CN108106894A - Insect cell expression sST2 is as the calibration object for measuring sST2 concentration in serum - Google Patents

Insect cell expression sST2 is as the calibration object for measuring sST2 concentration in serum Download PDF

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CN108106894A
CN108106894A CN201711358221.6A CN201711358221A CN108106894A CN 108106894 A CN108106894 A CN 108106894A CN 201711358221 A CN201711358221 A CN 201711358221A CN 108106894 A CN108106894 A CN 108106894A
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sst2
dna
flag
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minute
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CN108106894B (en
Inventor
王珺楠
李宝民
杜培革
刘斌
关大伟
赵雪
应瑛
隋宝真
王玉华
史永峰
王贺
关恒
安丽萍
苑广信
王曼力
林林
张继海
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Changchun Hengxiao Biotechnology Co Ltd
Jilin University
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Changchun Hengxiao Biotechnology Co Ltd
Jilin University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N2001/2893—Preparing calibration standards
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

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Abstract

The invention belongs to biological technical fields.Disclose stimulates calibration object process of the expressing protein 2 as sST2 concentration in ELISA measure serum with insect cell expression Soluble growth, and the method step is as follows：T47D cell RNAs are extracted, for synthesizing cDNA, go out sST2DNA with PCR amplification；PFastBac Flag sST2 carriers are formed with sST2 DNA, then are transduceed into HD10Bac bacterial strains, generate restructuring Flag sST2 insect viruses plasmids；Flag sST2AcNPV Plasmid DNA is transduceed into insect cell, generates restructuring Flag sSt2AcNPV；SF9 cells are infected with Flag sST2 AcNPV, Flag sST2 albumen is given expression to, is purified into Anti Flag M2 affinity agarose with natural structure and antigenicity Flag sST2；By the use of Flag sST2 as calibration object, double fastener heart enzyme-linked immunization is established, measures sST2 concentration in serum or other liquid, the invention is prepared with albumen natural structure and antigenicity Flag sST2, as the calibration object for measuring sST2 concentration in liquid, for sST2 concentration in clinical detection serum.

Description

Insect cell expression sST2 is as the calibration object for measuring sST2 concentration in serum
Technical field
The invention belongs to biological technical fields.
Background technology
Soluble growth stimulation expressing protein 2 (Soluble suppression of tumorigenicity 2, SST2 interleukin-1 receptor family member) is belonged to.ST2 gene expressions are in mast cell, Th2 cells（Th2）, into fibre Tie up cell, cardiac muscle cell etc..3 kinds of subtype protein matter of ST2 gene codes in human body, when expressed in people's intestinal tissue ST2V.Second is that film combination receptor Protein S T2L, by the immunoglobulin domains of 3 series connection, transmembrane domain outside cell membrane It is formed with intracellular TIR (Toll/Interleukin-lreceptor) structural domains three parts.ST2L selective expressions exist Th2 lymphocytic cell surfaces can be used as Th2 cell surface marker molecules.ST2L can be with extracellular ligand molecule interleukin-33 (IL-33) interleudin-1 receptor accessory Protein and on cell membrane forms compound, and a plurality of signal path in active cell promotes The relevant inflammatory factors of Th2（IL-4, IL-5, IL-13）Expression participates in the regulation process of panimmunity relevant disease.ST2 bases Because encoding the third albumen as sST2, compared with ST2L, sST2 lacks ST2L transmembrane domains and intracellular domain, in C- ends There are 5 different amino acid fragments, for secretory, soluble type albumen（sST2）, it is secreted into extracellular blood, sST2 is in the heart It plays an important role during force failure occurrence and development.
Research shows IL-33 by being combined with myocardial cell membrane ST2L, has that anti-myocardial is loose, anti-cardiac muscle fibre The effects that change.When myocardial hypertrophy and failure, Cardiac Fibroblasts and vascular endothelial cell are being subject to mechanical stretch to stimulate Afterwards, IL-33 is released to increase；At the same time, vascular endothelial cell, fibroblast and cardiac muscle cell also discharge more sST2 and arrive In blood.SST2 can be used as Decoy receptor, and can dissociate in competitive binding blood IL-33, be tied so as to inhibit IL-33 in blood ST2L receptors on myocardial cell membrane are closed, weakens the anti-myocardial hypertrophies of IL-33 and Fibrosis parameters, aggravates heart failure.Thus see Go out, sST2 concentration, which increases, in blood represents heart failure.
SST2 concentration in patients serum is measured, as heart failure Novel marker, for judging the generation of heart failure More after.It needs to develop the immunological method kit for measuring sST2 concentration in serum, while needs to purify sST2 as examination The calibration object of agent box.
The content of the invention
The purpose of the present invention expresses sST2 using insect cell system, available for diagnosis of heart failure insect cell expression SST2 is as the calibration object for measuring sST2 concentration in serum.
Step of the present invention is：
A, T47D RNA are extracted, T47D cDNA gene pools is synthesized, goes out sST2 DNA, nucleotide sequence 55-987, structure with PCR amplification Build pFastBac-Flag-sST2 carriers；
B, the transduction of pFastBac-Flag-sST2 carrier DNAs generates restructuring Flag-sST2AcNPV into HD10Bac bacteriums Plasmid；
C, recombinate the transduction of Flag-sST2 AcNPV Plasmid DNA and enter SF9 cells, generate restructuring Flag-sST2 AcNPV；
D, Flag-sST2 AcNPV infection SF9 cells are recombinated and are given expression to 8 amino acid residue --- -- DYKDDDDK, it is short The Flag-sST2 albumen of peptide Flag, sST2-----19-328 amino acid residue；
E, it is purified into Anti-Flag M2 affinity agarose with albumen natural structure and antigenicity Flag-sST2 Albumen.
The present invention with Flag-sST2 as calibration object, establish double fastener heart enzyme-linked immunization ----ELISA, measure serum or SST2 concentration in other body fluid.
Present invention synthesis cDNA and PCR amplification sST2 DNA：
（1）Synthesize cDNA random primings
T47D cell RNAs 2ul（600ng）
10mM dNTP1ul
Random primer (50ng/ul) 2ul
H2O 5ul
It is placed in 0.5ml microcentrifugal tubes, mixing；It is placed under the conditions of 65 DEG C, 5 minutes, is placed in ice cube 5 minutes；
（2）After centrifugation, add following substance into above-mentioned 0.5ml microcentrifugal tubes
5XSSIV buffer 4ul
100mM DTT 1ul
RNase out 1ul
RT 1ul
H2O 4ul；
（3）Mixing after centrifugation, is placed under the conditions of 50 DEG C, is reacted 10 minutes；
（4）It is placed under the conditions of 80 DEG C, inactivates 10 minutes；
（5）Add 1ul E.coilRNaseH, under the conditions of 37 DEG C, react 20 minutes, obtain cDNA；
（6）Synthetic primer, primer -1,5 '-tttttcatatgaagtttagtaaacaatcatggg-3 '
Primer -2,5 '-tttttctcgagtcagaaacactccttacttggatt-3 '；
（7）Carry out PCR
Reaction buffer 10ul
High GC enhancer10ul
10mMdNTP1ul
25pm/ul primers -11ul
25pm/ul primers -11ul
cDNA2ul
Q5 HF-DNA Polymerase 0.5ul
H2O 25ul；
（8）PCR conditions are as follows：
A. 95 DEG C 2 minutes
B. 95 DEG C 1 minute
C. 56 DEG C 1 minute
D. 72 DEG C 1 minute
Repeat B-D steps 30 time
E. 95 DEG C 30 seconds 1 minute
F. 56 DEG C 30 seconds 1 minute
G. 72 DEG C 6 minutes
25 DEG C are cooled to, completes PCR；
（9）5ul PCR products are drawn, add 2ul 5X DNA loading buffer；
（10）It is placed in 1% