全 文 ：于膜分离是一个相对的过程，在大分子的部位中存
在小分子量的成分，因此表现出一定的 ACE 抑制活
性;对水解前后的结果进行比较发现，水解后地龙匀
浆液的 ACE抑制活性明显增强，说明酶解有利于提
高地龙蛋白产物的 ACE抑制活性。
参 考 文 献
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novel antihypertensive peptides in milk fermented with
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收稿日期:2011-04-19
基金项目:安徽省教育厅自然科学基金重点项目资助(KJ2010A328;KJ2011A272)
作者简介:韦传宝(1961-) ，男，博士，副教授，主要从事中药生物技术研究;E-mail:weichuanbao@ sina. com。
·资源·
Prokaryotic Expression of CP Gene of Fritillary virus
Y Infecting Thunberg Fritillary and
Antiserum Preparation
WEI Chuan-bao1，2，WEI Yang-yang2，YANG Yu2，LIU Shi-liang2，HU Hao-yu2，HE Yue2
(Western Anhui University，1. Anhui Provincial Laboratory of Biomimetic Sensor and Detecting Technology;
2. Biological and Pharmaceutical Engineering College，Liuan 237012，China)
Abstract Objective:To prepare antiserum against Fritillary virus Y(FVY)CP for detecting FVY and study serological relation-
ships 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 pS-
BET and expressed in Escherichia coli BL21(DE3)plys E strain. The object protein was purified by 12% SDS-PAGE firstly and subse-
quently 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% nucleo-
tide 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 spe-
cial 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.
Key words Thunberg fritillary;Fritillary virus Y;CP gene;Prokaryotic expression;Antiserum preparation
CLC number:Q785;S436. 421 Document code:A Article ID:1001-4454(2011)10-1498-05
·8941· Journal of Chinese Medicinal Materials 第 34 卷第 10 期 2011 年 10 月
DOI:10.13863/j.issn1001-4454.2011.10.003
侵染浙贝母的浙贝母病毒 Y(FVY)CP基因的原核表达及抗血清制备
韦传宝1，2，隗洋洋2，杨 宇2，刘士亮2，胡皓雨2，何 月2
(皖西学院 1. 安徽仿生传感与检测技术实验室;2. 生物与制药工程学院，安徽 六安 237012)
摘要 目的:制备抗侵染浙贝母的浙贝母病毒 Y(Fritillary virus Y，FVY)CP 血清，为大田检测 FVY 和研究
FVY与其他病毒之间的血清学关系奠定基础。方法:根据 Genbank报道的 FVY CP基因序列设计引物，扩增其 CP
基因。Blast分析 FVY CP基因核苷酸序列与其他马铃薯 Y病毒属成员 CP基因的同源性。将 CP基因插入表达载
体 pSBET，转化大肠杆菌 BL21(DE3)Plys E菌株，通过 IPTG诱导表达。经 12% SDS-PAGE和 5% ～20% 梯度 SDS-
PAGE两次纯化 CP，免疫小鼠获得抗 CP血清，采用 Western blot分析抗体的特异性。用原核表达的 17 种马铃薯 Y
病毒属病毒 CP蛋白作为抗原，检测抗 FVY CP血清是否能与其进行交叉反应。采用 ELISA 分析抗体能否与天然
FVY病毒离子结合。结果:FVY CP基因与油点草病毒 Y(TrVY)CP 基因、大豆花叶病毒半夏分离物 CP 基因和小
西葫芦黄花叶病毒六安分离物 CP基因分别有 81. 2%、68. 1%和 67. 2%的同源性。制备的抗体对 FVY CP 具有高
度特异性，同时能与大豆花叶病毒半夏分离物(SMV-P)CP 蛋白有中等强度的结合，与小西葫芦黄花叶病毒 CP 蛋
白有弱的结合。结论:抗体能够与天然 FVY病毒离子结合，因此可以作为该病毒的大田检测。
关键词 浙贝母;浙贝母病毒 Y;CP基因;原核表达;抗血清制备
Thunberg fritillary(Fritillaria thunbergii Miq. ) ，
the genus Fritillaria，family Liliaceae，is a precious
traditional Chinese medicinal plant. In China，Thun-
berg fritillary was originally cultivated in Xiangshan
country，Zhejiang province and now is widely planted
in Zhejiang，Jiangsu，Anhui，Hunan and Jiangxi prov-
inces. The plant is harvested when it withers at the be-
ginning of summer and the bulb is dried for medicinal
use as a cure for sore throats and frequent coughing〔1〕.
