感染浙贝母的贝母病毒Y的CP基因原核表达及抗体制备

Prokaryotic expression of CP gene of Fritillary virus Y infecting Thunberg fritillary and antiserum preparation.

作者信息

Wei Chuan-Bao, Wei Yang-Yang, Yang Yu, Liu Shi-Liang, Hu Hao-Yu, He Yue

机构信息

Western Anhui University, Anhui Provincial Laboratory of Biomimetic Sensor and Detecting Technology, Liu'an 237012, China.

出版信息

Zhong Yao Cai. 2011 Oct;34(10):1498-502.

PMID:22372135

Abstract

OBJECTIVE

To prepare antiserum against Fritillary virus Y (FVY) CP for detecting FVY and study serological relationships with other viruses.

METHODS

Specific primer was designed according to Genbank (accession: AM039800) to amplify CP gene of FVY infecting Thunberg fritillary. Sequence relationship with other potyviruses was made by Blast. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subsequently 5% - 20% gradient SDS-PAGE. The antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis. The reactivity of the antiserum produced to FVY CP was tested by Western blot against the over-expressed coat proteins of 17 potyviruses. The ability to combine with nature FVY particles was confirmed by ELISA analysis.

RESULTS

It shared 81.2% nucleotide acids identities with TrVY (Tricyrtis virus Y, AY 864850) CP gene, 68.1% with SMV-P (Soybean mosaic virus Pinellia strain, AJ507388. 2) CP gene and 67.2% with ZYMV (Zucchini yellow mosaic virus Luan isolate) CP gene. The prepared antiserum was special to FVY CP, also reacted moderately to the expressed CP of SMV-P (Soybean mosaic virus Pinellia strain) and weakly to that of ZYMV (Zucchini yellow mosaic virus Luan isolate).

CONCLUSION

The antibody could combine to nature FVY particles and the antiserum is suitable for FVY detection by ELISA in large scale.

摘要

目的

制备抗浙贝母病毒Y（FVY）外壳蛋白（CP）的抗血清用于检测FVY，并研究其与其他病毒的血清学关系。

方法

根据Genbank（登录号：AM039800）设计特异性引物，扩增感染浙贝母的FVY的CP基因。通过Blast分析其与其他马铃薯Y病毒属病毒的序列关系。将CP基因插入pSBET并在大肠杆菌BL21（DE3）plys E菌株中表达。目的蛋白先经12% SDS-PAGE纯化，随后经5% - 20%梯度SDS-PAGE纯化。用小鼠制备抗CP的抗血清，并通过Western blot分析确认其特异性。通过Western blot检测所产生的抗血清对17种马铃薯Y病毒属病毒过表达外壳蛋白的反应性。通过ELISA分析确认其与天然FVY颗粒结合的能力。

结果

其与TrVY（藜芦病毒Y，AY 864850）CP基因的核苷酸同一性为81.2%，与SMV-P（大豆花叶病毒半夏株系，AJ507388. 2）CP基因的同一性为68.1%，与ZYMV（小西葫芦黄花叶病毒栾分离株）CP基因的同一性为67.2%。所制备的抗血清对FVY CP具有特异性，对SMV-P（大豆花叶病毒半夏株系）表达的CP也有中度反应，对ZYMV（小西葫芦黄花叶病毒栾分离株）表达的CP有弱反应。