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CN108048339A - A kind of bacterial strain for recombinantly expressing phosphatidase and its application - Google Patents

A kind of bacterial strain for recombinantly expressing phosphatidase and its application Download PDF

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Publication number
CN108048339A
CN108048339A CN201711280976.9A CN201711280976A CN108048339A CN 108048339 A CN108048339 A CN 108048339A CN 201711280976 A CN201711280976 A CN 201711280976A CN 108048339 A CN108048339 A CN 108048339A
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phosphatidase
enzyme activity
aspergillus
application
arg
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CN108048339B (en
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王艺璇
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Wuxi Weilan Biotechnology Co ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

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Abstract

The invention belongs to gene engineering technology fields, and in particular to a kind of bacterial strain for recombinantly expressing phosphatidase and its application.The pichia pastoris engineered strain that the present invention is built can high efficiency recombinant expressed aspergillus source in vitro phosphatidase, shake-flask fermentation enzyme activity is up to 470U/mL, so as to compensate for the deficiencies in the prior art, realizes the secreted in vitro expression of aspergillus phosphatidase.

Description

A kind of bacterial strain for recombinantly expressing phosphatidase and its application
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of bacterial strain for recombinantly expressing phosphatidase and its application.
Background technology
Phosphatidase is the class of enzymes of metabolism and the biosynthesis of being responsible for phosphatide in organism, can the various water of catalyzing glycerol phosphatide Solution reaction, can fall into 5 types according to the difference of its hydrolytic phosphatide position：Phospholipase A1, A2, B, C, D.
The biological function of phosphatidase can be summarized as three classes：The maintenance and reparation of membrane structure；Endocellular metabolism mechanism and The adjusting of signal transduction and the digestion of internal phosphatide.Such as phospholipase A2（PLA2）Largely it is present in snake venom, bee venom, scorpion venom, animal In pancreas and plant tissue, it can mediate generation that there is phosphatide transhipment, film reparation, extracellular hydrolysis and neuron transfer factor etc. Lipid medium.And phospholipase C（PLC）Play a part of second messenger in the vital movement of biology, be widely present in each Among kind protokaryon, eucaryote, simply slightly has difference on molecular structure.
Phosphatidase not only has critically important physiological function in vivo, but also with very high application value, it can be wide All various aspects such as scientific research, medicine, feed improvement and food industry are used in generally.Such as PLA2 is applied in terms of medicine The production of anti-inflammatory drug, PLC are applied to development of antitumor drug etc.；In terms of feed improvement, phosphatidase is added in feeding Glycerophosphatide is hydrolyzed in material, the utilization ratio of feed can be improved and promotes growth of animal；In terms of food industry, phospholipase A1 （PLA1）, phospholipase A2（PLA2）And phospholipase C（PLC）It can be widely used in fat degumming, simultaneously because phosphatidase can make Dough forms colloidal complex, it is possible to reduce starch retrogradation, therefore it is also very extensive baking sector application.In addition phospholipase D （PLD）It is also widely used for glycerophosphatide modification.
Phosphatidase is prevalent in animal, plant and microorganism.Since microbe-derived phosphatidase is numerous with species It is more, be mostly excretion, easily monomeric protein, a large amount of quick the features such as preparing and is at low cost, have become current food work The most important approach of industry application.The microorganism of common production phosphatidase mainly have Pseudomonas alcaligenes ( Pseudomonasalcaligenes), Vibrio harveyi (Vibrio harveyi), serratia marcescens ( Serratiamarcescens), streptomycete (Streptomyces) and aspergillus oryzae (Aspergillus oryzae) etc..
But the phosphatidase production capacity of existing wild strain is often very limited, can not meet industrial requirement.Cause This, in order to adapt to industrial processes high temperature, high pressure, highly acidity, high ion concentration and exceeded extreme of heavy metal ion Environment is, it is necessary to further screen the new gene and its production bacterial strain with the phosphatidase for improving performance.
The content of the invention
The object of the present invention is to provide one kind to derive from aspergillus（Aspergillus sp.）Phosphatidase and its recombination expression Engineered strain.The present invention is transformed into Pichia pastoris by building the expression vector containing phospholipase gene（Pichia pastoris）In, structure obtains pichia pastoris engineered strain, can efficient secretory expression phosphatidase.
One aspect of the present invention provides a kind of Pichia yeast engineering, carries the recombinant plasmid that can express phosphatidase.
The phosphatidase, amino acid sequence are SEQ ID NO: 1.
The phosphatidase, the nucleotides sequence of encoding gene are classified as SEQ ID NO: 2.
One aspect of the present invention provides application of the Pichia yeast engineering in phosphatidase is produced.
Advantageous effect
The pichia pastoris engineered strain that the present invention is built can high efficiency recombinant expressed aspergillus source in vitro phosphatidase PD, shaking flask hair Ferment enzyme activity is up to 470U/mL, so as to compensate for the deficiencies in the prior art, realizes the secreted in vitro expression of aspergillus phosphatidase.And And the Optimun pH of phosphatidase PD of the present invention is 8.5, optimum temperature is 45 DEG C, quiet in 4-50 DEG C of water-bath Put 1 hour, enzyme activity is more stable, has almost no change, in 55-65 DEG C of water-bath stand 1 it is small when after remain to retain more than 80% Enzyme activity, stood in 70 DEG C of water-baths 1 it is small when after still can retain vigor more than 50%, heat-resisting effect is notable.The phosphatidase can It is widely used in vegetable oil enzymatic degumming, remaining phosphorus is only 13.29ppm in degummed oil, and degumming effect is good, and application prospect is wide It is general.
Specific embodiment
The routine techniques and method that the present invention has used genetic engineering and biology field uses, such as MOLECULAR CLONING:A LABORATORY MANUAL, 3nd Ed. (Sambrook, 2001) and CURRENT Recorded method in PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These generality are with reference to text It offers and provides definition well known by persons skilled in the art and method.But this be not meant to limit the invention to it is described Any specific method, experimental program and reagent, because they can change.
Unless being separately construed as limiting herein, whole technical term and scientific terminology used herein have neck belonging to the present invention The identical meanings that the those of ordinary skill in domain is generally understood.DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 3nd Ed. (Singleton et al., 2006) and COLLINS DICTIONARY BIOLOGY (Hale Et al., 2003) the general explanation of many terms used in the present invention is provided for technical staff.
In following embodiments of the present invention, unless otherwise indicated, the detection of phospholipase activity uses molybdenum blue method.
Molybdenum blue method is to detect the method for phosphatidase enzyme activity by detecting the Phos that hydrolysis releases, specifically Process is as follows：
Reagent：
10% (w/v) ascorbic acid：2g is taken to be dissolved in 20mL water, can slightly heat promotion dissolving, 4 DEG C preserve (Fresh).
2.5% (w/v) ammonium molybdate solution：2.5g ammonium molybdates is taken to be dissolved in 30% sulfuric acid solution of 100mL, room temperature；
1% mixed phosphatide：0.5g powdered soybean phospholipids are dissolved in 50mL deionized waters, are stored again after being rotated uniformly in beaker, 4 DEG C It preserves (Fresh)；
500mM Tris-Cl(pH7.5)：The mother liquor of the 1M Tris-Cl of 100mL is taken, adds water to 150mL, adjusts pH to 7.5, it is fixed Hold to 200mL, room temperature preserves；
