全 文 ：鸡α干扰素基因遗传转化烟草研究?
宋 莉1 ,2 , 赵德刚1 , 3
??
, 吴拥军3
( 1 贵州省农业生物工程重点实验室 , 贵州 贵阳 550025; 2 教育部绿色农药与农业生物工程重点实验室 ,
贵州 贵阳 550025; 3 贵州大学生命科学学院 , 贵州 贵阳 550025)
摘要 : 利用植物生物反应器生产外源药用蛋白近年来备受关注 , 本研究通过农杆菌介导法 , 将人工合成的
鸡α干扰素基因 ( ChIFN-α) 转化烟草 ( Nicotiana tabacum) 无菌苗叶盘。对抗性植株进行的 GUS活性鉴定 ,
PCR 和 RT-PCR 检测表明 , ChIFN-α基因已整合到烟草基因组中并具有转录活性 , ELISA 检测和细胞病变
( CPE) 抑制试验表明转基因烟草表达的干扰素蛋白具有抗病毒活性。
关键词 : 鸡α干扰素 ; 烟草 ; 表达 ; 生物学活性
中图分类号 : Q 943 , Q 78 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 03 - 247 - 06
Expression of Bioactive Chicken Alpha Interferon
in Transgenic Tobacco
SONG Li
1 , 2
, ZHAO De-Gang
1 , 3 * *
, WU Yong-Jun
3
(1 Guizhou Key Laboratory of Agricultural Bioengineering, Guiyang 550025 , China; 2 Key Laboratory of Green
Pesticideand Agricultural Biological Engineering, Ministry of Education, Guiyang 550025 , China;
3 Collegeof LifeSciences, Guizhou University, Guiyang 550025 , China)
Abstract : Expression of avian cytokine in tobacco or other plants had theeconomicallypotential usefor producing pharma-
cological applications products and applying easily in practice . The cDNA encoding chicken alpha interferon ( ChIFN-α)
was introduced into Nicotiana tabacumXanthin by Agrobacterium transformation . The transgenic plants were detected by
histochemical stain assay and molecular analysis . Recombinant ChIFN-αwas confirmed by enzyme- linked immunosorbent as-
say (ELISA) and cytopathic effect ( CPE) inhibition assay . The results showed that ChIFN-αgene was successfully intro-
duced into tobacco plants and stably expressed at various level from0 .0003% to0.0033% of total soluble protein . The bio-
logical activity of ChIFN-αwas 5 .01×103 IU?gof tissue in vitro . Since interferon was involved in host antiviral response,
our results provided the possibility for further study of plant oral adjuvant with ChIFN-αfor improving immunity in birds .
Key words: ChIFN-α; Tobacco; Expression; Bioactivity
Alpha interferon ( IFN-α) is apleiotropic cytokine
that possess powerful and wide-range of antiviral prop-
erties ( Schultz et al. , 1995a, b; Marcus et al. ,
1999; Levy et al. , 1999; Mo et al. , 2001; Ruttan-
apumma et al. , 2005 ) , antiproliferative ( Plach? et
al. , 1999 ) , and immunoregulatory functions ( Mati-
kainen et al. , 2001; Malmgaard, 2004 ) through mul-
tiple pathways . It is also the most important antiviral
drug in clinical practice . The resistance to viruses is
especially important in innate immune and the produc-
tionof interferon ( IFN) is amajor factor for regulating
thepathogenesis, virulence, and transmissionof infect-
云 南 植 物 研 究 2009 , 31 (3) : 247～252
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.09019
?
?? ?Author for correspondence; E-mail : dgzhao@ gzu. edu. cn
Received date: 2009 - 02 - 04 , Accepted date: 2009 - 04 - 01
作者简介 : 宋莉 ( 1971 - ) 女 , 在读博士研究生 , 主要从事生物技术制药研究。 ?
Foun ?dation items: The National Key Projects in the Science & Technology Pillar Program of China ( No . 2007BAD59B06 ) , and the International
Science and Technology Cooperation Program of China ( No . 2007DFA31260)
edvirus (Kochs et al. , 2007; Cauthen et al. , 2007)
by expressing interferon regulatory factor 3 target genes
( IRF3) , IFN-stimulated genes, alpha IFNs and IFN-
dependent antiviral gene to establish antiviral state
( Brenda et al. , 2008 ) . Chicken alpha interferon
( ChIFN-α) is synthesized by the body in response to a
viral attack . The broad-spectrum antiviral value of
ChIFN-αhas beenwell demonstrated by its capacity to
defense against and treat viral infection in many avian
viruses such as avian influenza virus (AIV ) ( Xia et
al. , 2004; Wei et al. , 2006 ) , which is difficult to
control for that migratory birds are known to act as the
vectors in AIV dispersal ( Kilpatrick et al. , 2006;
Jourdain et al. , 2007) . But in fact, use of ChIFN-α
was limited due to the difficulty in manufacturing the
protein in large enough quantities by fermentation
method or extraction from animal tissue cell . So, it is
necessary and imperative to establish an efficient pro-
duction system for making interferon economically to
produce or expediently to use .
Transgenic plants could offer a valuable alterna-
tive to produce foreignproteins, such asbiopharmaceu-
ticals (Gutiérrez-Ortega et al. , 2005; Bellucci et al. ,
2007) , vaccine antigens (Gómez et al. , 2000; Kehm
et al. , 2001 ) and antibodies ( Khoudi et al. , 1999;
Vaquero et al. , 1999; Negrouk et al. , 2005 ) . Com-
pared to other in vitro expression systems, plant com-
bines the key advantages of eukaryotic, including pro-
tein secretion, folding, posttranslational modification,
relatively higher protein production levels and low risk
of contamination by animal viruses or bacterial endotox-
ins ( Melnick et al. , 1992; Denecke et al. , 1995;
Boston et al. , 1996; Ohya et al. , 2001 ) . In addit-
ion, transgenic plants are the most economical produc-
tion system of recombinant protein by large-scale and
cost-effective production as well as the avoidance of
protein separation and purification during downstream
processing ( Düring et al. , 1990; Khoudi et al. ,
1999; Vaquero et al. , 1999 ) by direct oral adminis-
tration ( e.g ., plant-derived oral vaccine or plant
drug) . Despite considerable evidences havebeen accu-
mulated to indicate that animal IFN can be correctly
expressed in plant cells (Zhen et al. , 1994; Tzahi et
al. , 2001; Sawahel , 2002; Ohya et al. , 2005; Li et
al. , 2006; Song et al. , 2008) , there has been no re-
port on the stable expression of ChIFN-α in transgenic
plant . In this study, we for the first time introduced
ChIFN-α gene into the model plant tobacco via
Agrobacteriumtransformation, which resulted in a suc-
cessful expression of the bioactive product .
1 Material and methods
1 .1 Constructs, Plant Transformation and Histochemical
Identification
ChIFN-αcDNA (GenBank accession no . U07868) domain
was amplified from plasmid pBSK-BNANSI with a ChIFN-αgene
(synthesized by Generay Ltd . , Shanghai , China) by using the
following primers: forward primer 5′-GTTCTAGAATGGCT-
GTTCCAGCTTCTC-3′, and reverse primer: 5′-CGGGTAC-
CCTATTAGTGGTGGTGGTGGTGGTGGGATCCAGATCCTCCCTA
GGTCCTGGTGTTTCCG-3′. The forward primer contained the
recognition sequence for XbaI , and the reverse primer contained
the recognition sequence for KpnI . PCR reaction condition of the
ChIFN-αwas: denaturation at 94℃ for 5 min; 5 cycles at 94℃
for 40 s, 55℃ for 40 s and72℃ for 40 s; 25 cycles at 94℃ for
40 s, 58℃ for 40 s and 72℃ for 40 s; and finally extension at
72℃ for 8 min . IFN geneobtained was cloned intoplasmidpSH
(KpnI-XbaI fragment) to make the expression vector pSFRLH
and its presence in the colonies was confirmed by either restric-
tion digest or direct sequencing . The resulted plasmid comprises:
the ChIFN-αgene, reporter geneβ-glucoronidase ( GUS) and
selectableneomycin phosphotransferase ( NPT) genefor kanamy-
cin resistance . These geneswere placed under the control of the
cauliflower mosaic virus (CaMV) 35S promoter andpoly A termi-
nator (Fig. 1) . The plasmid pSFRLH was transfered to Agrobac-
terium tumefaciens strain EHA105 by freeze-thaw method ( Hol-
sters et al. , 1978) . Leaf disc transformation of tobacco ( Nicoti-
ana tabacumL . Xanthin) was then conducted following a previ-
ously established protocol using Agrobacterium transformation
(Horsch et al. , 1985) . GUS activity of the transformed tobacco
was analysed histochemically following a previously established
procedure (Song et al. , 2008) .
Fig . 1 pSFRLH construct containing ChIFN-α
gene used for plant transformation
842 云 南 植 物 研 究 31 卷
1 .2 PCR and RT - PCR Analyses
The presenceand expression of the ChIFN-αgenewerean-
alyzed by PCR and RT-PCR assay . Genomic DNA and total RNA
were isolated fromtransformed and control leaves in T0 plants of
GUS-positive . The following primer sets were used-forward 5′-
GCTGTTCCAGCTTCTCCAC-3′and reverse 5′-CCTGGTGTTTC-
CGGTAAGG-3′in PCR assay . The PCR cycling conditions were
once94℃ for 5 min; 30 cycles at 94℃ for 30 s, 56℃ for 30 s
and 72℃ for 30 s; and one cycle at 72℃ for 8 min . The ob-
tained total RNA was used in RT-PCR analysis with a one-step
RNA PCR kit (TaKaRa, Dalian, China) according to manufac-
turer′s instruction by using the primers 5′-GTTCTAGAATGGCT-
GTTCCAGCTTCTC-3′and 5′-GGGGTACCCTATTACTAGGTCCT
GGTG-3′. TheRT-PCR conditionswere denaturation at 94℃ for
5 min; 5 cycles at 94℃ for 40 s, 57℃ for 40 s and72℃ for 40
s; 25 cycles at 94℃ for 40 s, 59℃ for 40 s and 72℃ for 40 s;
and finally extension at 72℃ for 8 min . PCR and RT-PCR prod-
uctswere separated on a 1 .0 % (w?v) agarosegel .
1 . 3 ELISA Assay
Enzyme-linked immunosorbent assay (ELISA) was found to
be a convenient, economical , and efficient method for antibody
detection against antigen . Expression of recombinant IFN from
transgenic tobacco was measured using a ChIFN-α ELISA kit
(RapidBioLab, California, USA) following the manufacturer′s
instruction . Soluble protein was extracted from transgenic and
control leaves ( fresh wt) by using 3 ml of extraction buffer ( 100
mmol?L Tris-HCl ( pH 8 .0 ) , 10 mmol?L EDTA , 50 mmol?L
ascorbic acid, 10 .0 % (v?v) glycerol , 10 .0 mmol?Lβ-mercapto-
ethanol and 0 .5 % (w?v) SDS) per gram of leaf material . Cell
debriswas removed by tworoundsof centrifugation (12 000 r?min,
20 min, 4℃ ) . The quantitative determination of leaf protein ex-
tractswas proceeded according to Bradford (1976) . Experiments
of standard curve and every sample were all repeated thrice .
