

图3 CD2v-TF重组蛋白纯化结果A. SDS-PAGE分析；B.蛋白质印迹法分析。M：蛋白质相对分子质量；1～3：用20 mmol/L咪唑洗脱的杂蛋白；4～6：用50 mmol/L咪唑洗脱的目的蛋白CD2v-TF；7：纯化的目的蛋白CD2v-TF；8：诱导后菌体；9：未诱导菌体（对照）。

图4 CD2v-His重组蛋白纯化结果A. SDS-PAGE分析；B.蛋白质印迹法分析。M：蛋白质相对分子质量；1、7：未诱导菌体（对照）；2、6：诱导后菌体；3：洗脱的杂蛋白；4：洗脱的目的蛋白CD2v-His；5：纯化的目的蛋白CD2v-His。

图5 利用间接ELISA方法检测2种CD2v重组蛋白的免疫反应性*表示与CD2v-His重组蛋白组相比在P＜0.05水平差异有统计学意义；n=3。

1	YOON K J, ZIMMERMAN J J, MORILLA A. Trends in Emerging Viral Infections of Swine[M]. New York, USA: John Wiley & Sons, 2008.
2	ZHOU X T, LI N, LUO Y Z, et al. Emergence of African swine fever in China, 2018[J]. Transboundary and Emerging Diseases, 2018, 65(6): 1482-1484. DOI: 10.1111/tbed.12989 doi: 10.1111/tbed.12989
3	KARGER A, PÉREZ-NÚÑEZ D, URQUIZA J, et al. An update on African swine fever virology[J]. Viruses, 2019, 11(9): 864. DOI: 10.3390/v11090864 doi: 10.3390/v11090864
4	WANG Y, KANG W F, YANG W P, et al. Structure of African swine fever virus and associated molecular mechanisms underlying infection and immunosuppression: a review[J]. Frontiers in Immunology, 2021, 12: 715582. DOI: 10.3389/fimmu.2021.715582 doi: 10.3389/fimmu.2021.715582
5	WANG N, ZHAO D M, WANG J L, et al. Architecture of African swine fever virus and implications for viral assembly[J]. Science, 2019, 366(6465): 640-644. DOI: 10.1126/science.aaz1439 doi: 10.1126/science.aaz1439
6	KAY-JACKSON P C, GOATLEY L C, COX L, et al. The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7[J]. Journal of General Virology, 2004, 85(Pt 1): 119-130. DOI: 10.1099/vir.0.19435-0 doi: 10.1099/vir.0.19435-0
7	何庆,邹亚文,王东亮,等.非洲猪瘟病毒跨膜蛋白CD2v的结构与功能研究进展[J].病毒学报,2021,37(5):1244-1251. DOI:10.13242/j.cnki.bingduxuebao.004023 HE Q, ZOU Y W, WANG D L, et al. Research advances in structure and function of transmembrane protein CD2v of African swine fever virus[J]. Chinese Journal of Virology, 2021, 37(5): 1244-1251. (in Chinese with English abstract) doi: 10.13242/j.cnki.bingduxuebao.004023
8	RUIZ-GONZALVO F, RODRÍGUEZ F, ESCRIBANO J M. Functional and immunological properties of the baculovirus-expressed hemagglutinin of African swine fever virus[J]. Virology, 1996, 218(1): 285-289. DOI: 10.1006/viro.1996.0193 doi: 10.1006/viro.1996.0193
9	JANCOVICH J K, CHAPMAN D, HANSEN D T, et al. Immunization of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African swine fever virus immunogenic and protective proteins[J]. Journal of Virology, 2018, 92(8): e02219-17. DOI: 10.1128/JVI.02219-17 doi: 10.1128/JVI.02219-17
10	RKOVÁ E, MÜLLER P, VOJTĚEK B. Protein expression and purification[J]. Klinicka Onkologie, 2014, 27(): 92-98. DOI: 10.14735/amko20141s92 doi: 10.14735/amko20141s92
11	李勇坤.非洲猪瘟病毒CD2v蛋白杆状病毒表达及其单克隆抗体制备[D].陕西,杨凌:西北农林科技大学,2022. DOI:10.17660/ejhs.2022/005 LI Y K. Expression of African swine fever CD2v protein by baculovirus system and preparation of monoclonal antibody[D]. Yangling, Shaanxi: Northwest A&F University, 2022. (in Chinese with English abstract) doi: 10.17660/ejhs.2022/005
12	LÜ C J, ZHAO Y, JIANG L L, et al. Development of a dual ELISA for the detection of CD2v-unexpressed lower-virulence mutational ASFV[J]. Life, 2021, 11(11): 1214. DOI: 10.3390/life11111214 doi: 10.3390/life11111214
13	蒋智勇,蔡汝健,李艳,等.基于CHO-K1细胞稳定表达的非洲猪瘟病毒CD2v蛋白的间接ELISA抗体检测方法的建立[J].中国兽医科学,2022,52(2):135-142. DOI:10.16656/j.issn.1673-4696.2022.0029 JIANG Z Y, CAI R J, LI Y, et al. Development of an indirect ELISA based on African swine fever virus CD2v protein stably expressed in CHO-K1 cells[J]. Chinese Veterinary Science, 2022, 52(2): 135-142. (in Chinese with English abstract) doi: 10.16656/j.issn.1673-4696.2022.0029
14	HUANG C J, LIN H, YANG X M. Industrial production of recombinant therapeutics in Escherichia coli and its recent advancements[J]. Journal of Industrial Microbiology & Bio-technology, 2012, 39(3): 383-399. DOI: 10.1007/s10295-011-1082-9 doi: 10.1007/s10295-011-1082-9
15	ASSENBERG R, WAN P T, GEISSE S, et al. Advances in recombinant protein expression for use in pharmaceutical research[J]. Current Opinion in Structural Biology, 2013, 23(3): 393-402. DOI: 10.1016/j.sbi.2013.03.008 doi: 10.1016/j.sbi.2013.03.008
16	ROSANO G L, CECCARELLI E A. Recombinant protein expression in Escherichia coli: advances and challenges[J]. Frontiers in Microbiology, 2014, 5: 172. DOI: 10.3389/fmicb.2014.00172 doi: 10.3389/fmicb.2014.00172
17	胡巧云,黄小久,唐小明,等.非洲猪瘟CD2v截短蛋白的原核表达及间接ELISA方法的建立[J].中国兽医学报,2022,42(6):1103-1108. DOI:10.16303/j.cnki.1005-4545.2022.06.01 HU Q Y, HUANG X J, TANG X M, et al. Prokaryotic expression of CD2v truncated protein of African swine fever virus and establishment of an indirect CD2v-ELISA[J]. Chinese Journal of Veterinary Science, 2022, 42(6): 1103-1108. (in Chinese with English abstract) doi: 10.16303/j.cnki.1005-4545.2022.06.01
18	任肖,吴竞,于海男,等.非洲猪瘟病毒Georgia 2007/1株CD2v蛋白的原核表达及多克隆抗体制备[J].中国畜牧兽医,2019,46(12):3698-3706. DOI:10.16431/j.cnki.1671-7236.2019.12.029 REN X, WU J, YU H N, et al. Prokaryotic expression and polyclonal antibody preparation of African swine fever virus Georgia 2007/1 strain CD2v protein[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(12): 3698-3706. (in Chinese with English abstract) doi: 10.16431/j.cnki.1671-7236.2019.12.029
19	于浩洋,吴绍强,王彩霞,等.非洲猪瘟CD2v胞外区基因片段的真核表达及其多克隆抗体的制备[J].中国兽医科学,2021,51(1):45-52. DOI:10.16656/j.issn.1673-4696.2021.0012 YU H Y, WU S Q, WANG C X, et al. Eukaryotic expression the CD2v extracellular gene fragment of African swine fever virus and characterization of its polyclonal antibodies[J]. Chinese Veterinary Science, 2021, 51(1): 45-52. (in Chinese with English abstract) doi: 10.16656/j.issn.1673-4696.2021.0012
20	钟秋萍,于婉琪,薄宗义,等.非洲猪瘟病毒外膜蛋白CD2v的原核表达及其免疫特性的研究[J].中国兽医科学,2020,50(6):689-695. DOI:10.16656/j.issn.1673-4696.2020.0089 ZHONG Q P, YU W Q, BO Z Y, et al. Prokaryotic expression and immunological characterization of African swine fever virus out membrane CD2v protein[J]. Chinese Veterinary Science, 2020, 50(6): 689-695. (in Chinese with English abstract) doi: 10.16656/j.issn.1673-4696.2020.0089