Thunberg fritillary is typically propagated from bulb
and half of the total bulb has to be used for propaga-
tion. After vegetative propagation for hundreds of
years，severe degeneration with virus infections is a
problem， resulting in poor quality and decreasing
yields〔2〕. It was found that Thunberg fritillary was in-
fected by three kinds of viruses，including Thunberg
fritillary mosaic virus(tentatively named TFMV)〔3〕，
Fritillary virus Y(tentatively named，FVV)〔4〕and Lily
mottle virus(LmoV)〔5〕. It is hoped that propagation u-
sing virus-free Thunberg fritillary bulbs would be feasi-
ble to resolve its characteristics. ELISA analysis is an
effective method to detect virus and antiserum against
the virus is prerequisite for ELISA analysis. In this pa-
per，for the first time，we report the studies on pro-
karyotic expression of Fritillary virus Y CP gene，prep-
aration its antiserum and study on serological relation-
ship with other virus.
1 Materials
Fresh Thunberg fritillary leaves infected with FVY
were collected from the fields in Panan city，Zhejiang
Province where Thunberg fritillary is planted on a large
scale. They were routinely stored at － 80℃ before
use. pGEM-T vevtor，pSBET vevtor，Escherichia coli
TG1 and BL21(DE3)pLys S were products of Pro-
mega. Taq DNA polymerase system，Nde I，Bam HI
were products of TaKaRa. Ribonuclease Inhibitor，
AMV Reverse Transcriptase and DNA Ligation Kit
ver. 2 were products of Life Technologies Ltd. SDS-
PAGE Middle Range Protein Molecular Weight Stand-
ards and GeneRuler TM 1 kb DNA Ladder were prod-
ucts of MBI. RNeasy Plant Mini Kit and Gel Extraction
Kit was products of QIAGEN(Valencia，CA，USA).
Anti-mouse IgG(coupled with alkaline phophatase) ，
BCIP (5-bromo-4-chloro-3-indolyl-phosphate) ，NBT
(Nitroblue tetrazolium)and IPTG (Isopropyl-β-D-thio-
galactopyranoside)were products of Sigama. Primers
were synthesised by Shanghai Sangon Biological Engi-
neering Technology ＆ Services Co. ，Ltd，Shanghai，
China. All other reagents used were of analytical
grade.
2 Methods
2. 1 Viral RNA extraction and cDNA synthesis
Viral RNA was isolated from FVY-infected Thunberg
fritillary leaves using the RNeasy Plant Mini Kit and
first-strand cDNA was then synthesised using M-MLV-
reverse transcriptase according to the manufacturer s
instructions and with M4T(5-GTT TTCCCA GTC ACG
ACA C(T)16-3)as the initial primer.
2. 2 FVY CP gene cloning and sequence analysis
The LA Taq DNA polymerase system was used ac-
·9941·Journal of Chinese Medicinal Materials 第 34 卷第 10 期 2011 年 10 月
cording to the manufacturers protocols. FVY CP gene
was cloned by PCR amplification using primer pairs:
FVY-CP(+) :5-CCA TAT GTC AGG ATC AGG TGA
AGT-3(with restriction endonuclease Nde I side)and
FVY-CP(－) :5-GGG ATC CCT GCA TGG GGT TCA
TCC CAA-3(with restriction endonuclease Bam H I
side). PCR was conducted with a final volume of 25
μL containing 15. 6 μL ddH2O，2 μL cDNA，2. 5 μL
of a 10 mmol /L dNTP mixture，0. 4 μL Taq DNA pol-
ymerase，2. 5 μL 10 × Taq buffer and 1 μL of 10
pmole for each primer. Conditions for amplification in-
cluded an initial denaturation at 94 ℃ for 4 min，30
cycles of denaturation at 94 ℃ for 30 s，annealing at
51. 5 ℃ for 30 s and extension at 72 ℃ for 50 s，fol-
lowed by a final extension at 72 ℃ for 10 min. Suc-
cessful amplification of fragments of the expected size
was confirmed by electrophoresis through 1% (w /v)
agarose gel. PCR fragments were purified using the Gel
Extraction Kit according to the manufacturers instruc-
tions. FVY CP gene was cloned into pGEM-T vector
following the manufacturers protocols. Three independ-
ent clones were auto-sequenced in both directions using
ABI PRISMTM 3770 DNA Sequencer by Shanghai
Sangon Biological Engineering Technology ＆ Services
Co. ，Ltd. Sequence relationship with other potyviruses
was made by Blast.
2. 3 Prokaryotic expression of FVY CP gene
The CP gene was cleavaged with Bam H I and Nde I
and was inserted into pSBET cleavaged with the same
restriction endonuclease. The pSBET with FVY CP
gene was transformed to Escherichia coli BL21(DE3)
plys S strain and the stain expressing CP gene was con-
firmed by 12% SDS-PAGE analysis.
2. 4 Antiserum preparation CP gene transformed
stain bodies were lysed by 2 × SDS-laoding bufffer(0. 1
mol /L Tris-HCl pH 6. 8，0. 2 mol /L dithiothreitol，
4% SDS，0. 2% bromophenol blue and 20% glycerol)
at 100℃ for 10 min and the lysate was centrifugated for
10 min at 10，000 g. The expressed CP was purified by
12% SDS-PAGE and dyed in 0. 25 mol /L KCl solution
at 4 ℃ . The CP was taken by cutting the objective
band. The CP in polyacrylamide gel was extracted by 2
× SDS laoding buffer through grinding and centrifugat-
ing. The CP in the supernatant was further purified by
5% ～ 20% gradient SDS-PAGE and dyed in 0. 25
mol /L KCl solution at 4 ℃ . The objective band con-
taining CP was taken and grinded in normal saline.
Mice were immuned by injecting CP，containing in
polyacrylamide，for 4 times and the antiserum against
the CP was obtained.
2. 5 Western blot and ELISA analysis The speci-
ficity of the antiserum produced was confirmed by
Western blot analysis. Proteins were frst resolved by
12% SDS-PAGE and then electrophoretically trans-
ferred to a nitrocellulose filter. After incubation with
the antiserum at a dilution of 1∶ 500(V /V) ，antibody-
binding CP was visualized by incubating the filter with
anti-mouse IgG that were coupled with alkaline
phophatase and by subsequent staining with BCIP /
NBT〔6〕. ELISA analysis was done using published
methods〔7〕. Thunberg fritillary leaves infected with
FVY were used as sample and health Thunberg fritillar-
y leaves were used as negative control.
2. 6 Serological characteristics analysis The reac-
tivity of the antiserum produced to FVY CP was tested
by Western blot against the over-expressed coat pro-
teins of 17 potyviruses，including Onion yellow dwarf
virus(OYDV) ，Leek yellow stripe virus(LYSV) ，Shal-
lot yellow stripe virus(SYSV) ，Zucchini yellow mosaic
virus Luan isolate(ZYMV) ，Lily mottle virus(LMoV) ，
Potato virus Y(PVY) ，Soybean mosaic virus Pinellia
strain(SMV-P) ，Dasheen mosaic virus(DsMV) ，Nar-
cissus late season yellows virus(NLSYV) ，Sugarcane
mosaic virus(SCMV) ，Sorghum mosaic virus(SrMV) ，
Narcissus yellow stripe virus(NYSV) ，Tuberose mild
mottle virus (TuMMoV) ，Tuberose mild mosaic virus
(TuMMV) ，Ornithogalum mosaic virus (OrMV) ，
Thunberg fritillary mosaic virus (TFMV)and Scallion
mosaic virus (ScaMV) ，produced using published
methods〔6〕.