100mM CaCL2：It weighs 0.555g CaCL2 and is dissolved in 50mL water, 4 DEG C of preservations；
1M Tris-Cl(pH9.0)：It weighs 24.228g Tris to be dissolved in 180mL water, adjusts pH to 9.0, be settled to 200mL, often Temperature preserves；
500mM MgCl2：It weighs 9.521g MgCl2 to be dissolved in 200mL water, room temperature preserves；
500U/mL alkaline phosphatases (AP, purchased from Takara)：10U/ μ L are 500 U/mL after 20 times of dilution, and 4 DEG C preserve.
The formulation of the standard curve of P-Mo blue detection Phos method:
The dipotassium hydrogen phosphate reagent powder 0.1431g of drying is taken, deionized water is dissolved in, is settled to 100mL, that is, is configured to 1mg/mL PO4 3-Dipotassium hydrogen phosphate solution.From 1mg/mL PO4 3-Dipotassium hydrogen phosphate solution take out 0,2,5,8,10,15,20 respectively, 50 μ L add in deionized water and complement to 940 μ L, add 10% (w/v) ascorbic acid solution of 20 μ L, 40 are added in after shaking up 30s 2.5% (w/v) ammonium molybdate solution (solvent is 30% sulfuric acid solution) of μ L, is finally settled to 1mL, final PO4 3-Concentration is 0, 2,5,8,10,15,20,50 μ g/mL.Each concentration point is parallel does 3 repeat samples.After 37 DEG C of water-bath 10min, in absorbance Light absorption value detection is carried out under 700nm, linear fit is carried out to get the standard curve detected to P-Mo blue to obtained data.
1 gene cloning of embodiment
Applicant extracts aspergillus first（Aspergillus sp.）WLP（The bacterial strain is sieved by inventor Wang Yi fine jades in January, 2017 Selected from the fallen leaves surface in Qingdao of Shandong province Laoshan District forest farm）Genome DNA.Then using genome DNA as template, profit It is expanded with upstream and downstream primer.
PCR amplification condition is 95 DEG C of 4min；94℃ 30S；30 Xun Huans of 55 DEG C of 40S, 72 DEG C of 1min；72℃ 7min.Pcr amplification product is recycled using gel reclaims kit, send to Huada Gene Research Center, Beijing and carries out sequencing analysis.
The results show that the nucleotides sequence of amplified production is classified as SEQ ID NO:2, the amino acid sequence of coding is SEQ ID NO:1.It compares and finds through NCBI Blast, SEQ ID NO:1 with aspergillus fumigatus (Aspergillus fumigatus) phosphatidase Amino acid sequence similarity is only 68%, and therefore, what the present invention obtained is a new allele, is named as PD.
The structure of 2 expression vector of embodiment
The amplified production of recycling is connected respectively to pMD18-T carriers, obtains cloning vector pMD-PD.Using plasmid pMD-PD as mould Plate, design primer carry out PCR amplification, and amplification condition is 95 DEG C of 4min；94 DEG C of 40s, 56 DEG C of 40s, 72 DEG C of 1.5min, totally 30 A cycling；72℃ 7min.Gel recycles amplified production, carries outEco RI and Not I double digestions.Equally, to expression plasmid PPIC9K is also carried outEco RI and Not I double digestions.With T4 ligases 4 DEG C of double digestion product, that is, clone gene and expression vector Connection is overnight.Finally, connection product is imported e. coli bl21.Corresponding positive colony expression plasmid is named as pPIC-PD.
Embodiment 3 recombinantly expresses the structure of engineering bacteria
Expression plasmid pPIC-PD is usedSal It after the identification of I restriction enzyme digestion and electrophoresis, is concentrated through ethanol precipitation, DNA concentration is measured, with 3 μ g/ μ L Concentration dilution plasmid fragments save backup.Pichia pastoris GS115 Electroporation-competent cells are prepared, are finally resuspended in 1 mL precoolings Electrophoretic buffer in (MgCl containing 1mM2, 10mM HEPES, 250mM sucrose, pH 7.8).In 80 μ L competent cells Add in 5 μ L linearisation recombinant plasmids；Electricity conversion（Condition is 1500V, 200 Ω, 25 μ F）；Finally it is coated on MM tablets（MM is cultivated Base component：1.34%YNB, 4 × 10-5% biotins, 0.5% methanol）, one of positive transformant is selected, is named as Pichia pastoris PD（Pichia pastorisPD）.
Embodiment 4 is fermented and enzyme activity determination