1 . 4 Assay of ChIFN-αActivity
Total soluble protein was extracted from the transgenic to-
bacco by usingammonium sulphate precipitation method ( 100%
saturation) and lyophilized in a freeze dryer (Eyela, Tokyo, Ja-
pan) for 48 h and stored at - 20℃ until further used . The bio-
logical activity of transgenic plants of # 3 clone wasmeasured for
lethality tovesicular stomatitis virus (VSV) usingacytopathic ef-
fect ( CPE ) inhibition assay as described ( Schultz et al. ,
1995b) . Inbrief , themonolayer of chickenembryonic fibroblasts
(CEFs) from 9-day-old chicken embryos were challenged with
VSV at a 100 times 50% tissue culture infectious dose ( 100
TCID50) , then weretreatedwith IFN preparationsfromtransgen-
ic tobacco and incubated at 37℃ until destructionof the untreat-
ed virus- infected cellswas apparent, the rate of monolayer 50%
virus protection was estimated and compared with reference
ChIFN-a preparation and expressed in international units of IFN
activity ( IU) . TheCPE inhibition assaywas repeated thrice .
2 Results
2 .1 Plasmid Construction, Transformation and
Detection of Transformed Plants
The ChIFN-αcDNA was initially cloned into pSH
and resultant plasmid pSFRLH was subsequently intro-
duced into tobacco leaf discs by Agrobacterium-mediat-
ed transformation . After appropriate culture and selec-
tion, 11 independent transgenic plantletswereobtained
for the expression of reporter GUS gene by histochemi-
cal staining reaction (Fig. 2) . To confirmthe presence
of ChIFN-α gene in plants, genomic DNA isolated
from kanamycin-resistant and GUS-positive transforma-
nts were detected by PCR amplification method . The
expected PCR product of genomic DNA from selected
transformed tobacco plant was a 573 base pair
( Fig. 3A ) . The same DNA fragment was amplified
when plasmids pSFRLH was used as template . Howev-
er, no band was observed for the non-transformed pla-
nts . ChIFN-α from transformed tobacco had marked
mRNA expression, and there was a specific fragment
near 635 bp indicating the expected gene by RT-PCR
amplification (Fig. 3B) .
Fig . 2 The histochemical analysis of GUS gene expression
in leaf ( A ) , roots ( B) and flower (C) tissues of # 3
transgenic line respectively
2 .2 Expression of the ChIFN-α Gene in Trans-
genic Tobacco Plants
Transgenic plants containing ChIFN-αcDNA were
examined for the expression levels by ELISA method .
Results showed that recombinant protein was able to neu-
tralize chook IFN-α antibody in sandwich ELISA . The
amounts of IFN protein detected varied from 0 .0003%
9423 期 SONG Li et al. : Expression of Bioactive Chicken Alpha Interferon in Transgenic Tobacco
Fig . 3 PCR and RT-PCR analysis
(A ) PCR analysis of ChIFN-αgene in transformed tobacco plants . The
arrow indicated the 573 bp position of product of expected mass . M : DL
2000 marker ; C1 : Plasmid of pSFRLH; T1 -T3 : DNA from transformed
plants; C2 : Water; C3 : DNA from no-transformation control plant . ( B)
RT-PCR analysis of the expression of ChIFN-α mRNA in transformed
tabacco . M: DL 2000 marker ; C1 : Non-transgenic tabacco plant; C2 :
Plasmid of pSFRLH; T1 -T3 : Transformed tabacco plants with pSFRLH;
C3 : RNA of transgenic tabacco with no reverse-transcriptase .
to0 .0033% of total solubleprotein for ChIFN-α-trans-
formed plants, with most found in # 5 plant, which
was estimated to be 0 .16μgprotein?kg tissueof trans-
formed leaves . The least was 0 .0003% in # 3 line
(corresponding to 0 .01μg IFN?kg tissue) .
