CN106046136B - - 2 mutant EtMIC2-2119 of one breeder Eimeria Tenella microwire egg - Google Patents
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- CN106046136B CN106046136B CN201610390480.6A CN201610390480A CN106046136B CN 106046136 B CN106046136 B CN 106046136B CN 201610390480 A CN201610390480 A CN 201610390480A CN 106046136 B CN106046136 B CN 106046136B
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Abstract
The present invention relates to genetic engineerings and field of genetic engineering, provide -2 mutant EtMIC2-2119 of a breeder Eimeria Tenella microwire egg, the mutant is to carry out lactam enzyme by directional anagenesis in vitro with the EtMIC2 of wild type Eimeria tenella SD-01 (GenBank:KC333870.1) for basic protein, and establish gene mutation library, use yeast surface display protein mutation library, using the means such as selected by flow cytometry apoptosis and the evaluation of animal immune protecting effect, what the variant protein matter that screening immunogenicity and immune protective efficiency significantly improve finally obtained, the mutant variation protein has 11 amino acid mutation sites；The more natural EtMIC2 immune protective effect ACI of EtMIC2 after variation improves about 25.06%, can be used as vaccine and applies in resisting the fields such as chicken eimeria tenella infection.
Description
Technical field
The present invention relates to genetic engineerings and field of genetic engineering, and it is prominent to provide a breeder Eimeria Tenella microwire egg -2
Variant EtMIC2-2119.
Background technique
Chicken coccidiasis is a kind of Intestinal protozoan disease as caused by the Eimeria of special sexual cell endoparasitism.It is every according to estimates
Year whole world can be more than 3,000,000,000 dollars for preventing and treating globidiosis cost, and Chinese there are about more than 30 hundred million RMB every year.At present
The prevention and control of globidiosis rely primarily on drug and vaccine, the appearance with drug-resistant worm plant and the legislation limitation for coccidiostat,
People more pay attention to immunoprophylaxis.Vaccine in the market is mainly the mixing of a variety of chicken Eimerias egg capsule living, is had fine
Immune protective effect, but since live vaccine is there are safety problem and higher production cost, researchers will be more
Development of the energy for the novel molecular vaccine based on subunit.
Novel molecular vaccine development resists by the antigen with preferably antigenicity and immunogenicity since coccidia is complicated
The limitation of original structure and the history of life, difficulty and the antigen immunogenicity screening of Antigen Identification hinder subunit vaccine development
Progress.Researchers have carried out a large amount of research also to improve the immune protective effect of antigen, the development including new adjuvant, benefit
The nospecific immunity etc. of raw bacterium induction, but these all cannot fundamentally improve the immunogenicity of antigen.Therefore, how to obtain
A kind of efficient novel vaccine becomes one of urgent problem to be solved.
Summary of the invention
Aiming at the problems existing in the prior art, the present invention provides a breeder Eimeria Tenella microwire egg -2 mutation
Body EtMIC2-2119 and its encoding gene, the mutant are with the EtMIC2 of wild type Eimeria tenella SD-01
(GenBank:KC333870.1) lactam enzyme by directional anagenesis in vitro is carried out for basic protein, and establishes gene mutation library, use yeast table
Face display protein mutation library screens immunogene using the means such as selected by flow cytometry apoptosis and the evaluation of animal immune protecting effect
What the variant protein matter that property and immune protective efficiency significantly improve finally obtained, which has 11 amino acid
Mutational site.The more natural EtMIC2 immune protective effect ACI of EtMIC2-2119 after variation improves about 25.06%, can be used as
Vaccine is applied in resisting the fields such as chicken eimeria tenella infection.
The dominant mechanism that inventor applies in the present invention is as follows:
Directed evolution is widely used in protein engineering field, wherein important one of application is exactly to improve being immunized for antigen
Originality.The change of key amino acid has a significant impact for the immune response of antigen and Immune discrimination, such as reversible inhibition host
Immune response, influence T cell activation, improve protein immunogenic etc., be using random mutation building gene mutation library
One of common method of directed evolution.
