马铃薯卷叶病毒与S病毒重组双CP多克隆抗体的制备

doi:10.7668/hbnxb.20193055

摘要/Abstract

摘要：

为了制备出马铃薯卷叶病毒(PLRV)与S病毒(PVS)重组双CP多克隆抗体并用于PLRV与PVS这2种病毒的间接与DAS-ELISA检测,构建了PLRV与PVS融合双CP基因的原核表达载体pET22b-LRCP/SCP,用超声破碎法代替溶菌酶法裂解重组菌BL21(pET22b-LRCP/SCP)并提取菌体蛋白,用镍离子亲和层析纯化获得了分子质量为51.2 ku的高纯度的重组双CP。用该重组双CP作抗原免疫家兔获得了高效价(1∶128 k)抗血清。纯化的重组双CP多克隆抗体与PLRV或PVS阳性标准物特异性结合,与另外4种马铃薯主要病毒(PVX、PVY、PVA与PVM)无交叉反应。间接ELISA反应中,稀释度为1∶3 200的重组双CP多克隆抗体与PLRV或PVS阳性物都呈现阳性反应;在DAS-ELISA反应中,稀释度均为1∶100的重组双CP多克隆抗体及其碱性磷酸酶标记物与PLRV或PVS的阳性标准物也分别呈现阳性反应。结果表明,制备的重组双CP抗体既可用于PLRV与PVS这2种病毒的间接ELISA检测,也可用于DAS-ELISA检测。

关键词: 马铃薯卷叶病毒, S病毒, 重组双CP, 多克隆抗体, 间接ELISA, DAS-ELISA

Abstract:

The purpose was to prepare polyclonal antibody against the recombinant double CP of Potato leafroll virus (PLRV)and Potato virus S (PVS),and apply the polyclonal antibody to indirect ELISA and DAS-ELISA detections of PLRV and PVS.Prokaryotic expression vector pET22b-LRCP/SCP of the fused double CP gene of PLRV and PVS was constructed.After replacement of lysozyme treatment by ultrasonic disruption,inclusion body protein was extracted from the recombinant strain BL21(pET22b-LRCP/SCP),the target protein(recombinant double CP)was purified by nickel ion affinity chromatography and high-purity target protein of 51.2 ku was obtained.The high-purity recombinant double CP was used as antigen to immunize rabbits to prepare an antiserum with a titer of 1∶128 k.Specific reactions were respectively observed between the purified polyclonal antibody (IgG) against the recombinant double CP and the positive standard of PLRV or PVS and no cross-reaction was found between the purified IgG and other four potato major viruses (PVX, PVY, PVA and PVM). The purified IgG against the recombinant CP with the diluted concentration of 1∶3 200 still positively reacted with PLRV or PVS in indirect ELISA detection.The purified IgG and the alkaline phosphatase-conjugated IgG both with the diluted contraction of 1∶100 also positively reacted with PLRV or PVS positive standard in DAS-ELISA detection.The results showed that one type of the prepared IgG against the recombinant double CP could detect two viruses of PLRV and PVS by DAS-ELISA or indirect ELISA.

Key words: Potato leafroll virus, Potato virus S, Recombinant double CP, Polyclonal antibody, Indirect ELISA, DAS-ELISA

吉祥, 宋志成, 韦晓玲, 杨煜, 隋炯明, 郭宝太. 马铃薯卷叶病毒与S病毒重组双CP多克隆抗体的制备[J]. 华北农学报, 2022, 37(4): 212-219. doi: 10.7668/hbnxb.20193055.

JI Xiang, SONG Zhicheng, WEI Xiaoling, YANG Yu, SUI Jiongming, GUO Baotai. Preparation of Polyclonal Antibody Against Recombinant Double CP of Potato leafroll virus and Potato virus S[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4): 212-219. doi: 10.7668/hbnxb.20193055.

http://www.hbnxb.net/CN/Y2022/V37/I4/212

图/表 10

图1 pET22b-LRCP/SCP原核表达载体的酶切鉴定 M.DNA Marker 15000;1.表达载体pET22b-LRCP/SCP;2.质粒pET22b-LRCP/SCP+HindⅢ(+);3-4.质粒pET22b-LRCP/SCP+Hind Ⅲ+Xho Ⅰ。

Fig.1 Restriction identification of prokaryotic expression vector pET22b-LRCP/SCP M.DNA Marker 15000;1.Expression vector pET22b-LRCP/SCP;2.Plasmid pET22b-LRCP/SCP+Hind Ⅲ(+);3-4.Plasmid pET22b-LRCP/SCP+Hind Ⅲ+XhoⅠ.

图2 PLRV与PVS重组双CP的诱导表达、溶菌酶法提取及纯化 M.蛋白分子量标准;1.未诱导的菌体;2.诱导后的菌体;3.包涵体;4.纯化的重组双CP。

Fig.2 Expression induction,lysozyme lysis extraction and purification of recombinant double CP of PLRV and PVS M.Protein Marker;1.Uninduced bacterial cells;2.Bacterial cells induced with IPTG;3.Inclusion body;4.Purified recombinant double CP.

图3 重组双CP的诱导表达、超声破碎法提取及纯化 M.蛋白分子量标准;1.未诱导的菌体;2.诱导后的菌体;3.包涵体蛋白溶解液;4-5.纯化的重组双CP。

Fig.3 Expression induction,ultrasonic disruption extraction and purification of recombinant double CP M.Protein Marker;1.Uninduced bacterial cell;2.Bacterial cell induced with IPTG;3.Dissolved inclusion body protein;4-5.Purified recombinant double CP.

图4 重组双CP多克隆抗体的分离与纯化 M.蛋白分子量标准;1.重组双CP抗血清;2.分离的抗体;3. 纯化的抗体。

Fig.4 Isolation and purification of IgG against recombinant double CP M.Protein Marker;1.Antiserum against recombinant double CP; 2.Isolated IgG;3.Purified IgG.

表1 PLRV与PVS重组双CP多克隆抗体(IgG)活性的间接ELISA测定(PLRV阳性物,

O D ¯ 450

)

Tab.1 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PLRV positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶3 200	1∶6 400	1∶12 800	阴性对照 Negative control
0.071	0.734	0.679	0.626	0.529	0.439	0.300	0.176	0.121

表1 PLRV与PVS重组双CP多克隆抗体(IgG)活性的间接ELISA测定(PLRV阳性物,

O D ¯ 450

)

Tab.1 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PLRV positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶3 200	1∶6 400	1∶12 800	阴性对照 Negative control
0.071	0.734	0.679	0.626	0.529	0.439	0.300	0.176	0.121

表2 PLRV与PVS重组双CP多克隆抗体(IgG)活性的间接ELISA测定(PVS阳性物,

O D ¯ 450

)

Tab.2 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PVS positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶ 3 200	1∶ 6 400	1∶12 800	阴性对照 Negative control
0.071	0.632	0.583	0.497	0.329	0.270	0.206	0.174	0.093

表2 PLRV与PVS重组双CP多克隆抗体(IgG)活性的间接ELISA测定(PVS阳性物,

O D ¯ 450

)

Tab.2 Activity determination of IgG against double CP of PLRV and PVS by indirect ELISA(PVS positive standard,

O D ¯ 450

)

空白对照 Blank control	重组双CP抗体(IgG)的稀释度 Diluted concentration of IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶200	1∶400	1∶800	1∶1 600	1∶ 3 200	1∶ 6 400	1∶12 800	阴性对照 Negative control
0.071	0.632	0.583	0.497	0.329	0.270	0.206	0.174	0.093

图5 重组双CP多克隆抗体特异性的间接ELISA测定

Fig.5 Specificity detection of IgG against double CP by indirect ELISA

表3 PLRV与PVS重组双CP抗体碱性磷酸酶标记物活性的直接ELISA测定(PLRV阳性物,

O D ¯ 405

)

Tab.3 Activity determination AP-conjugated IgG of double CP of PLRV and PVS by direct ELISA(PLRV positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.077	0.500	0.387	0.268	0.183	0.145	0.120	0.108	0.091

表3 PLRV与PVS重组双CP抗体碱性磷酸酶标记物活性的直接ELISA测定(PLRV阳性物,

O D ¯ 405

)

Tab.3 Activity determination AP-conjugated IgG of double CP of PLRV and PVS by direct ELISA(PLRV positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against the double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.077	0.500	0.387	0.268	0.183	0.145	0.120	0.108	0.091

表4 PLRV与PVS重组双CP抗体碱性磷酸酶标记物活性的直接ELISA测定(PVS阳性物,

O D ¯ 405

)

Tab.4 Activity determination of AP-conjugated IgG against double CP of PLRV and PVS by direct ELISA(PVS positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.071	0.555	0.430	0.285	0.202	0.152	0.124	0.117	0.098

表4 PLRV与PVS重组双CP抗体碱性磷酸酶标记物活性的直接ELISA测定(PVS阳性物,

O D ¯ 405

)

Tab.4 Activity determination of AP-conjugated IgG against double CP of PLRV and PVS by direct ELISA(PVS positive standard,

O D ¯ 405

)

空白对照 Blank control	重组双CP抗体(IgG)AP标记物的稀释度 Diluted concentration of AP-conjugated IgG against double CP							阴性对照 Negative control
空白对照 Blank control	1∶ 200	1∶ 400	1∶ 800	1∶ 1 600	1∶ 3 200	1∶ 6 400	1∶ 12 800	阴性对照 Negative control
0.071	0.555	0.430	0.285	0.202	0.152	0.124	0.117	0.098

表5 PLRV与PVS阳性标准物的DAS-ELISA检测(

O D ¯ 405

)

Tab.5 DAS-ELISA detection of PLRV and PVS positive standards(

O D ¯ 405

)

重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	抗原类型Antigen type
重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	双重组CP Recombinant double CP	PVS阳性标准物 PVS positive standard	PLRV阳性标准物 PLRV positive standard
IgG(1∶100)	0.082	0.096	0.490	0.262	0.271
IgG(1∶200)	0.078	0.089	0.433	0.207	0.203
IgG(1∶300)	0.081	0.091	0.395	0.166	0.166

表5 PLRV与PVS阳性标准物的DAS-ELISA检测(

O D ¯ 405

)

Tab.5 DAS-ELISA detection of PLRV and PVS positive standards(

O D ¯ 405

)

重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	抗原类型Antigen type
重组双CP抗体 IgG against double CP	空白对照 Blank control	阴性对照 Negative control	双重组CP Recombinant double CP	PVS阳性标准物 PVS positive standard	PLRV阳性标准物 PLRV positive standard
IgG(1∶100)	0.082	0.096	0.490	0.262	0.271
IgG(1∶200)	0.078	0.089	0.433	0.207	0.203
IgG(1∶300)	0.081	0.091	0.395	0.166	0.166