2 . 3 Biological Activity of Plant Extracts Contain-
ing ChIFN-α
To determine the IFN activity expressed by tobac-
co plants, theCEFs were challenged by VSV and then
treated with ChIFN-α from one independent transfor-
mant ( # 3 line) . The results showed that recombinant
ChIFN-α could induce antiviral activity in CEFs, and
the level of IFN activity in plant extracts was about
5 .01×103 IU?g of tissue . No protection to the CEFs
appeared using the crude extract from non-transformed
tobacco plant . The result of represent experiment was
shown in Fig. 4 .
3 Discussion
In this study, ChIFN-αgenewas first successful-
ly introduced into plant cells using A. tumefaciens me-
diated transformation system . Histochemical staining of
tobacco tissues displayed the produce of transformed
plants . The insertion and transcription of ChIFN-α
gene in transformants were confirmed by PCR and RT-
Fig . 4 Microscopic appearanceof control ( A ) and treated with
recombinant IFN ( B) CEF cells, demonstrating cytopathic effects
PCR analysis, respectively . Results of ELISA andCPE
inhibition assay indicated the correct expression and
bioactivity of recombinant IFN in plant cells .
In the current study, the highest expression level
of ChIFN-αwas0 .16μg?kgof freshweight leaf materi-
al, which is equivalent to 0 .0033% of total soluble
protein in # 5 transformant . This productivitywas sim-
ilar to theproductionof human EGF peptide in tobacco
(Higo et al. , 1993 ) . In that research the introuduced
hEGF gene was expressed as an active protein at the
highest level of about 0 .001% of total soluble protein
in transgenic plant . However, compared to other for-
eign genes introduced into plant cells, the IFN expres-
sion was lower (Sawahel , 2002; Ohya et al. , 2002 ) .
The reasonmay be that the introduced IFN mRNA was
not efficiently translated or the synthesized proteins
were not enough to steady . The IFN productivity may
not be sufficient for a plant bioreactor to produce for-
eign protein . So, further studies areneeded to develop
amore robust systemsuch as useof tobacco mosaic vir-
usΩ sequence (Gallie et al. , 1991) , proper promoter
( Chen et al. , 2004) , appropriatesignal peptide (Peng
et al. , 2006 ) , removal of mRNA destabilizing se-
quences ( Perlok et al. , 1991 ) , codon optimization
(Fujimoto et al. , 1993; Peng et al. , 2006) and plant
chloroplast transformation ( Leelavathi and Reddy,
2003) .
Here, the recombinant ChIFN-α from transgenic
tobacco plant was confered protection against VSV in-
fection in CEF cells by using a CPE inhibition assay .
The IFN bioactivity was about 5.01×103 IU?g of to-
bacco tissue, which was higher than HuIFN-alpha (560
052 云 南 植 物 研 究 31 卷
IU?gof tissue) in plant extracts reportedbyOhya et al .
(2001) . However, the specific activity of recombinant
ChIFN-αwas for less than it in E. coli or baculovirus
( Schultz et al. , 1995b; Ruttanapumma et al. ,
2005) . Maybe the actual activity presented in the
study was likely higher than this value . It should be
noted, the CPE sample was the lowest expression line
( # 3 ) . The low productivity of IFN may reflect the
specific properties, or there were some pernicious in-
gredients for CEFs in the crude protein, thereby influ-
enced the accuracy of experimental results . The evalu-
ation and upgrade bioactivity of recombinant IFN in
transgenic plant should still be further studied .
In summary, ChIFN-α gene was correctly tran-
scribed and translated into polypeptide in tobacco
cells . Synthesized ChIFN-α showed the evident bioac-
tivity of 5 .01×103 IU?g of tissue at the expression of
0 .0003% . These results would prove helpful to pro-
duce functional animal cytokines with potential pharma-
ceutical applications in transgenic plants, which may
lead a new way to prevent infectious diseases or im-
prove the immune function of birds by direct oral ad-
ministration in poultry fields .
Acknowledgements : Wethank TheCenter of StemCell andTis-
sue Engineering Reaserch, Guiyang Medical College, for techni-
cal assistance with thecytopathic effect inhibition analysis .
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