木尔坦棉花曲叶病毒编码4个蛋白的抗血清制备

林丹; 林文忠; 尚鹏祥; 林文武; 丁新伦; 吴祖建; 张洁

doi:10.19303/j.issn.1008-0384.2021.11.014

木尔坦棉花曲叶病毒编码4个蛋白的抗血清制备

doi: 10.19303/j.issn.1008-0384.2021.11.014

福建农林大学植物病毒研究所/福建农林大学闽台作物有害生物生态防控国家重点实验室, 福建　福州　350002

基金项目: 福建省自然科学基金项目（2019J01656）；福建农林大学科技创新专项（CXZX2020017A、CXZX2019018G、CXZX2018099）

详细信息

作者简介:
林丹（1993−），女，硕士生，研究方向：植物病理学（E-mail:964107593@qq.com）

通讯作者:
张洁（1983−），女，博士，讲师，研究方向：植物病理学（E-mail:Jiezhang3553@163.com）

中图分类号: S432.1
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出版历程
- 收稿日期: 2021-08-16
- 修回日期: 2021-09-28
- 网络出版日期: 2021-12-30
- 刊出日期: 2021-11-28

Preparation of Antisera Against Four Proteins Encoded by Cotton Leaf Curl Multan Virus

Institute of Plant Virology, Fujian Agriculture and Forestry University/State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fuzhou, Fujian　350002, China

摘要

摘要: 目的对入侵我国的木尔坦棉花曲叶病毒（Cotton leaf curl Multan virus，CLCuMuV）编码的V1、V2、C1和C4蛋白抗血清进行制备，为CLCuMuV的检验检疫及其致病机制的研究奠定基础。方法利用原核表达技术对V1、V2、C1和C4蛋白进行IPTG诱导表达，western blot分析表达蛋白的特异性后，V1、V2、C1蛋白免疫新西兰大白兔，C4蛋白免疫小白鼠制备抗血清，利用western blot对抗血清的特异性进行分析。结果通过PCR分别扩增V1（768 bp）、V2（363 bp）、C1（1 089 bp）和C4（300 bp）目的基因片段，分别连接于原核表达载体后，通过SDS-PAGE和western blot分析分别在34、16、44 、13 kDa处存在与预期蛋白大小一致的条带，表明该4种蛋白均成功表达。Western blot分析发现获得的4种抗血清均具有较高的特异性。结论成功制备了CLCuMuV V1、V2、C1和C4蛋白的抗血清，可用于后续建立CLCuMuV的检疫方法及研究其致病机制。
- 棉花 /
- 木尔坦棉花曲叶病毒 /
- 原核表达 /
- 免疫印迹 /
- 抗血清
Abstract: Objective Antisera of V1, V2, C1, and C4 proteins encoded by the invasive cotton leaf curl Multan virus (CLCuMuV) were prepared for quarantine at entry ports and study on the pathogenic mechanism of the virus. Method Using a prokaryotic expression technology and IPTG induction, V1, V2, C1, and C4 were expressed and the specificity analyzed by western blot. Separately, New Zealand white rabbits were immunized with V1, V2, and C1, while mice with C4 to prepare the antisera. Specificity of the antisera was subsequently verified by western blot. Result The target gene fragments of V1 (768 bp), V2 (363 bp), C1 (1 089 bp), and C4 (300 bp) were amplified by PCR and connected to the respective prokaryotic expression vectors. The SDS-PAGE and western blot showed that the 4 proteins were successfully expressed at 34, 16, 44, and 13 kDa locations with high specificity on the respective antiserum. Conclusion The antisera of CLCuMuV V1, V2, C1, and C4 were successfully prepared for quarantine inspection and study on the pathogenic mechanism of the invasive virus.
- cotton /
- cotton leaf curl Multan virus /
- prokaryotic expression /
- western blot /
- antiserum

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图 1 目的基因的PCR扩增

注：M ，DNA Marker；1~2， V1基因；3~4，V2基因；5~6：C4基因；7~8，C1基因。

Figure 1. PCR amplification of target genes

Note: M, DNA marker; 1-2, V1 gene; 3-4, V2 gene; 5-6, C4 gene; 7-8, C1 gene.　　　　

下载: 全尺寸图片幻灯片

图 2 目的蛋白的SDS-PAGE分析

注：①M：蛋白Marker；1～2：V1蛋白；3～4：V2 蛋白；5～8：C1蛋白；9～11：C4 蛋白。②1、3、5～7、9～10：IPTG诱导表达；2、4、8、11：未加IPTG诱导表达。

Figure 2. SDS-PAGE analysis on target proteins

Note: ① M: Protein marker; 1-2: V1 protein; 3-4: V2 protein; 5-8: C1 protein; 9-11: C4 protein. ② 1, 3, 5-7, 9-10: IPTG induced expressions; 2, 4, 8, 11: non-IPTG induced expressions.

下载: 全尺寸图片幻灯片

图 3 目的蛋白的Western blot分析

注：①M：蛋白Marker；1～2：V1蛋白；3～6：V2蛋白；7～8：C1蛋白；9～11：C4蛋白。②1、3～5、7、9～10：IPTG诱导表达；2、6、8、11：未加IPTG诱导表达。

Figure 3. Western blot analysis on target proteins

Note: ① M: protein marker; 1-2: V1 protein; 3-6: V2 protein; 7-8: C1 protein; 9-11: C4 protein. ② 1, 3-5, 7, 9-10: IPTG induced expressions; 2, 6, 8, 11: non-IPTG induced expressions.

下载: 全尺寸图片幻灯片

图 4 Western blot分析血清的特异性

注：①M：蛋白Marker；1~2：V1 抗血清；3~4：V2抗血清；5~6：C1抗血清；7~8：C4抗血清；②1、3、5、7：感病本氏烟；2、4、6、8：健康本氏烟。

Figure 4. Specificity of antisera as detected by western blot

Note: ①M: protein marker; 1-2: V1 antiserum; 3-4: V2 antiserum; 5-6: C1 antiserum; 7-8: C4 antiserum. ②1, 3, 5, 7: infected N. benthamiana; 2, 4, 6, 8: healthy N.benthamiana.

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表 1 引物名称及其序列

Table 1. Names and sequences of primers

引物名称 Primer name	序列^a(5′-3′) Sequence^a(5′-3′)
gateV1-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGAAGCGAGCTGCAG
gateV1-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCATTCGTTACAGAGTCATAAAAATATATCC
gateV2-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTGGGATCCATTGTTAAACG
gateV2-R	GGGACCACTTTGTACAAGAAAGCTGGGTCCAACCCTTTGGAACATCCG
C1-F	ATGGCTCCCCCCAAACGTTTTAAAATAC
C1-R	AGATCTACTCTCCTCCTCCTGTTGCG
gateC4-F	GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGAGCCCTCATCTCC
gateC4-R	GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTCCTTAATGACTCTAAGAGC
注：下划线处代表重组位点 Note: Recombination sites are underlined.

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