21	COSTA S, ALMEIDA A, CASTRO A, et al. Fusion tags for protein solubility, purification and immunogenicity in Escherichia coli: the novel Fh8 system[J]. Frontiers in Microbiology, 2014, 5: 63. DOI: 10.3389/fmicb.2014.00063 doi: 10.3389/fmicb.2014.00063
22	周星怡,牛冬,李玉峰.非洲猪瘟病原学与传播特点及防控方法的研究进展[J].中国兽医科学,2022,52(9):1172-1180. DOI:10.16656/j.issn.1673-4696.2022.0138 ZHOU X Y, NIU D, LI Y F. Advancement in etiology, transmission and prevention and control of African swine fever[J]. Chinese Veterinary Science, 2022, 52(9): 1172-1180. (in Chinese with English abstract) doi: 10.16656/j.issn.1673-4696.2022.0138
23	GLADUE D P, O’DONNELL V, RAMIREZ-MEDINA E, et al. Deletion of CD2-like (CD2v) and C-type lectin-like (EP153R) genes from African swine fever virus Georgia-∆9GL abrogates its effectiveness as an experimental vaccine[J]. Viruses, 2020, 12(10): 1185. DOI: 10.3390/v12101185 doi: 10.3390/v12101185
24	MONTEAGUDO P L, LACASTA A, LÓPEZ E, et al. BA71ΔCD2: a new recombinant live attenuated African swine fever virus with cross-protective capabilities[J]. Journal of Virology, 2017, 91(21): e01058-17. DOI: 10.1128/JVI.01058-17 doi: 10.1128/JVI.01058-17
25	FENG Z H, CHEN J H, LIANG W W, et al. The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice[J]. Virology Journal, 2020, 17(1): 180. DOI: 10.1186/s12985-020-01450-7 doi: 10.1186/s12985-020-01450-7
26	TSUMOTO K, EJIMA D, KUMAGAI I, et al. Practical considerations in refolding proteins from inclusion bodies[J]. Protein Expression and Purification, 2003, 28(1): 1-8. DOI: 10.1016/s1046-5928(02)00641-1 doi: 10.1016/s1046-5928(02)00641-1
27	NARAYAN G, AGRAWAL A, JOSHI N, et al. Protein production and purification of a codon-optimized human NGN3 transcription factor from E. coli [J]. The Protein Journal, 2021, 40(6): 891-906. DOI: 10.1007/s10930-021-10020-x doi: 10.1007/s10930-021-10020-x
28	SMITH D B, JOHNSON K S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase[J]. Gene, 1988, 67(1): 31-40. DOI: 10.1016/0378-1119(88)90005-4 doi: 10.1016/0378-1119(88)90005-4
29	房康.通过使用MBP标签高水平原核表达纯化死亡结构域家族蛋白及炎症小体NLRP1与ASC的结构生物学研究[D].安徽,合肥:中国科学技术大学,2018. DOI:10.7754/clin.lab.2017.170922 FANG K. High-level prokaryotic expression and purification of death domain superfamily with MBP tag and structural basis of NLRP1 and ASC inflammasome[D]. Hefei, Anhui: University of Science and Technology of China, 2018. (in Chinese with English abstract) doi: 10.7754/clin.lab.2017.170922
30	尹鑫,刘澜澜,贾莹,等.鸡氨肽酶N的高效可溶性表达及生物学功能分析[J].生物工程学报,2010,26(4):470-475. DOI:10.13345/j.cjb.2010.04.014 YIN X, LIU L L, JIA Y, et al. Expression and biological function analysis of chicken aminopeptidase N[J]. Chinese Journal of Biotechnology, 2010, 26(4): 470-475. (in Chinese with English abstract) doi: 10.13345/j.cjb.2010.04.014