3 Results and analysis
3. 1 FVY CP gene cloning and sequence analysis
～800 bp DNA fragment of the expected CP gene was
obtained by PCR amplification using primer pairs:FVY-
CP(+)and FVY-CP(－). PCR fragment was cloned
into pGEM-T vector and transformed into Escherichia co-
li TG1. Sequence analysis results indicated that FVY
CP gene consists of 822 bp and has corresponding 274
amino acids. It shared 81. 2% nucleotide acids identi
ties with TrVY (Tricyrtis virus Y，AY 864850)CP gene，
·0051· Journal of Chinese Medicinal Materials 第 34 卷第 10 期 2011 年 10 月
Fig. 1 Multiple aligment of FVY CP gene with other related virus CP gene
Fig. 2 12% (m/V)SDS-PAGE analysis of the lysat-
es of FVY CP gene transformed BL21 (DE3)
M. protein marker 1. FVY CP gene transformed BL21 (DE3)
2. BL21 (DE3)plys S induced by IPTG (CK-)
68. 1% nucleotide acids identities with SMV-P (Soy-
bean mosaic virus Pinellia strain，AJ507388. 2)CP
gene and 67. 2% nucleotide acids identities with
ZYMV (Zucchini yellow mosaic virus Luan isolate)CP
gene (Fig. 1).
3. 2 FVY CP gene prokaryotic expression The
CP gene was expressed in Escherichia coli BL21
(DE3)plys S strain. CP gene transformed stain was
induced by IPTG for 2 h and confirmed by 12% SDS-
PAGE analysis (Fig. 2). The deduced CP had a pre-
dicted molecular weight of 30. 28 kD，which was simi-
lar to that indicated by 12% SDS-PAGE.
3. 3 Western blot analysis The polyclonal antise-
rum produced reacted only with FVY CP but not with
any of Escherichia coli BL21 (DE3)plys S protein and
·1051·Journal of Chinese Medicinal Materials 第 34 卷第 10 期 2011 年 10 月
the antiserum is special to combine with expressed
FVY CP (Fig. 3).
Fig. 3 Western blot analysis of FVY CP antiserum
M. protein marker 1. BL21 (DE3)plys S induced by IPTG
(CK-) 2. FVY CP gene transformed BL21 (DE3)
3. 4 ELISA detection of FVY particles Five
healthy and five FVY-infected Thunberg fritillary plants
were used as samples for ELISA test. The results indi-
cated that all the healthy plants are negative and all the
FVY-infected are positive in ELISA test，which corre-
sponds to the results by PCR analysis. It was shown
that the antibody could combine to nature FVY parti-
cles and the antiserum is suitable for FVY detection.
3. 5 Serological relationship with other viruses
The FVY CP antiserum also reacted moderately to the
expressed CP of SMV-P and weakly to that of ZYMV
but not to other potyviruses tested.
4 Conclusion
Thunberg fritillary was infected by three kinds of
viruses (TFMV，LMoV and FVY)〔3-5〕. TFMV CP and
LMoV CP have been expressed in Escherichia coli
BL21 (DE3)plys S strain in previous studies. In this
study FVY CP was expressed and the polyclonal antise-
rum reacted only with FVY CP has been produced suc-
cessfully. From the results in this study，conclusions
have been made as followings. Firstly，antiserum pre-
pared by injecting expressed CP has advan-tange of
high specificity and stable quality，because there is no
other protein antigen exception to the CP. The ex-
pressed CP are purified to a very high purity by 12%
SDS-PAGE firstly and subsequently 5% ～ 20% gradi-
ent SDS-PAGE. Secondly，the results from previous
studies and this study indicate that there are no rela-
tionship in serology among TFMV，LMoV and FVY.
Thirdly， the antibody in prepared antiserum could
combine to nature FVY particles. The result by ELISA
analysis corresponds with the results by RT-PCR analy-
sis and the antiserum is suitable for FVY detection in
large sample.
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櫥
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毣
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1136-1146.
《中药材》杂志为国家首批公布的可登处方药广告的专业媒体。
·2051· Journal of Chinese Medicinal Materials 第 34 卷第 10 期 2011 年 10 月