全 文 ：植物病理学报
ACTA PHYTOPATHOLOG ICA SIN ICA　35(3):256-261(2005)
Received date: 2004-02-28;Revised date: 2004-07-28
Foundation item:The K ey Pro ject o f th e Science D epartmen t o f Fu jian Prov ince, Ch in a (2000H004;2001Z127)
Co rrespond ing author:Professor, speciali ty:p lan t v iro logy;E-m ail: linq iy ing@ 126. com
Biog raphy:LIU Zhen-yu (1969 - ), male, D octo r, sp ecial ity:plan t patho logy;E-m ail:cosmos liu@ 163. com
Purification and Characterization of an Anti-TMV
Protein from aMarin A lgaeU lva pertusa
LIU Zhen-yu1, 2 , XIE Li-yan1 , WU Zu-jian1 ,
LIN Q i-y ing1* , XIE Lian-hu i1
(1 Ins titute of Plan tV iro logy, Fu jian A griculture and Forestry Un ivers ity, Fuzhou 350002, Ch ina;
2Co llege of Plan tProtection, ShandongA gricul turalUn ivers ity, Taian 271018, Ch in a)
Abstract:An antivira l protein UPCM40was isolated and purified from the algaeUlva pertusa K je llm through
ammon ium sulfate prec ip itation and CM-Sepharose FF ion-exchange colum n chrom atography. It was shown
that this k ind of protein is a sing le bandw ith molecularwe igh t of about 36 kD v ia SDS-PAGE electrophoresis.
And it is of only single componen t under PAGE analysis. W hole waves scanning on UPCM40 showed charac-
ter istic peaks at 190 - 220 nm and 250 -300 nm , and them axim um absorption on latter peak was 275 nm. The
activ ity ofUPCM40 aga inst the infec tion ofTobacco mosaic virus(TMV)was tested. The result showed that
the inhibition ra tes were 85.6% and 90.2% onN icotiana glu tinosa andChenopodium amaran ticolor respec-
tive ly. It also showed that theUPCM40 had certain inhibition activity toFusarium oxysporum f. sp. cucumeri-
num , Rhizoctonia solani andG loeosporium musarum a t the concen trat ion of 150 μg /mL through Dua l-culture
experiments.
Keywords:pro tein; antivirus;Ulva pertusa K jellm;purification;positive ion-exchange co lumn chrom a-
tography;TMV
孔石莼(U lva pertusa)中一种抗 TMV活性蛋白的纯化及其特性　刘振宇 1, 2 , 谢荔岩 1 , 吴祖
建 1 , 林奇英 1 , 谢联辉1　(1福建农林大学植物病毒研究所 , 福州 350002;2山东农业大学植物保护学院 , 泰安
271018)
摘要:采用硫酸铵盐析和阳离子交换柱层析(CM-Sepha rose Fast F low), 从孔石莼(U lva pertusa K jellm)藻体中分离纯化得
到 1个蛋白 , 命名为 UPCM 40。经 SDS-PAGE确定其分子量约为 36 kD, Na tive-PAGE可知其为单一组分;该蛋白不含糖;其
全波长扫描结果显示 ,该蛋白在 190 ～ 220 nm和 250 ～ 300 nm处有特征吸收峰 ,在 250 ～ 300 nm范围中的最大吸收峰在 270
～ 275 nm处。经测定发现该蛋白具较好的抗烟草花叶病毒(Tobacco mosaic v irus , TMV)的活性 ,当蛋白质浓度为 50 μg /
mL时 ,对 TMV 的抑制效果为:在枯斑寄主心叶烟上的侵染抑制率达 85. 6%, 在苋色藜上为 90.2%。测定该蛋白对 6种供
试真菌的抑制效果发现 ,对镰刀菌(Fusarium oxysporum f. sp. cucum erinum)、立枯丝核菌(Rhizocton ia solani )和香蕉炭疽菌
(G loeosporium musarum)均有一定程度的抑菌作用 , 但抑制活性很低。
关键词:孔石莼;阳离子交换层析;蛋白纯化;抗病毒;TMV
中图分类号:S432. 41;S968. 411　　　文献标识码:A　　　文章编号:0412-0914(2004)-03-0256-06
DOI牶牨牥牣牨牫牴牪牰牤j牣cnki牣apps牣牪牥牥牭牣牥牫牣牥牨牨
In troduction
Plan t viruses as a significant pathogen ic agen t
cause some severe ly dam age to crops and plants.
S ince the first plan t v irus resistance prote in — PAP
was isola ted fromPhy tolacca amercana
[ 1] , many sci-
entists m ade attemp ts at finding out these kinds of
prote in sim ilarly and som e of the prote ins had been
obtained from differen t organ isms
[ 2, 3]
. These kinds of
algae protein no t only can be used in con trolling viru-
ses, but a lso be used as sources for transgene[ 4, 5] .