[1]	戴远兴,郭留明,何婧,沈峥嵘,耿艳飞,吕明芳,袁正杰,李静,张恒木. 中国小麦花叶病毒富含半胱氨酸蛋白多克隆抗体的制备与应用[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(5): 677-686.
[2]	王子仪,金子安,卢辰赫,乔治,王圣文,颜焰,周继勇,郑肖娟. 猫冠状病毒非结构蛋白3的多克隆抗体制备及亚细胞定位[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(5): 744-754.
[3]	张丹,卢舒婷,卢辰赫,王子仪,徐丽华,陈伊玄,王圣文,金子安,倪成章,周继勇,郑肖娟. 猫冠状病毒核衣壳蛋白的可溶性表达与多克隆抗体反应性鉴定[J]. 浙江大学学报（农业与生命科学版）, 2023, 49(3): 424-434.
[4]	张彤,汪一萍,葛洋,NTIRI Eric,周文武. 结球甘蓝α-法呢烯合成酶基因的鉴定与功能分析[J]. 浙江大学学报（农业与生命科学版）, 2021, 47(2): 182-192.
[5]	阳毅敏, 潘灵韬, 庄浩瀚, 孙洪超, 杨怡, 陈学秋, 杜爱芳. 微小隐孢子虫黏蛋白CGD5_2060 原核表达及其黏附功能[J]. 浙江大学学报（农业与生命科学版）, 2018, 44(2): 230-236.
[6]	王静,刘丽,张志明,赵茂俊,潘光堂. 玉米病程相关蛋白1基因的克隆与表达分析[J]. 浙江大学学报(农业与生命科学版), 2012, 38(1): 35-42.
[7]	丁美会,金凤,沈立荣,陈正贤. 中华蜜蜂王浆抗菌肽Acc- royalisin基因原核表达及多克隆抗体制备[J]. 浙江大学学报(农业与生命科学版), 2010, 36(6): 609-614.
[8]	戈林泉, 周国鑫, 王祺, 祝树德, 娄永根. 水稻β-石竹烯合成酶基因OsCAS的克隆鉴定、原核表达及其遗传转化[J]. 浙江大学学报(农业与生命科学版), 2009, 35(4): 365-371.
[9]	谢芳, 韩文瑜, 雷连成, 韩俊友, 胡博. 牛蛙皮肤抗菌肽Ranalexin 基因的原核表达及其抑菌活性[J]. 浙江大学学报(农业与生命科学版), 2009, 35(1): 27-32.
[10]	于翠,邓凤林,杨翠云,吴建祥. 番茄斑萎病毒外壳蛋白原核表达及Dotblot ELISA检测方法的建立[J]. 浙江大学学报(农业与生命科学版), 2008, 34(6): 597-601.
[11]	姜磊　吴建祥　周雪平. 与烟草曲茎病毒相伴随的DNAβ的βC1基因原核表达及蛋白的单抗制备[J]. 浙江大学学报(农业与生命科学版), 2008, 34(2): 132-136.
[12]	黄俊　张灵霞　胡松年于军. LZ-8蛋白的原核密码子优化表达及免疫调节功能的初步检测[J]. 浙江大学学报(农业与生命科学版), 2008, 34(1): 7-12.
[13]	潘滨　吴建祥　李桂新等. 烟草曲茎病毒复制相关蛋白基因原核表达条件优化[J]. 浙江大学学报(农业与生命科学版), 2007, 33(1): 24-28.
[14]	金伟波　吴方丽　郭蔼光. 小麦高分子量谷蛋白14亚基基因的PCR体系及原核表达研究[J]. 浙江大学学报(农业与生命科学版), 2007, 33(1): 45-50.
[15]	于涟　谢荣辉　卢觅佳　黄耀伟　李建荣. 鸡白细胞介素2原核表达及多克隆抗体的制备[J]. 浙江大学学报(农业与生命科学版), 2003, 29(1): 10-13.

Viewed

Full text

Abstract

Cited

Shared

Discussed