And EtMIC family protein has important role in terms of coccidia migrates, invades host cell and existence intracellular,
EtMIC2 protein can all express within the entire life cycle of Eimeria tenella, research shows that EtMIC2 gene or
Protein can inducing moiety immunoprotection reaction, be good vaccine candidate antigen.
Therefore the present inventor is oriented evolution for EtMIC2 using random mutation technology, establishes gene mutation
Purpose variant protein matter is screened using yeast surface display technology combination FACS and animal immune protection test, to change in library
The immunogenicity of kind EtMIC2 protein provides technical support to resist chicken eimeria tenella infection.
The specific technical solution of the present invention is as follows:
The EtMIC2 nucleotide sequence (GenBank:KC333870.1) that the present invention is announced according to GenBank first manually closes
At natural EtMIC2 sequence, nucleotide sequence is as shown in Seq ID No:3, by the EtMIC2 nucleotide sequence and pEASY-T1
Carrier connection modeling EtMIC2-T1.
Inventor is using the above-mentioned EtMIC2-T1 of fallibility PCR amplification and obtains mutant, establishes gene mutation library later, makes
With yeast surface display protein mutation library, the hands such as selected by flow cytometry apoptosis and the evaluation of animal immune protecting effect are then applied
The variant protein matter that section, screening immunogenicity and immune protective efficiency significantly improve, is finally obtained mutation of the present invention
Body is named as EtMIC2-2119, and the mutant nucleotide sequence is as shown in Seq ID No:1, and the amino acid sequence of coding is such as
Shown in Seq ID No:2, by the way that compared with unmutated EtMIC2, which has 11 amino acid mutation positions
Point, specific mutated site are as follows:
EtMIC2-2119 different amino acids and its site compared with EtMIC2
Inventor has carried out immune protective effect verifying to it after obtaining above-mentioned mutant, as a result, it has been found that compared to
Unmutated EtMIC2 protein, mutant EtMIC2-2119 Western Immuno group body weight increase are significant；Mutant EtMIC2-
The caecum average lesion score of 2119 Western Immuno groups is significantly lower than unmutated EtMIC2 immune group；Mutant EtMIC2-
2119 Western Immuno groups in terms of gram excrement egg sac number compared with native protein EtMIC2, though Oocyst Production of Three is reduced,
But do not show notable difference；The ACI of mutant EtMIC2-2119 Western Immuno group is 172.48, than unmutated
EtMIC2 protein immunization group is high by about 25.06%, has good anticoccidial effect, it is seen that mutant provided by the present invention
EtMIC2-2119 can be used as vaccine and apply in resisting the fields such as chicken eimeria tenella infection.
In conclusion present invention obtains a kind of completely new -2 mutant EtMIC2- of chicken Eimeria Tenella microwire egg
2119, which improves about 25.06% compared with for mutant immune protective effect ACI, and it is soft in resistance chicken to can be used as vaccine
It is applied in the fields such as tender eimeria tenella infection.
Detailed description of the invention
Fig. 1 is yeast surface display protein EtMIC2 and mutation EtMIC2 protein s DS-PAGE testing result gray scale
Figure,
M is Marker in figure；1 is EBY100 negative control；2 be EtMIC2 protein；3 be EtMIC2-2119 variation egg
White matter；
Fig. 2 is yeast surface display protein EtMIC2 and mutation EtMIC2 protein Western blot testing result
Grayscale image,
M is Marker in figure；1 is EtMIC2 protein；2 be EtMIC2-2119 variant protein matter；
Fig. 3 is that selected by flow cytometry apoptosis shows the yeast cells schematic diagram for having EtMIC2 albumen.
Specific embodiment
Test material and reagent
1, bacterial strain, carrier: various bacterial strains according to the present invention are conventional bacterial strain, can directly be reached from the market；Institute
The cloning vector pEASY-T1 of use is purchased from Quan Shi King Company；Expression vector pET-30a (+) is obtained using this field routine techniques
It obtains or buys.