参考文献 24

[1]	Gong H L, Igiraneza C, Dusengemungu L. Major in vitro techniques for potato virus elimination and post eradication detection methods.A review[J]. American Journal of Potato Research, 2019, 96(4):379-389. doi: 10.1007/s12230-019-09720-z. doi: 10.1007/s12230-019-09720-z URL
[2]	Wright N S. The effect of separate infections by Potato viruses X and S on Netted Gem potato[J]. American Potato Journal, 1977, 54(4):147-149. doi: 10.1007/BF02852870. doi: 10.1007/BF02852870 URL
[3]	白艳菊, 李学湛, 吕典秋, 何云霞, 张儒喜, 朱财. 应用DAS-ELISA法同时检测多种马铃薯病毒[J]. 中国马铃薯, 2000, 14(3):143-144.
	Bai Y J, Li X Z, Lü D Q, He Y X, Zhang R X, Zhu C. Simultaneous detection of multiple potato viruses by DAS-ELISA[J]. Chinese Potato Journal, 2000, 14(3):143-144.
[4]	Plchova H, Moravec T, Dedic P, Cerovska N. Expression of recombinant Potato leafroll virus structural and non-structural proteins for antibody production[J]. Journal of Phytopathology, 2011, 159(2):130-132. doi: 10.1111/j.1439-0434.2010.01740.x. doi: 10.1111/j.1439-0434.2010.01740.x URL
[5]	王韬远, 张春雨, 王忠伟, 李闯, 张胜利, 李建平, 李小宇, 王永志. 马铃薯卷叶病毒衣壳蛋白原核表达及多克隆抗体的制备[J]. 东北农业科学, 2021, 46(4):20-23. doi: 10.16423/j.cnki.1003-8701.2021.04.005. doi: 10.16423/j.cnki.1003-8701.2021.04.005
	Wang T Y, Zhang C Y, Wang Z W, Li C, Zhang S L, Li J P, Li X Y, Wang Y Z. Prokaryotic expression and polyclonal antibody preparation of the Potato leafroll virus coat protein[J]. Journal of Northeast Agricultural Sciences, 2021, 46(4):20-23.
[6]	隋炯明, 何心凤, 郭真, 李广存, 王晶珊, 郭宝太. 马铃薯卷叶病毒缺失突变CP基因的原核表达及抗血清的制备[J]. 园艺学报, 2012, 39(10):1949-1957. doi: 10.16420/j.issn.0513-353x.2012.10.002. doi: 10.16420/j.issn.0513-353x.2012.10.002
	Sui J M, He X F, Guo Z, Li G C, Wang J S, Guo B T. Prokaryotic expression of deletion mutation CP gene of Potato leafroll virus and preparation of antiserum[J]. Acta Horticulturae Sinica, 2012, 39(10):1949-1957.
[7]	李楠楠, 左玉玲, 隋炯明, 盖树鹏, 樊连梅, 李广存, 郭宝太. 重组CP多克隆抗体在马铃薯卷叶病毒DAS-ELISA检测中的应用[J]. 华北农学报, 2011, 26(6):85-88. doi: 10.7668/hbnxb.2011.06.016. doi: 10.7668/hbnxb.2011.06.016
	Li N N, Zuo Y L, Sui J M, Gai S P, Fan L M, Li G C, Guo B T. Application of polyclonal antibody against the recombinant CP to the DAS-ELISA detection of Potato leafroll virus[J]. Acta Agriculturae Boreali-Sinica, 2011, 26(6):85-88.
[8]	李广存, 杨煜, 王秀丽, 杨元军, 毕玉平. 马铃薯S病毒外壳蛋白基因的克隆及其在大肠杆菌中的表达[J]. 园艺学报, 2004, 31(4):517-519. doi: 10.16420/j.issn.0513-353x.2004.04.028. doi: 10.16420/j.issn.0513-353x.2004.04.028
	Li G C, Yang Y, Wang X L, Yang Y J, Bi Y P. Cloning of Potato virus S coat protein gene and its expression in E.coli JM109[J]. Acta Horticulturae Sinica, 2004, 31(4):517-519.
[9]	宋志成, 费新敏, 杨煜, 乔利仙, 王晶珊, 李广存, 郭宝太. 马铃薯A病毒重组CP多克隆抗体的制备及其在DAS-ELISA检测中的应用[J]. 华北农学报, 2017, 32(1):41-46. doi: 10.7668/hbnxb.2017.01.007. doi: 10.7668/hbnxb.2017.01.007
	Song Z C, Fei X M, Yang Y, Qiao L X, Wang J S, Li G C, Guo B T. Preparation of polyclonal antibody against recombinant CP of Potato virus A and its application to DAS-ELISA detection[J]. Acta Agriculturae Boreali-Sinica, 2017, 32(1):41-46.
[10]	Balogun O S, Xu L X, Teraoka T, Hosokawa D. Effects of single and double infections with Potato virus X and Tobacco mosaic virus on disease development,plant growth,and virus accumulation in tomato[J]. Fitopatologia Brasileira, 2002, 27(3):241-248. doi: 10.1590/s0100-41582002000300001. doi: 10.1590/s0100-41582002000300001 URL
[11]	Jeevalatha A, Kaundal P, Sharma N N, Thakur P, Chakrabarti S K, Singh B P. Expression of coat protein of an ordinary strain of Potato virus Y in Escherichia coli and production of polyclonal antibodies for diagnosis[J]. Journal of Phytopathology, 2013, 161(9):671-674. doi: 10.1111/jph.12113. doi: 10.1111/jph.12113 URL
[12]	魏旭言, 白艳菊, 张威, 高艳玲, 徐丽萍, 罗秋香. 马铃薯A病毒的研究进展[J]. 中国马铃薯, 2021, 35(1):75-80. doi: 10.19918/j.cnki.1672-3635.2021.01.011. doi: 10.19918/j.cnki.1672-3635.2021.01.011
	Wei X Y, Bai Y J, Zhang W, Gao Y L, Xu L P, Luo Q X. Research progress in Potato virus A[J]. Chinese Potato Journal, 2021, 35(1):75-80.
[13]	张威, 魏旭言, 高艳玲, 范国权, 邱彩玲, 孙旭红, 武新娟, 张俐俐, 白艳菊. 马铃薯A病毒不同分离物的侵染鉴定及致病力分析[J]. 中国马铃薯, 2021, 35(6):568-574. doi: 10.19918/j.cnki.1672-3635.2021.06.012. doi: 10.19918/j.cnki.1672-3635.2021.06.012
	Zhang W, Wei X Y, Gao Y L, Fan G Q, Qiu C L, Sun X H, Wu X J, Zhang L L, Bai Y J. Infection identification and pathogenicity analysis of different Potato virus A isolates[J]. Chinese Potato Journal, 2021, 35(6):568-574.
[14]	He Z, Gan H F, Liang X Y. Analysis of synonymous codon usage bias in Potato virus M and its adaption to hosts[J]. Viruses, 2019, 11(8):752. doi: 10.3390/v11080752. doi: 10.3390/v11080752 URL
[15]	Su X X, Wu K, Zhang L Z, Rahman M S, Zheng K Y, Li T, Zhang Z K, Dong J H. Complete genome sequence of a new isolate of Potato virus M in Yunnan,China[J]. Archives of Virology, 2017, 162(8):2485-2488. doi: 10.1007/s00705-017-3380-7. doi: 10.1007/s00705-017-3380-7 URL
[16]	Cerovská N, Moravec T, Velemínský J. Polyclonal antibodies to a recombinant coat protein of Potato virus A[J]. Acta Virologica, 2002, 46(3):147-151. pmid: 12580376
[17]	张春雨, 李小宇, 万千, 夏黎明, 王忠伟, 王韬远, 王永志. 马铃薯M病毒衣壳蛋白原核表达和多克隆抗体制备[J]. 东北农业科学, 2017, 42(3):27-30. doi: 10.16423/j.cnki.1003-8701.2017.03.006. doi: 10.16423/j.cnki.1003-8701.2017.03.006
	Zhang C Y, Li X Y, Wan Q, Xia L M, Wang Z W, Wang T Y, Wang Y Z. Prokaryotic expression and polyclonal antibody preparation of the Potato virus M coat protein[J]. Journal of Northeast Agricultural Sciences, 2017, 42(3):27-30.
[18]	Kapoor R, Mandal B, Paul P K, Chigurupati P, Jain R K. Production of cocktail of polyclonal antibodies using bacterial expressed recombinant protein for multiple virus detection[J]. Journal of Virological Methods, 2014, 196:7-14. doi: 10.1016/j.jviromet.2013.09.016. doi: 10.1016/j.jviromet.2013.09.016 URL
[19]	Kapoor R, Mandal B, Paul P K, Jain R K. Simultaneous detection of Potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli[J]. Journal of Plant Biochemistry and Biotechnology, 2014, 23(3):332-335. doi: 10.1007/s13562-013-0251-5. doi: 10.1007/s13562-013-0251-5 URL
[20]	Zhang Y, Gao Y L, He W Q, Wang Y Q, Qian Y J, Zhou X P, Wu J X. Monoclonal antibody-based serological detection of Potato virus M in potato plants and tubers[J]. Journal of Integrative Agriculture, 2020, 19(5):1283-1291. doi: 10.1016/S2095-3119(19)62755-3. doi: 10.1016/S2095-3119(19)62755-3 URL
[21]	Song G, Wu J Y, Xie Y, Liu Y, Qian Y J, Zhou X P, Wu J X. Monoclonal antibody-based serological assays for detection of Potato virus S in potato plants[J]. Journal of Zhejiang University Science B, 2017, 18(12):1075-1082. doi: 10.1631/jzus.B1600561. doi: 10.1631/jzus.B1600561 URL
[22]	Rettcher S, Jungk F, Kühn C, Krause H J, Nölke G, Commandeur U, Fischer R, Schillberg S, Schröper F. Simple and portable magnetic immunoassay for rapid detection and sensitive quantification of plant viruses[J]. Applied and Environmental Microbiology, 2015, 81(9):3039-3048. doi: 10.1128/AEM.03667-14. doi: 10.1128/AEM.03667-14 URL
[23]	Panferova N A, Panferov V G, Safenkova I V, Varitsev Y A, Zherdev A V, Dzantiev B B. Development of enzyme-linked immunosorbent assay with tiramine amplification for the detection of Potato virus X[J]. Applied Biochemistry and Microbiology, 2019, 55(4):434-440. doi: 10.1134/s0003683819040136. doi: 10.1134/S0003683819040136
[24]	梁雨欣, 吴建祥, 李小宇, 张春雨, 侯吉超, 周雪平, 王永志. 马铃薯Y病毒衣壳蛋白抗原表位分析及其快速ELISA检测方法的建立[J]. 中国农业科学, 2021, 54(6):1154-1162. doi: 10.3864/j.issn.0578-1752.2021.06.007. doi: 10.3864/j.issn.0578-1752.2021.06.007
	Liang Y X, Wu J X, Li X Y, Zhang C Y, Hou J C, Zhou X P, Wang Y Z. Mapping of epitopes and establishment of rapid DAS-ELISA for Potato virus Y coat protein[J]. Scientia Agricultura Sinica, 2021, 54(6):1154-1162.

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