It had been found that the biological activities of
the substance from marine algae gave a multi-effica-
cious in both medical and health use:anti-tum ur,
oxida tion prevention, anti-bacteria, -fungi, and
-v irus, e tc, consequen tly, study ing on the active
substance of a lgae has becom e one of the ho t spots in
th is area. It is known that cultivative algae are so
popular in the world currently and its area has been
approxim a ted to 200 km
2[ 6 - 9]
. Obviously, to reveal
and deve lop the va lue of the antiviral property of
prote ins from a lgae is in a vast significance.
Ulva pertusa K jellm is a kind of green algae be-
longing to Chlorophyta, U lvaceae , distributes all
over theworld and has a large b iom ass. M any inves-
tigations on chem ica l com pounds and pharm aceutical
activ ities ofU. pertusa K je llm have been m ade re-
cen tly and shown that it possess a vast range of pro-
spects on pharm acy besides its ed ib ility. Butm ost of
these investigations focus on lase and sma llm olecu-
lar com pounds, few of them invo lved in ac tive pro-
tein
[ 10, 11]
. Recen tly an agglutin in purification and
properties fromU. pertusa and an alka line phospha-
tase activities from U. pertusa have been repor-
ted
[ 12, 13]
. As we know , TMV is one of the m ost
popular and destructive virus in the world. A k ind of
anti-TMV prote in from U. pertusa has been iso lated
and tested, tha t is whatwe concern in this paper.
1　M ater ia ls andM ethods
1.1　Materia ls
Fresh samples ofU. pertusa Kje llm were ob-
tained from the sea areas of Nan-R i island in Putian
c ity, Fujian province. A fter samples were picked out
and chosen, washed and rinsed thoroughly in d is-
tilled water, then preserved in a freezer ( - 70℃).
Tobacco mosaic virus (TMV)was purified and
preserved in our laboratory. N icotiana glutinosa and
Chenopodium amarantico lor were propagated and
were used for anti-TMV ac tiv ity evalua tion. N icotia-
na tabacum var. K326 was used for TMV preservation
and mu ltiplication.
CM-Sepharose Fast F low (Pharm acia), acry-
lam ide, b is-acry lam ide, T ris and SDS (Shanghai
bio-eng ineering techno logy L td. , Ch ina); BSA
(Sigm a); low m olecular weight standard prote in
(Shangha i biochem ical institute);all other reagents
(Chinese products, AR).
1. 2　Methods
1.2.1　Sam ple digestion and pro teins salting-out:
Sam ples of U. pertusa K je llm were hom ogen ized
w ith 0.02m ol L -1 PB buffer, at pH 7.2, placed
at 4℃ for 12 -24 h, then cen trifuged at 10 000 g for
20m in at 4℃. The ammon ium sulfate was added in-
to the supernatant to 40% saturation and kept at
4℃ overn ight, then centrifuged at 10 000 g for 20
m in at 4℃. The supernatan t was re treated w ith
(NH4)2 SO4 , itwas added to 80% for saturation other
than the sam e processes as above. A fter centrifuged
(10 000 g, 20m in, 4℃), the superuan tantwas d is-
carded and the precipitation was collected and d is-
solved in 0.01 m ol L- 1 PB buffer, pH 7.2, and
then d ialyzed against distilled water then 0.01mol
L
-1
PB buffer, pH 7.2.
1.2.2 　 CM-Sepharose Fast F low ion-exchange
chrom a tography:Three m illiliter of 40%, 80% sal-
ting-out prote in was loaded on CM-Sepharose Fast
F low column(10 cm ×1.6 cm , id, 20 mL bed vol-
um e)wh ichwas pre-equilibra tedw ith the same buff-
er. E luting liquid was collec ted for every 2.7 mL
under the flow speed of 32 mL h- 1 , and then it
were mon itored by theA280 measuring. A fter the OD
2573期 L IU Zhen - yu, e t al:Purification and Charac teriza tion o f an Anti- TMV Pro te in from aM arin A lgae U lva pe rtusa
value had lowed to the baseline, the further e lution
was carried outw ith the buffer conta in ing aNaC l lin-
ear gradien t from 0 to 1.0 mol L -1 at the flow rate
of 32 mL h-1. Every fraction after tested to be
w ith anti-TMV activity were pooled and purified
through d ia lyzing extensively against distilled water
then PB buffer ( pH 7.2 ) at 4℃. and then the pro-
teins preparation were stored at - 20℃ for further
study.
1.2.3　De term ination for purity andm olecularm ass
of the algae pro tein:The purity and molecular m ass
of iso lated proteins were de term ined by SDS-PAGE
according to the procedure described by Laem-
m li
[ 14]
. The protein bands on running gels were vi-
sualized by staining w ith Coomassie brilliant blue.
1.2.4　Determ ination on protein concen tration, to-
tal carbohydrate conten t and absorp tion spectrum:
The concen tration of purified prote in was determ ined
by the Bradford m ethod, using BSA as the standard
check
[ 15]
. Total carbohydrate con tent was determ ined
by the phenol /sulfuric ac id procedure w ith the AR
glucose as the standardm easuring
[ 16]
. The g lycopro-
tein bands on the gel of SDS-PAGE and Native-
PAGE were visualized by sta in ing w ith period ic ac id
Schiffs reagent[ 17] . The absorption spectrum of the
prote in was de tec ted by the spectropho tom eterw ith in
190 - 600 nm (U ltrospec 2 100 Pro. , Amersham
Pharm ac ia B iotech product).
1.2.5　 Inh ib ition to the TMV infec tion :TMV
were purified according to the Gooding
[ 18]
. A ll of
the a lgae prote in solution tested were diluted in a se-
ries of concentration and m ixed w ith the same vo-
lum e of inoculum-TMV (20 μg /mL) individua lly.
The m ixtures of inoculum were rubbed on the one
side of basal leaves ofN. glutinosa andCh. ama-
ranticolor using carborundum (600 mesh). And the
ano ther opposite side leaves were rubbed w ith TMV
solution d iluted w ith the sam e volum e of PB buffer
as con trol
[ 19]
. E ach treatmen twas repeated for 5 or 6
times and was carried on in greenhouse. The necrotic
lesions were counted after the infection sym ptom s
appeased. Inh ibition rate(%) =[ 1 - ( num ber of
necro tic lesions tested /num ber of necrotic lesions
con trolled )] ×100.
1.2.6　 Inhibition to plant pathogene tic fung i:3
pieces ( =5 mm) of plant pathogene tic fung i pure
cu lture were placed on a PDA pla te in an equ ila teral
triangle position. A fter 24 - 28 h incuba ted, colon ies
had been grown up to 10 - 15mm in d iam eter, then
a p iece of filter paper dish saturated w ith 15 μL of
algae prote in solution was placed at the cen ter of the
triangle. A fter it was inculated under 28 - 30℃ for
24 - 72 h, the inh ib ition zones be tween fungus colo-
n ies and paper dish were m easured. E ach treatment
was in 3 repeated. The tested fung iwere listed in the
Table 2.
2　Resu lts
2. 1　Pu rification of UPCM40
The preparation of prote ins from U. pertusa
were reso lved via CM-Sepharose co lumn. In Fig.1,
the OD value was plotted to the fractions of prote in
elution.
Fig.1　Optical dens ity curve of pro tein from
U lva pertusa
258 植物病理学报 35卷
　　It is consp icuous that five prote in peaks can be
seen in the elution curve, the peak A and B are cor-
responding to algae pro te ins wh ich could not be ad-
hered to CM-Sepharose FF but can be eluted by PB
buffer, C , D and E were pro te ins which could be
adhered to CM-Sepharose FF and also can be eluted
by NaC l linear gradient from 0 to 1.0 m ol L- 1.
Consequently, pro teins of algae appeared in the
peaks ofC , D and E m a inly, and it was considered
to be som e prote ins w ith positive charge.
2.2 　 SDS-PAGE and Native-PAGE analysis
and protein characteristics
The prote in fraction collected from peak E was
reso lved by SDS-PAGE and Native-PAGE. It was
only one single band appeared in SDS-PAGE ge l,
which implied that th is protein is ofmonom eric pro-
tein and in e lec trophoretic purity. This k ind of a lgae
prote in was nam ed as UPCM40 and the m olecular
m ass of itwas estim ated to be about 36 kD accord ing
to the standard markerm easuring (Fig.2).
No o ther consp icuous band had been observed
while the electrophoretic gel was sta ined w ith perio-
dic acid Schiffs reagen t, which meant no carbohy-
dra te con tain in the prote in. I twas further confirm ed
by the phenol /su lfur ic ac id procedure.
The result also showed tha t there were two typi-
ca l absorption peaks existed, under u ltraviole t spec-
trophotome ter, one is in 190 - 220 nm , another in
250 -300 nm and itsm axim al peak was in 270 - 275
nm , not in 280 nm (Fig.3, Fig.4). Accordingly,
the UPCM40 was considered to be different from or-
d inary prote in fromU. pertusa algae.
2.3　The inhib ition effec tof UPCM 40 on TMV
TMV was purified and then exam ined by ultra-
vio let spectrophotom eter, in wh ich it presented a
typ ica l absorp tion feature, the m ax im al peak is in
260 nm and the m in im al peak is in 240 nm. The
concen tra tion of the purified TMV was also estim ated
to be 60m g /mL.
Fig.2　SDS-PAGE　electropho tog ram　of
UPCM40
1:UPCM 40; 2: The rude prote in of 40% and 80%
(NH4)2SO4 saturation salting-out prote ins;M:The low mo-
lecularmass marker
Table 1　The inh ib ition rate of UPCM 40 to
TMV
Concentration of
UPCM 40(μg /mL)
Inhibition rate (%)
N.glutinosa Ch.
am aranticolor
12. 5 37. 8 42. 1
25. 0 76. 4 74. 9
50. 0 85. 6 90. 2
150. 0 90. 5 93. 1
The purified TMV were d iluted w ith PBS, and
m ixed w ith the UPCM40 of pro tein, andm ade a final
concentra tion of TMV in 10 μg /mL constantly and
forUPCM in a series of range. Two k inds of host
(necrosis response to TMV ) were inoculated by
TMV w ith d ifferen t concen tration of UPCM40. As
the result showed in Tab le 1 the inh ib ition rate onN.
glutinosa and Ch. amarantico lor was 85.6% and
90.2%, respec tively wh ile the concentration of UP-
CM40 was at 50 μg /mL. The result a lso indicated
that the h igher concentration of the UPCM40 is, the
2593期 L IU Zhen - yu, e t al:Purification and Charac teriza tion o f an Anti- TMV Pro te in from aM arin A lgae U lva pe rtusa
h igher inh ib ition it w ill take. But the inhibition rate
increased steady after it got close to 50 μg /mL.
Fig.3　The UV absorption spec trum of
UPCM40 in 190 - 225 nm
Fig.4 　 The UV absorp tion spectrum of
UPCM 40 in 220 - 340 nm
2.4　 The inh ib ition ac tiv ity of UPCM 40 to
some pathogenetic fung i of p lan t
Six k inds of plan t pathogene tic fungi were eva-
luated for the inh ib ition activity of UPCM 40 inc 150
μg /mL concen tra tion. The result showed that the
UPCM40 had a slight inh ibition activity toF. o. f.
sp. cucumerinum , R . solani andG .musarum. L it-
tle effects had been observed toA lternaria alternata ,
Botry tis cinerea andPhy tophthora capsic i(Tab le 2).
Table 2　The inhibition activ ity o fUPCM 40 to
six pathogenetic fungi o f p lant
Fungi tested Inh ib ition activity
A lternaria a lternata -
Bo trytis c inerea -
F. o. f. sp. cucum erinum +
Phytoph thora capsici -
G loeosporium musarum ++
Rhizoctonia solani +
++, +:M ean differen t leve l of inh ib ition activ ity respec-
tively; -:Means no inh ib ition ac tivity
3　D iscussion
A purified UPCM40 protein from theU . pertu-
sa w ith anti-TMV activity was first reported. The
pro te in could be adhered to cat ion ic exchange agent
under the cond ition of pH 7.2. It was am onom er ic
pro te in w ith them olecularm ass abou t 36 kD and no
carbohydrate conta ined in i.t A slightly effect onF.
o. f. sp. cucumerinum , R. solani andG. musa-
rum. Th is kind of prote in was supposed to be a new
kind of a lgae pro tein found inU. pertusa even inUl-
raceae before. Due to its properties were d ifferent
from other proteins derived from U. pertusa , w ith
these differences after checked to the prote in database
of Sw iss-Prot, it is reasonab le to consider that UP-
CM40 is a new k ind of prote in fromU. pertusa.
A ccording to the low conten t of active com po-
nen t from U. pertusa , and particu lar grow th condi-
tions it acquired, it is d ifficult to get enough active
substances fromU. pertusa , direc tly. But possib ly,
it w ill be ab le to get enough supplem ent of active
substance via gene tic eng ineering. Because of its an-
tiv iral properties, these k inds of genes a lso possess
som e potentia l in increasing the plant d isease resis-
tan.t The gene clon ing of the prote in UPCM 40 is in
progress.
In dual facilities that TMV was chosen as a tar-
get for evaluating the antiviral activity ofUPCM40 in
th is study, for its destructive significance in crops
and p lants, precedent and ava ilable bo th in inform a-
tion and pro tocol in TMV study. And from the re-
260 植物病理学报 35卷
sults we ga in, it is hopeful to develop the UPCM40
as a factor in virus diseases con trol through d isease
resistan t transgen ic research. In the consequence of
the an tiv iral ac tiv ity of the UPCM 40was revealed an
advanced study on the m echan ism of inh ib ition acti-
vity and som e extensive study on an im al v iruses w ill
be also carried on.
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责任编辑:杨晓昱
2613期 L IU Zhen - yu, e t al:Purification and Charac teriza tion o f an Anti- TMV Pro te in from aM arin A lgae U lva pe rtusa