2, main agents and drug: rTaq enzyme, dNTPs, DNA Marker, Protein Marker, PCR product purifying examination
Agent box, DNA plastic recovery kit and plasmid extraction kit are purchased from Beijing Quanshijin Biotechnology Co., Ltd；Restriction enzyme
Enzyme BamH I, Nhe I, Hind III and Sal I are purchased from Dalian treasured bioengineering Co., Ltd；T4DNA ligase is purchased from
Fermentas；FITC marks sheep anti-mouse igg, HRP label sheep anti-mouse igg and IgA to be purchased from the limited public affairs of Beijing Bo Aosen biotechnology
Department；Quick StartTMBradford 1x Dye Reagent is purchased from Bio-Rad company；His· Purificat
Ion Kit is purchased from Merck company；Complete Freund's adjuvant and incomplete Freund's adjuvant etc. are purchased from Sigma company；Dithiothreitol (DTT)
(DTT) it is purchased from Sigma company；Yeast plasmid rapidly extracting kit is purchased from the green skies Bioisystech Co., Ltd in Shanghai, other
Conventional reagent is that domestic analysis is pure.
3, the preparation of culture medium:
(1) LB liquid medium: weighing yeast extract 5g using electronic balance, tryptone 10g, sodium chloride 10g, molten
Solution is in 1L deionized water, 121 DEG C of high pressure sterilization 20min.
(2) solid LB media: preparation LB liquid medium 100mL by the above process, addition 1.5g agar powder, 121 DEG C
High pressure sterilization 20min is poured into culture dish after cooling.
(3) solid LB/AMP (Kan) culture medium: LB liquid medium 100mL is prepared by the above process, is down to not to temperature
When scalding one's hand, 100 μ L Amp (Kan) are added and store liquid, after mixing, pour into culture dish, 4 DEG C of preservations after cooling.
(4) yeast extract 10g, tryptone 20g, glucose 20g YPD fluid nutrient medium: are weighed using electronic balance
It is dissolved in 1L deionized water, 121 DEG C of sterilizing 15min.
(5) solid YPD culture medium: preparation YPD fluid nutrient medium 100mL by the above process, addition 1.5g agar powder, 121
DEG C high pressure sterilization 20min is poured into culture dish after cooling.
(6) yeast nitrogen 6.7g, sour hydrolyzed casein 5g, glucose SD-CAA fluid nutrient medium: are weighed using electronic balance
20g is dissolved in 1L deionized water, is sufficiently dissolved using magnetic stirring apparatus, 121 DEG C of high pressure sterilization 15min.
(7) solid SD-CAA culture medium: 1.5-2g agar powder will be added in 100ml SD-CAA fluid nutrient medium, high pressure is gone out
After bacterium, it is poured into culture dish.
(8) SG-CAA fluid nutrient medium: no amino acid is weighed without ammonium sulfate yeast nitrogen 6.7g, sour water using electronic balance
Solution casein 5g is dissolved in 1L deionized water, is sufficiently dissolved using magnetic stirring apparatus, 121 DEG C of high pressure sterilization 15min are added later
4 DEG C of galactolipin preservations of the filtration sterilization of 20g.
4, the experimental methods of molecular biology illustrated in detail is not done in the present embodiment, referring to " Molecular Cloning: A Laboratory
Guide " specific method listed in book of (third edition) J. Pehanorm Brooker one carries out, or according to kit and product description
It carries out.
The acquisition of 1 mutant EtMIC2-2119 of embodiment
The EtMIC2 nucleotide sequence (GenBank:KC333870.1) that basic invention is announced according to GenBank first is artificial
Synthesis of natural EtMIC2 sequence, EtMIC2 nucleotide sequence obtained, nucleotide sequence as shown in Seq ID No:3, it
It is connect to modeling EtMIC2-T1 with pEASY-T1 carrier afterwards；Using EtMIC2-T1 as template, using EtMIC2-951-F and
EtMIC2-951-R is primer, using Divers ify PCR Random Mutagenes is Kit (Clontech, USA) into
Row fallibility PCR amplification EtMIC2 mutant obtains fallibility PCR product；Wherein the primer is as shown in table 1:
Mutant EtMIC2-2119 specific primer described in table 1.