Embodiment 3 is built to obtained Pichia yeast engineering PD and is inoculated in 5ml BMGY (1% yeast extract, 2% albumen It is old, 1. 34 % YNB, 4 × 10-5% biotins, l% glycerine）, 30 DEG C of overnight incubations, thalline were collected by centrifugation, and thalline is added in 50ml BMMY inducing cultures（1% yeast extract, 2% peptone, 1. 34 % YNB, 4 × 10-5% biotins, 0.5% first Alcohol）, it is every 12 it is small when add 50 μ L methanol, Fiber differentiation 7 days, zymotic fluid centrifugation takes supernatant to carry out SDS-PAGE electrophoresis detections, It turns out that have a protein band at 27kDa, it is consistent with the molecular size range of prediction.
By 0.5ml fermented supernatant fluids, the 0.1M pH value 8.0Tris-HCL buffer solutions of 2.0ml, 2.5ml 2% phosphatidyl courage Alkali（The use of purity is more than 98% phosphatidyl choline, purchased from Aladdin reagent Co., Ltd）Mixing, in 150rpm, 37 DEG C of water-baths, When oscillating reactions 24 is small.
After reaction, 1ml is sampled, adds in n-hexane extraction in equal volume, vibrates mixing, 12000rpm is centrifuged 2 minutes, taken Go out upper organic phase part, add in into a new pipe.Lower water is taken mutually to repeat extraction, centrifugally operated, collects, merge twice just Hexane extract is uncapped, and is placed in draught cupboard organic phase volatilization is complete.Per Guan Zhongjia 15ul isopropanols, part dissolving is filled.It takes 5ul point chromatoplates.Carry out TLC detections（TLC detection methods referring to document Toida J., Arikawa Y., Kondou K., Fukuzawa M..Purification and characterization of triacylglycerol lipase from Aspergillus oryzae.1998.Bioscience Biotechnology Biochemistry,62(4):759-763）.
TLC testing results show occur diglyceride in hydrolysate, thus judge new gene PD provided by the invention The albumen of coding is phosphatidase.
In the reaction system of 200 μ L, containing powdered soybean phospholipid 0.5% (w/v), buffer system (25mM Tris-Cl, PH7.5), 5mM CaCl2 add in 20 μ l (3 μ g/ml) of phosphatidase PD, and reaction carries out 30 min, and the chloroform for adding in 200 μ L shakes Mixing 30s is swung, 12,000rpm centrifugation 1min is carried out, 80 μ L supernatants is taken to be added in phosphatidase reaction system, final volume 200 μ L, the system also contain 50mM Tris-Cl (pH9.0), 10mM MgCl2, CIAP10U/ μ L.Reaction in 37 DEG C of water-baths into Row 30min.Each deionized water for adding in 740 μ L, adds the ascorbic acid of 10% (w/v) of 20 μ L and 2.5 % of 40 μ L (w/v) ammonium molybdate solution.37 DEG C of colour developing 10min.The solution after colour developing is taken to carry out absorbance 700nm detections.It is examined with reference to P-Mo blue The standard curve of survey carries out analysis recurrence to data, is multiplied by after extension rate to get energy value.
Measurement result is shown：Phosphatidase enzyme activity is 470U/mL in above-mentioned fermented supernatant fluid, so as to illustrate structure of the present invention Pichia yeast engineering PD really can secreting, expressing phosphatidase PD in vitro.
5 phosphatidase characterization analysis of embodiment
1st, most suitable action pH analysis
With pH value be respectively 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0,12.0 buffer solution to above-mentioned Fermented supernatant fluid is diluted, and the enzyme activity of above-mentioned fermented supernatant fluid is measured under the conditions of 35 DEG C of temperature, using highest enzyme activity as 100%, Opposite enzyme activity is calculated, is pH- with respect to enzyme activity curve.The results show：The Optimun pH of phosphatidase PD of the present invention is 8.5。
, optimum temperature analysis
Respectively at 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 60 DEG C, 65 DEG C, 70 DEG C, enzyme activity is measured under conditions of pH5.5, Using highest enzyme activity as 100%, opposite enzyme activity is calculated, does temperature-opposite enzyme activity curve.The results show：Phosphatidase PD of the present invention Optimum temperature be 45 DEG C.
, temperature stability
500ul is taken to be distributed into aliquot above-mentioned fermented supernatant fluid, be respectively placed in 4 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, After keeping the temperature 1h in 50 DEG C, 55 DEG C, 60 DEG C and 70 DEG C of water-bath, enzyme activity is detected, using the enzyme activity of sample under 4 DEG C of water-baths as 100%, The enzyme activity of other temperature spots divided by the enzyme activity, so as to obtain it with respect to enzyme activity value.
The results show that phosphatidase PD provided by the invention stands 1 hour in 4-45 DEG C of water-bath, enzyme activity is more stable, Have almost no change, in 50-60 DEG C of water-bath stand 1 it is small when after remain to retain more than 83% enzyme activity, stand 1 in 70 DEG C of water-baths Still the vigor more than 50% can be retained after hour, heat-resisting effect is notable.
Application of 6 phosphatidase of embodiment in vegetable oil degumming
Enzymatic degumming is that the phospholipid hydrolysis in crude oil is generated oil-soluble diglyceride and water using phosphatidase in oil and fat refining The phosphate of dissolubility, diglyceride are dissolved in the part for becoming edible oil in oil, can improve the yield of degummed oil.Also, by The amphipathic property of phospholipid molecule is destroyed in hydrolysis, so as to reduce the formation for the colloid that big gauging is combined in scouring processes, Also the yield of oil is improved from another point of view.Therefore many advantages, such as enzymatic degumming, which has, reduces production cost, increases oil yield.
The phosphatidase PD that applicant prepares gained using embodiment 4 carries out enzymatic degumming：
200g crude oil of soybean is heated to 70-80 DEG C under stiring, adds 45% citric acid solutions of 0.16g, mixture is sheared 1 Minute, at a temperature of 70-75 DEG C, with magnetic stirrer 30 minutes, the oil is cooled down, until oil temperature is 55-65 DEG C, Ran Houtian Add 8% sodium hydroxide solutions of 0.5g, by mixture shear-mixed 30 seconds, temperature is maintained 50-55 DEG C, addition 5mL contains phosphatide The fermented supernatant fluid of enzyme, then mixing shearing 1 minute, temperature at 50-55 DEG C, be stirred to react 5 it is small when, be then centrifuged for the enzyme The oil of processing collects separated oily and wet glue, and remaining phosphorus is only 13.29ppm in degummed oil, provided by the invention so as to illustrate Phosphatidase PD has preferable degumming effect.
Sequence table
<110>Wang Yixuan
<120>A kind of bacterial strain for recombinantly expressing phosphatidase and its application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 241
<212> PRT
<213>Aspergillus (Aspergillus sp.)
<400> 1
Ser Ala Pro Asn Arg Ala Ser Lys Pro Gly Pro Gly Leu Arg Ile Leu
1 5 10 15
Thr Ala Thr Val Ile Lys Thr His Gly Leu Gly Asp Arg Lys Pro Leu
20 25 30
Ala Gln Asn Trp Arg Arg Arg Gly Met Tyr Gly Lys Val Ala Phe Met
35 40 45
Phe Pro Lys Ala Pro Ile Ile Pro Ile Arg Val Ile Phe Glu Met Thr
50 55 60
Met Leu Arg Asp His Asn Phe Lys Arg Arg Gly Arg Asp Leu Asp Trp
65 70 75 80
Lys Ser Ala Ile Arg His Gln Asp Glu Arg Gly Phe Arg Arg Cys Arg
85 90 95
Asp Tyr Phe Arg Thr Leu Asn Arg Lys Gln Ile Cys Arg Ser Ile Glu
100 105 110
Pro Ser Arg Ile Val Leu Gly Gly Phe Ser Gln Gly Ala Asn Val Phe
115 120 125
Val Phe Ser Gly Ile Thr Arg Lys Glu Lys Leu Gly Gly Val Phe Asp
130 135 140
Leu Val Ser Asn Leu Val Val Asn Lys Tyr Leu Lys Asp Asp Ile Glu
145 150 155 160
Glu Asn Trp Pro Asn Lys Lys Lys Pro Leu Phe Leu Ala His Gly Phe
165 170 175
Lys Asp Glu Val Gly Leu Phe Asp Phe Gly Glu Leu Leu Ala Asn Lys
180 185 190
Met Lys Glu Ile Gly Leu Glu Asp Ala Thr Phe Lys Ser Tyr Pro Asn
195 200 205
Leu Gly Pro Phe Ala Asp Pro Val Glu Ile Glu Val Trp Ala Arg Phe
210 215 220
Pro Gln Lys Val Ile Pro Pro Glu Asn Asp Gly Gln Ala Ser Ala Gly
225 230 235 240
Leu
<210> 2
<211> 726
<212> DNA
<213>Aspergillus (Aspergillus sp.)
<400> 2
agtgctccaa atcgcgcatc gaaacccggg ccggggctta gaatactcac tgcaacggta 60
atcaagaccc atggactggg tgacaggaag ccccttgctc agaactggcg tcgccggggg 120
atgtacggta aggttgcttt tatgttccct aaagcgccta taatcccgat cagggtgatc 180
tttgagatga cgatgcttag agatcacaat tttaagaggc gtggtcgcga tctcgattgg 240
aaatcagcca ttcggcacca agacgaacgg ggtttccgtc gatgtcggga ctacttcagg 300
acgttgaata ggaaacaaat ttgtaggagt atcgagccct cccggattgt tctgggtggt 360
ttctcccaag gggctaatgt gtttgtcttt tctggtatta ctcgtaagga gaagctcggt 420
ggtgtcttcg atttggtcag caatctggtg gtcaataaat atctgaagga cgatattgaa 480
gagaattggc cgaataagaa aaagcctttg ttcctcgctc atggctttaa agatgaagtc 540
gggctgttcg acttcggtga acttttggcg aacaagatga aagagatcgg cttggaggat 600
gccactttca aatcttatcc taacttgggc cccttcgccg atccagtaga gattgaggtt 660
tgggcgcgat ttcctcagaa agtcattcct ccagaaaacg acgggcaggc ttccgccgga 720
ttatga 726

Claims (4)

1. a kind of Pichia yeast engineering carries the recombinant plasmid that can express phosphatidase.
2. Pichia yeast engineering as described in claim 1, which is characterized in that the phosphatidase, amino acid sequence are SEQ ID NO: 1。
3. Pichia yeast engineering as claimed in claim 2, which is characterized in that the phosphatidase, the core of encoding gene Nucleotide sequence is SEQ ID NO: 2.
4. application of any Pichia yeast engineerings of claim 1-3 in phosphatidase is produced.
CN201711280976.9A 2017-12-07 2017-12-07 Bacterial strain for recombinant expression of phospholipase and application thereof Active CN108048339B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101070530A (en) * 2007-04-13 2007-11-14 新疆农业科学院微生物应用研究所 Low-temperature alkaline phosphatidase A1 and coding gene thereof
WO2006096527A3 (en) * 2005-03-04 2007-11-22 Diversa Corp Nucleic acids and proteins and methods for making and using them
CN104328095A (en) * 2014-09-26 2015-02-04 江南大学 Phospholipase A2 with most appropriate pH being in acid range and application thereof
CN106701712A (en) * 2015-11-13 2017-05-24 丰益(上海)生物技术研发中心有限公司 New phospholipase
WO2017120890A1 (en) * 2016-01-15 2017-07-20 江南大学 Method for increasing exogenous protein expression level by means of phospholipase fusion expression

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006096527A3 (en) * 2005-03-04 2007-11-22 Diversa Corp Nucleic acids and proteins and methods for making and using them
CN101070530A (en) * 2007-04-13 2007-11-14 新疆农业科学院微生物应用研究所 Low-temperature alkaline phosphatidase A1 and coding gene thereof
CN104328095A (en) * 2014-09-26 2015-02-04 江南大学 Phospholipase A2 with most appropriate pH being in acid range and application thereof
CN106701712A (en) * 2015-11-13 2017-05-24 丰益(上海)生物技术研发中心有限公司 New phospholipase
WO2017120890A1 (en) * 2016-01-15 2017-07-20 江南大学 Method for increasing exogenous protein expression level by means of phospholipase fusion expression

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
刘逸寒等: "毕赤酵母表面展示磷脂酶D高密度发酵优化", 《食品研究与开发》 *

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