CN105802979B - A kind of expression vector and expression of calf intestine alkaline phosphatase - Google Patents

A kind of expression vector and expression of calf intestine alkaline phosphatase Download PDF

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CN105802979B
CN105802979B CN201610200709.5A CN201610200709A CN105802979B CN 105802979 B CN105802979 B CN 105802979B CN 201610200709 A CN201610200709 A CN 201610200709A CN 105802979 B CN105802979 B CN 105802979B
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expression
alkaline phosphatase
calf intestine
intestine alkaline
fusion protein
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CN105802979A (en
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张鹭鹭
李先坤
何丽
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Zhengzhou Ke Ke Biotechnology Co., Ltd.
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

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Abstract

The present invention relates to field of biotechnology more particularly to the expression vectors and expression of a kind of calf intestine alkaline phosphatase.Expression provided by the invention can efficiently, accurately express CIAP albumen, and its bioactivity is unaffected.Experiment shows the culture by 24 hours, every OD600Cell can generate the CIAP fusion protein of 30mg.There is good sensitivity with the SAT reporter probe of calf intestine alkaline phosphatase fusion protein provided by the invention preparation, compared with the CIAP albumen extracted in the prior art, property has no significant difference.

Description

A kind of expression vector and expression of calf intestine alkaline phosphatase
Technical field
The present invention relates to the expression vectors and expression side of field of biotechnology more particularly to a kind of calf intestine alkaline phosphatase Method.
Background technique
Calf intestine alkaline phosphatase (Calf Intestinal Alkaline Phosphatase, abbreviation CIAP), ammonia Base acid sequence passes through chemical coupling agent S-HyNic (acyl succinimide ester acetone hydrazone), 4FB (amber as shown in SEQ ID NO:1 Amber acid imide 4- formylbenzoate) crosslinked action, ε amino and few nucleosides on calf intestine alkaline phosphatase amino acid side chain The amino at 3 ' end of acid is replaced by itrile group and aldehyde radical respectively, reacts so that calf intestine alkaline phosphatase be made to be combined together with oligonucleotide As compound second level protein-nucleic acid structural body, conduct after purification is purified or is concentrated by ultrafiltration through FPLC (high performance liquid chromatography) The reporter probe of SAT (nucleic acid signal amplifying technique) detection of nucleic acids platform uses.Therefore, calf intestine alkaline phosphatase is in biological skill Art field has a very wide range of applications.
In the prior art, it is the product extracted from animal tissue that CIAP, which mainly passes through, and this extraction prepares CIAP Mode low yield, it is time-consuming and laborious, cause cost of material high.Also, it extracts in the CIAP obtained and is constantly present portion Divide the impurity for being difficult to purify removal, unstable hidden danger is brought to later experiments.
Using the method for genetic engineering, carrying out prokaryotic expression to albumen is the current hot topic side for solving to synthesize outside aleuroplast One of method, yield is high, with short production cycle.But during prokaryotic expression, the selection of codon, the type of carrier, expression item Part etc. all can expression of results generate important influence, and there is no the report of the prokaryotic expression method for CIAP at present.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of expression vector of calf intestine alkaline phosphatase And expression, expression vector provided by the invention and expression can efficiently express CIAP.
The present invention provides a kind of expression vectors of calf intestine alkaline phosphatase, including nucleosides shown in SEQ ID NO:2 Acid sequence.
Preferably, the skeleton of expression vector is PMAL-c5x carrier.
Due to expressing the gene of mammal in vitro, especially with the gene of prokaryotic expression system expression mammal With unpredictability, and it is difficult.The selection of codon, expression vector skeleton type, codon and expression vector it Between cooperation, expression of results can all be had an impact.In the present invention, nucleotide sequence shown in SEQ ID NO:2 is according to CIAP Amino acid sequence it is preferred after Codon sequences use SEQ ID compared with the nucleotide sequence of NO:3~4 SEQ ID Nucleotide sequence shown in NO:2 expresses CIAP, can obtain higher expression quantity, and gained albumen not will form and forgive Body.
PMAL-c5x carrier is commercial vectors, is purchased from the Beijing NEB.
Preferably, the insertion point in PMAL-c5x carrier of amino acid sequence shown in SEQ ID NO:2 is located at NcolI- Between HindIII.
The preparation method of expression vector provided by the invention includes:
Step 1: by nucleotide sequence shown in SEQ ID NO:2 with NcolI-HindIII digestion, obtaining target fragment；
Step 2: by PMAL-c5x carrier with NcolI-HindIII digestion after, connect with target fragment, obtain expression carry Body.
In the present invention, nucleotide sequence shown in SEQ ID NO:2 is prepared by the way of artificial synthesized.SEQ ID NO: 3 be the expression codon of wild type CIAP, and SEQ ID NO:4 is optimized another codon, and experiment shows SEQ ID NO: After 3~4 connect carrier, expression effect is all not as good as SEQ ID NO:2.
The temperature of nucleotide sequence shown in digestion SEQ ID NO:2 is 37 DEG C, and the digestion time is 5h.
The temperature of digestion PMAL-c5x carrier is 37 DEG C, and the digestion time is 5h.
It is connect with target fragment after PMAL-c5x carrier digestion using T4DNA ligase, condition of contact is 16 DEG C, overnight.
It is transformed into bacillus coli DH 5 alpha by the plasmid of connection, with ammonia benzyl mycin screening positive clone, with bacterium colony PCR method It is verified.
The present invention the experimental results showed that, CIAP is expressed using PMAL-c5x carrier, biggish expression can be obtained Amount.
The present invention also provides a kind of host cells, are transfected by expression vector provided by the invention.
Preferably, host cell is e. coli bl21 cell.
Preferably, transfection uses heat shock method.
The present invention also provides a kind of expressions of calf intestine alkaline phosphatase fusion protein, with table provided by the invention Fiber differentiation after up to carrier transfection host cell.
Preferably, host cell is e. coli bl21 cell.
BL21 is common expression bacterial strain, and general cooperation is carried out the strongly expressed of target gene with strongly expressed carrier, is suitble to Express non-toxic albumen.The present invention has carried out efficient expression to CIAP using BL21 cell.
Although frequently can lead to generally, it is considered that the protein expression quantity with procaryotic cell expression zooblast is very high The secondary structure of mistake is formed, so that expression product is in terms of molecular structure, physicochemical property and biological function and native protein Matter has differences.The present invention is by the optimization to codon, host cell, expression vector, expression condition, so that fusion protein Expression quantity ensure that the formation of fusion protein secondary structure, and the life without influencing fusion protein under the premise of undiminished Object activity.
Preferably, the inducer of Fiber differentiation is IPTG；The concentration of the inducer is 0.5mmol/L.
Preferably, the culture solution of Fiber differentiation is LB culture medium.
Preferably, further including the steps that purifying, the purifying after Fiber differentiation are as follows: it is thin to collect the host through Fiber differentiation After the cracking of born of the same parents' ultrasound, reject cell fragment is eluted after being adsorbed with nickel column with imidazoles, is dialysed, and calf intestine alkaline phosphatase is obtained Fusion protein.
The condition of ultrasound cracking is 0 DEG C~4 DEG C, 6mm amplitude transformer, 35% power, 3.5s work, 7s rest, ultrasound 50min。
Protease inhibitors PMSF is added before ultrasonic.
The molecular weight of dialysis is 66kDa.
The calf intestine alkaline phosphatase fusion protein of expression preparation provided by the invention.
The N-terminal of calf intestine alkaline phosphatase fusion protein prepared by the present invention amino acid sequence shown in SEQ ID NO:1 It is connected with the label of 6 × his.Experiment shows that the presence of the label does not influence the bioactivity of the albumen of fusion.
The calf intestine alkaline phosphatase fusion protein of expression preparation provided by the invention is in preparation SAT reporter probe Application.
Calf intestine alkaline phosphatase fusion protein provided by the invention can be used in the preparation of SAT reporter probe.
Calf intestine alkaline phosphatase fusion protein provided by the invention is to the nucleic acid of coupling without particular/special requirement.
The present invention provides for the expression vector of CIAP, host, expression and application.Expression side provided by the invention Method can efficiently, accurately express CIAP albumen, and its bioactivity is unaffected.Experiment shows the culture by 24 hours, often OD600Cell can generate the CIAP fusion protein of 30mg.With calf intestine alkaline phosphatase fusion protein system provided by the invention Standby SAT reporter probe has good sensitivity, and compared with the CIAP albumen extracted in the prior art, property has no significance difference It is different.
Detailed description of the invention
Fig. 1 shows testing result signal-to-noise ratio.
Specific embodiment
The present invention provides a kind of expression vector of calf intestine alkaline phosphatase and expression, those skilled in the art can To use for reference present disclosure, it is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this It is it will be apparent that they are considered as being included in the present invention for the technical staff of field.Method and application of the invention is Be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to herein Methods and applications be modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
Material, reagent or the instrument that the present invention uses are all common commercially available product, can all be bought in market.
Below with reference to embodiment, the present invention is further explained:
The building of 1 expression vector of embodiment
1), nucleotide sequence shown in artificial synthesized SEQ ID NO:2 carries out digestion, digestion item with NdelI-HindIII enzyme Part is 37 DEG C.Product after digestion is subjected to agarose gel electrophoresis, glue recycles the nucleotide fragments that size is 1545bp.
2) PMAL-c5x carrier, is subjected to digestion with NdelI-HindIII enzyme, digestion condition is 37 DEG C.Digestion products warp Agarose gel electrophoresis, glue recycle the segment that size is 1500bp or so.
3), by after digestion linear carrier and target fragment with T4DNA ligase connection, connection temperature be 16 DEG C, connection Overnight.
4), connection product converts bacillus coli DH 5 alpha, with the LB culture medium culture containing ammonia benzyl antibiotic, picking positive bacteria Drop into row bacterium colony PCR identification.The bacterium colony that qualification result is the positive expand numerous.
5) after the bacterium colony after, expansion is numerous extracts plasmid, plasmid is transformed into e. coli bl21 competent cell, to contain ammonia The LB culture medium culture of benzyl antibiotic, picking positive bacteria drops into row culture presevation in case follow-up test.
The building of 1 expression vector of comparative example
1), nucleotide sequence shown in artificial synthesized SEQ ID NO:3 carries out digestion, digestion item with EcoRV-HindIII enzyme Part is 37 DEG C.Product after digestion is subjected to agarose gel electrophoresis, glue recycles the nucleotide fragments that size is 1545bp.
2) PMAL-c5x carrier, is subjected to digestion with EcoRV-HindIII enzyme, digestion condition is 37 DEG C.Digestion products warp Agarose gel electrophoresis, glue recycle the segment that size is 1500bp or so.
3), by after digestion linear carrier and target fragment with T4DNA ligase connection, connection temperature be 16 DEG C, connection Overnight.
4), connection product converts bacillus coli DH 5 alpha, with the LB culture medium culture containing ammonia benzyl antibiotic, picking positive bacteria Drop into row bacterium colony PCR identification.The bacterium colony that qualification result is the positive expand numerous.
5) after the bacterium colony after, expansion is numerous extracts plasmid, plasmid is transformed into e. coli bl21 competent cell, to contain ammonia The LB culture medium culture of benzyl antibiotic, picking positive bacteria drops into row culture presevation in case follow-up test.
The building of 2 expression vector of comparative example
1), nucleotide sequence shown in artificial synthesized SEQ ID NO:4 carries out digestion, digestion item with NcolI-HindIII enzyme Part is 37 DEG C.Product after digestion is subjected to agarose gel electrophoresis, glue recycles the nucleotide fragments that size is 1545bp.
2) PMAL-c5x carrier, is subjected to digestion with NcolI-HindIII enzyme, digestion condition is 37 DEG C.Digestion products warp Agarose gel electrophoresis, glue recycle the segment that size is 1500bp or so.
3), by after digestion linear carrier and target fragment with T4DNA ligase connection, connection temperature be 16 DEG C, connection Overnight.
4), connection product converts bacillus coli DH 5 alpha, with the LB culture medium culture containing ammonia benzyl antibiotic, picking positive bacteria Drop into row bacterium colony PCR identification.The bacterium colony that qualification result is the positive expand numerous.
5) after the bacterium colony after, expansion is numerous extracts plasmid, plasmid is transformed into e. coli bl21 competent cell, to contain ammonia The LB culture medium culture of benzyl antibiotic, picking positive bacteria drops into row culture presevation in case follow-up test.
The building of 3 expression vector of comparative example
1), nucleotide sequence shown in artificial synthesized SEQ ID NO:2 carries out digestion, digestion item with NcolI-HindIII enzyme Part is 37 DEG C.Product after digestion is subjected to agarose gel electrophoresis, glue recycles the nucleotide fragments that size is 1545bp.
2) pet28b carrier, is subjected to digestion with NcolI-HindIII enzyme, digestion condition is 37 DEG C.Digestion products are through fine jade Sepharose electrophoresis, glue recycle the segment that size is 1500bp.
3), by after digestion linear carrier and target fragment with T4DNA ligase connection, connection temperature be 16 DEG C, connection Overnight.
4), connection product converts bacillus coli DH 5 alpha, with the LB culture medium culture containing ammonia benzyl antibiotic, picking positive bacteria Drop into row bacterium colony PCR identification.The bacterium colony that qualification result is the positive expand numerous.
5) after the bacterium colony after, expansion is numerous extracts plasmid, plasmid is transformed into e. coli bl21 competent cell, to contain ammonia The LB culture medium culture of benzyl antibiotic, picking positive bacteria drops into row culture presevation in case follow-up test.
The building of 4 expression vector of comparative example
1), nucleotide sequence shown in artificial synthesized SEQ ID NO:2 carries out digestion, digestion item with EcoRV-HindIII enzyme Part is 37 DEG C.Product after digestion is subjected to agarose gel electrophoresis, glue recycles the nucleotide fragments that size is 1545bp.
2) pet32a carrier, is subjected to digestion with EcoRV-HindIII enzyme, digestion condition is 37 DEG C.Digestion products are through fine jade Sepharose electrophoresis, glue recycle the segment that size is 1500bp or so.
3), by after digestion linear carrier and target fragment with T4DNA ligase connection, connection temperature be 16 DEG C, connection Overnight.
4), connection product converts bacillus coli DH 5 alpha, with the LB culture medium culture containing ammonia benzyl antibiotic, picking positive bacteria Drop into row bacterium colony PCR identification.The bacterium colony that qualification result is the positive expand numerous.
5) after the bacterium colony after, expansion is numerous extracts plasmid, plasmid is transformed into e. coli bl21 competent cell, to contain ammonia The LB culture medium culture of benzyl antibiotic, picking positive bacteria drops into row culture presevation in case follow-up test.
2 expressing fusion protein of embodiment
1, the strain inducing expression destination protein for constructing embodiment 1 and comparative example 1~4, and detected, method are as follows:
1), bacterial strain is shaken in 3ml LB culture medium to OD600It is 0.6, IPTG is added, IPTG concentration is 0.3mmol/L, 37 DEG C, 140rpm overnight induction；
2), the expression of each strain destination protein of SDS-PAGE electrophoresis detection, and carry out the detection of inclusion body.
As the result is shown: embodiment 1, comparative example 1~2 strain after, be shown as in the supernatant that is formed after broken thallus Function expresses fusion protein；And although comparative example 3 and comparative example 4 also express fusion protein, its albumen forms inclusion body, It does not appear in supernatant.
2, the strain of embodiment 1, comparative example 1~2 is expanded culture, and is detected, method are as follows:
Strain is inoculated with 300ml and contains in the LB liquid medium of ammonia benzyl antibiotic 37 DEG C, and 250rpm shakes to OD600Value is 1.0 or so (records OD600 values), be added IPTG to IPTG concentration be 0.5mmol/L, 37 DEG C, 140rpm overnight induction；By bacterium solution 6000rpm, 4min, 4 DEG C after thalline were collected by centrifugation, detection obtains the amount of albumen.Wherein, the extraction of fusion protein is using nickel column parent With chromatography (kit is purchased from English promise biology), experiment is carried out referring to specification.It is specific:
1), thallus is under condition of ice bath, with 6mm amplitude transformer, 35% power, 3.5s work, 7s rest, ultrasonication 50min。
2), the solution being crushed is collected into 50ml centrifuge tube.10000rpm, 15min, 4 DEG C of centrifugations, take supernatant, It is added in the nickel column balanced.
3) after, eluting albumen, by bag filter dialysis, (dialyzate is 20mM PBS+50mM to the liquid eluted NaCl), the molecular weight of bag filter is 62kDa.
4) concentration of albumen after, measurement is dialysed, the quality of conversion gained albumen, as a result such as table 1:
1 Fiber differentiation result of table
Embodiment 1 Comparative example 1 Comparative example 2
OD600 value before inducing 0.55 0.60 0.65
OD600 value after induction 1.2 0.9 0.85
Gained albumen quality (mg) 30 15 6
Unit OD value obtains albumen quality 25mg 16.7mg 7mg
The result shows that strain obtained by embodiment 1 can be more efficient expression CIAP fusion protein.
The preparation and application of 3 probe of embodiment
To extract the CIAP albumen of acquisition in the prior art as control, the CIAP fusion of strain expression is made with embodiment 1 Albumen is experimental subjects, and the probe conjugate for being respectively AGGTCTCTGCTTAGAGGTACTT with sequence by the two, the probe is used for The report detection probe of SAT technology.Coupling method is referring to patent 6686461,6800728,7102024,7173125,7462689 Enclose product description.
Implementing procedure:
Application of the probe marker in SAT technology；
Step 1 designs a kind of coating primer nucleic acid sequence (5 '-NH2- GAGCGGATATATGCTTAGGAAT-3 '), synthesis The sequence is simultaneously dissolved in the TE buffer solution that PH is 8.0, and primer is diluted to 3~5 μ g/mL；
Step 2 prepares coating buffer, and according to each component in formula table, sodium dihydrogen phosphate, disodium hydrogen phosphate, poly rely ammonia Acid solution, sodium sulphate, sodium chloride, deionized water, it is noted that first plus sodium phosphate buffer, poly-D-lysine is added afterwards, it is clear to solution Diffluent sodium sulphate and reagent sodium chloride are added after clear, is protected from light room temperature magnetic agitation 2~3 hours or so；
Step 3 is dissolved in primer and the mixing of prepared coating buffer equal proportion in TE buffer, it is ensured that primer Final concentration is between 1.5~2.5 μ g/ml；
Step 4 is centrifuged using 96 hole polystyrene plastic plate three times of 1 × PBS pH, 8.2 solution cleaning blank 1200rpm 2min or so, it is ensured that noresidue liquid in plate is placed stand-by in drying box case；
Step 5 by the reagent coating machine prepared in step 3 or the Multi-channel liquid transfer device equal part calibrated into every hole, Coating 100~150 μ L of reagent or so is added in every hole, with masking foil or preservative film sealing plate, and is placed on 4 DEG C of refrigerator overnights Coating.
Step 6 is centrifuged using 96 hole polystyrene plastic plate three times of 1 × PBS pH, 8.2 solution cleaning blank 1200rpm 2min or so, it is ensured that noresidue liquid in plate is placed 5~6 hours dry in drying box.
Step 7 pack sealing, is added 2g desiccant.Go to warehouse for finished product storage.
Step 8 is coated with the verifying of plate, designs a kind of nucleic acid sequence (5 '-AGGTCTCTGCTTAGAGGTACTT-NH2- 3 '), The sequence and coated Primers complementary, synthesize the primer, by concentration dilution to 100pmol/ μ L.
Primer is diluted to 3 gradients by step 9, is 100pmol/ μ L, 10pmol/ μ L, 1pmol/ μ L respectively.It is separately added into In 100 μ L to coating plate hole.Experimental design such as table 2:
2 experimental design of table
Blank 100 10 1
Blank 100 10 1
Blank 100 10 1
Blank 100 10 1
Blank 100 10 1
Blank 100 10 1
Blank 100 10 1
Blank 100 10 1
The signal value of each plate hole is detected according to table 2, experiment obtains data such as table 3:
3 experimental data of table
800 536580 62500 5735
855 554850 61450 6555
765 578565 65135 5355
755 568745 64780 5675
920 567850 59865 6125
880 578695 68710 6345
910 605245 58740 5845
775 575850 61520 6005
Signal-to-noise ratio data such as Fig. 1 is tested, the results show that strain expression CIAP fusion protein is made with the embodiment of the present application 1 Probe obtained can accurately detect target signal value, which extracts the obtained spy of CIAP albumen obtained with the prior art The result of needle is consistent.
The above is only the preferred embodiment of the present invention, it is noted that those skilled in the art are come It says, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (6)

1. a kind of expression vector of calf intestine alkaline phosphatase, which is characterized in that by PMAL-c5x skeleton carrier and SEQ ID The composition of nucleotide sequence shown in NO:2.
2. a kind of host cell, which is characterized in that it is transfected by the expression vector of claim 1；The host cell is large intestine Bacillus BL21 cell.
3. a kind of expression of calf intestine alkaline phosphatase fusion protein, which is characterized in that with the expression vector of claim 1 Fiber differentiation after transfection host cell；The host cell is e. coli bl21 cell.
4. expression according to claim 3, which is characterized in that the inducer of the Fiber differentiation is IPTG；It is described The concentration of inducer is 0.5mmol/L.
5. expression according to claim 3, which is characterized in that further include the steps that purifying after the Fiber differentiation, The purifying are as follows: collect after the cracking of the host cell ultrasound of Fiber differentiation, reject cell fragment, with imidazoles after being adsorbed with nickel column Elution, is dialysed, and calf intestine alkaline phosphatase fusion protein is obtained.
6. the calf intestine alkaline phosphatase fusion protein of the described in any item expression preparations of claim 3~5 is in preparation SAT Application in reporter probe.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773226A (en) * 1992-03-10 1998-06-30 La Jolla Cancer Research Foundation Recombinant calf intestinal alkaline phosphatase
CN1334341A (en) * 2000-07-25 2002-02-06 霍夫曼-拉罗奇有限公司 Expression of alkaline phosphatase in yeast

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773226A (en) * 1992-03-10 1998-06-30 La Jolla Cancer Research Foundation Recombinant calf intestinal alkaline phosphatase
CN1334341A (en) * 2000-07-25 2002-02-06 霍夫曼-拉罗奇有限公司 Expression of alkaline phosphatase in yeast

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Cloning and expression of the bovine intestinal alkaline phosphatase gene:";Helge WEISSIG et al.,;《Biochem.J》;19931231;第290卷;第503-508页
"编码磷酸酯酶新基因的克隆、表达及产物的生化性质鉴定";罗奉奉;《中国优秀硕士学位论文全文数据库基础科学辑》;20130216(第3期);第24页第2.12.1节，第25-26页第2.13节

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