The preparation of mutant described in embodiment 2
By the fallibility PCR product obtained in embodiment 1 in unpurified situation, after being connect with pEASY-T1 carrier, turn
Enter 10 competent cell of Top, be coated with ammonia benzyl LB agar plate, the culture in 37 DEG C of incubators is until monoclonal colonies increase to 107For
Only, EtMIC2 gene mutation library is constructed；
Above-mentioned includes the recombinant plasmid of gene mutation library and pCTCON2 plasmid, is carried out using Nhe I and Sal I
Double digestion transfects saccharomyces cerevisiae EBY100 and (is purchased from after connecting in the gene mutation library of double digestion with pCTCON2 carrier
Invitrogen), obtain recombinant bacterial strain EBY100/EtMIC2, PCR identifies positive bacterium colony (design the primer is as shown in table 2)；
The bacterial strain EBY100 containing recombinant plasmid is taken, is coated on SD-CAA solid medium in 30 DEG C of incubators, cultivates 48h, until yeast
Bacterium bacterium colony is 108Until a, EtMIC2 mutein library recombinant bacterial strain EBY100/EtMIC2 is constructed；
Table 2
The ratio of the 1:100 by volume of the EBY100 bacterial strain containing recombinant plasmid is taken to be inoculated in the SD-CAA of 100mL, 30
DEG C, 220rpm cultivates about 8h, until OD600 about 1.0 or so stops culture, by 4 DEG C under sterile conditions of thallus, 3000r/min,
It is centrifuged 5min, discards supernatant, using sterile distilled water washing three times thoroughly to remove glucose, 3000r/min is centrifuged 5min,
Thallus is resuspended using the SG-CAA culture medium 100mL of Fresh, and is transferred in the triangular flask of sterilizing, 30 DEG C, 220rpm is lured
Lead culture about 12h；
The yeast bacteria concentration of inducing expression is adjusted to 5x107A/10 μ L, are added the DTT solution of final concentration of 100mM, and 4
DEG C effect overnight, during which constantly shake.The protein solution of acquisition is added in bag filter, it is molten using PBS under the conditions of 4 DEG C
Liquid dialyses and repeatedly (each 2h) removes DTT, and 10,000rpm, it is centrifuged 1min, supernatant is drawn, uses SDS-PAGE and Western
Blot analyzes expression, positive band (as illustrated in fig. 1 and 2) occurs in 50kDa or so as the result is shown；
The yeast cell for taking above-mentioned inducing expression washes 2 times using the PBS-BSA (1mg/mL) that pH is 7.2, and 10,000r/
Min is centrifuged 1min, and thallus is resuspended in the diluted anti-EtMIC2 in rabbit source of 40 μ L1:200 mostly anti-(using prior art preparation),
It mixes, ice bath 30min, is washed 2 times using the PBS-BSA (1mg/mL) that pH is 7.2,10,000r/min, centrifugation 1min, by thallus
It is resuspended in the diluted FITC label mouse anti-rabbit IgG monoclonal antibody of 50 μ L1:75, is mixed, ice bath 30min, the PBS-BSA for the use of pH being 7.2
(1mg/mL) washes 2 times, and 10,000r/min, it is centrifuged 1min, BD FACSAria Cel l-Sort ing System carries out sterile
Screening contains mutant strain, to show the difference of unmutated EtMIC2 protein Yu the anti-EtMIC2 polyvalent antibody affinity in rabbit source
For control, the weaker door P2 of delineation fluorescence signal (account for the 2.7% of total cell number, fluorescence intensity are as follows: 2757.18, represent with
How anti-EtMIC2 affinity is weaker) (as shown in Figure 3), after step sizing three times, coating SD-CAA solid agar plate, 30
DEG C culture 48h, collect 10 plants of yeast single colonie.
Routine techniques extracts the above-mentioned 8 saccharomycete pCTCON2 recombinant plasmid containing mutant gene, using the plasmid as mould
Plate uses PrimeSTAR DNA Polymerase using His-EtMIC2-F shown in table 2 and His-EtMIC2-R as primer
(Takara) it carries out PCR amplification EtMIC2 mutant gene and above-mentioned PCR product and pET30a is used into BamH I and Hind
III carries out double digestion, is ligated and transformed into BL21 bacterial strain and obtains recombinant bacterial strain BL21/EtMIC2-1130.Positive colony is by 1:100's
Ratio is inoculated in the LB culture medium of resistance containing kanamycin, cultivates 3h in 37 DEG C, the constant-temperature table of 220rpm, until
When OD600 ≈ 0.6, the IPTG solution inducing expression 4h of final concentration of 1mM is added into each pipe, 8,000rpm are centrifuged 5min, receive
Collect thallus, 1 × combination buffer washing thalline 2 times, use is added 4mL 1 × combination with thallus obtained by every 100mL culture medium and delays
Thallus is resuspended in fliud flushing ratio.After ultrasonic disruption (power 300W, opens 3sec, stops 6sec), 16,000g centrifugation 20min are collected
Supernatant and precipitating, precipitating add 5mL buffer ratio by every 100mL culture, and 1 × combination buffering of final concentration of 8M urea is added
Liquid thoroughly dissolves inclusion body, and 16,000g centrifugations collect supernatant in 30 minutes again, collect supernatant mixing twice, and adjusting pH is 8.0, makes
With 0.45um membrane filtration, HisBind protein purification is used for (according to HisPurificat ion Kit explanation
Book carries out), mutant protein is obtained, EtMIC2-2119 is named as, nucleotide sequence is as shown in Seq ID No:1, coding
Amino acid sequence as shown in Seq ID No:2, by the way that compared with unmutated EtMIC2, which has 11 amino
Acid mutation site.
According to Quick StartTMBradford 1x Dye Reagent specification, using 0.125,0.25,0.5,
0.75, each 5 μ L of the BSA standard items of 1.0,1.5 and 2.0mg/mL is added in the EP pipe containing 250 μ L dye reagents, sufficiently mixed
Even, room temperature acts on 5min, and each gradient does three repetitions；It takes each 5 μ L of testing protein quality sample to be added to try containing 250 μ L dyestuffs
It in the EP pipe of agent, mixes well, room temperature acts on 5min, and each gradient does three repetitions, mixed liquor is added to the hole of ELISA Plate
In, OD595 is measured using microplate reader；Standard curve is drawn, calculating mutant protein concentration according to the formula obtained is 1.36mg/
mL。
3 mutant EtMIC2-2119 immune protective effect of embodiment determines
The weighing of 1 Japanese instar chickling, rejects the biggish chicken of bodyweight difference.100 chick are divided into 4 groups, respectively PBS-I altogether
(not immune not infected group), (infected group is not immunized) in PBS-II, native protein EtMIC2, mutant EtMIC2 immune group, and every group
It 25, is raised in isolation cover.In 7 age in days, each group chicken carries out subcutaneous multiple spot respectively and Freund's complete adjuvant emulsification is immunized
The 100 μ L of protein or PBS of final concentration of 0.5mg/mL carries out the emulsification of booster immunization incomplete Freund's adjuvant in 14 age in days
The final concentration of 0.5mg/mL of correspondence 100 μ L of protein or PBS.21 Japanese instar chicklings equal oral vaccination 30 in addition to PBS-I group,
000 Sporulated Oocysts.According to immune chicken body weight increase situation, cecal lesion score, gram excrement egg sac number (OPG), anti-ball
The indexs such as worm index (ACI) determine mutant protein immune protective effect:
One, body weight increase situation measures: the specific method is as follows:
It carries out weighing by plumage when 21 ages in days (before attacking worm) and 31 ages in days (after attacking worm), records weighing results, and calculate flat
Weight gain (average weight gain (g)=- 21 age in days average weight (g) of 31 age in days average weight (g)) and the relative weight gain amount (the relative weight gain
Rate (%)=(weight gain of infected group/weight gain of not immune group is not infected) × 100%).The result shows that: compared with PBS-II group,
Immune native protein EtMIC2 group and mutant EtMIC2-2119 group body weight increase are significant, compared to unmutated EtMIC2 egg
White matter group, mutant EtMIC2-2119 group body weight increase are significant.
Two, cecal lesion score measures: the specific method is as follows:
According to (1970) 5 points of principles processed of Johnson and Reid, the 5d after infecting coccidia, every group is cutd open and kills 6 chickens, takes caecum
Appearance blutpunkte is looked first at, swelling situation, then longitudinal to split caecum, observed content object state is disposed using tap water
Content observes the bleeding of caecum inner wall, these comprehensive lesions carry out evaluation and score.The result shows that: compared with PBS-II group
The score of EtMIC2, EtMIC2-2119 immune group caecum average lesion significantly reduces, and the caecum of mutant EtMIC2-2119 group
Average lesion score is significantly lower than unmutated EtMIC2 immune group.
Three, gram excrement egg sac number (OPG): the specific method is as follows:
5d to 10d starts to collect each group excrement respectively daily after attacking worm, 4 DEG C of preservations after mixing.Every group takes 1g, adds
Enter 10mL tap water and carry out 10 times of dilutions, after stirring, is ground using 400 mesh filter screens and be filtered to remove big excrement
Particle.It takes on the 10 μ L drop of filtrate mixed well and glass slide, coccidian oocyst sum in the 10 μ L dilutions that count under low power lens,
According to formula (OPG=coccidian oocyst sum × 104) calculate OPG and protective rate.The result shows that: compared with PBS-II group, albumen
Matter EtMIC2, EtMIC2-2119 immune group Oocyst Production of Three significantly reduces, but variant protein matter EtMIC2-2119 group gram
In terms of excrement egg sac number compared with native protein EtMIC2 group, it is not significant that egg sac number reduces difference.
Four, calculate anticoccidial index (ACI): the specific method is as follows:
By Merck company of U.S. formula: ACI=(survival rate+body weight increase rate) × 100- (pathological changes value+oocyst value).Root
The curative effect of anticoccidial is determined according to ACI；ACI≤120 be it is invalid, 120 < ACI≤160 be it is medium effectively, 160 < ACI≤180 are
Well, ACI > I80 is outstanding.As the result is shown: the ACI of unmutated EtMIC2 Western Immuno group is 137.92, is had medium
Anticoccidial protecting effect, the ACI of mutant EtMIC2-2119 Western Immuno group is 172.48, than unmutated EtMIC2 albumen
Immune group is high by about 25.06%, has good anticoccidial effect.
Claims (2)
1. -2 mutant of a breeder Eimeria Tenella microwire albumen, which is characterized in that the mutant is named as EtMIC2-
2119, amino acid sequence encodes the core of -2 mutant of chicken Eimeria Tenella microwire albumen as shown in Seq ID No:2
Nucleotide sequence is as shown in Seq ID No:1.
2. -2 mutant of chicken Eimeria Tenella microwire albumen described in claim 1 resists chicken Eimeria Tenella in preparation
Application in the vaccine of infection.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0135712A2 (en) * | 1983-08-19 | 1985-04-03 | American Cyanamid Company | Antigens and monoclonal antibodies reactive against sporozoites of eimeria spp. |
| CN102816221A (en) * | 2012-08-06 | 2012-12-12 | 广东省农业科学院兽医研究所 | Chicken E. tenella MA1 (EtMA1) gene, vector, recombinant strain, EtMA1 protein, EtMA1-Outside domain, and use of the EtMA1 protein and the EtMA1-Outside domain |
| CN103276003A (en) * | 2013-06-02 | 2013-09-04 | 吉林农业大学 | Immunological regulation type chicken E.tenella resistant recombinant lactobacillus and preparation method thereof |
-
2016
- 2016-06-03 CN CN201610390480.6A patent/CN106046136B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0135712A2 (en) * | 1983-08-19 | 1985-04-03 | American Cyanamid Company | Antigens and monoclonal antibodies reactive against sporozoites of eimeria spp. |
| CN102816221A (en) * | 2012-08-06 | 2012-12-12 | 广东省农业科学院兽医研究所 | Chicken E. tenella MA1 (EtMA1) gene, vector, recombinant strain, EtMA1 protein, EtMA1-Outside domain, and use of the EtMA1 protein and the EtMA1-Outside domain |
| CN103276003A (en) * | 2013-06-02 | 2013-09-04 | 吉林农业大学 | Immunological regulation type chicken E.tenella resistant recombinant lactobacillus and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis;Hui Sun et al.;《Vaccine》;20140211;第32卷;第1869-1876页 |
| 柔嫩艾美耳球虫EtMIC2的酵母表面展示;孙慧等;《中国兽医学报》;20140228;第34卷(第2期);第231-235页 |
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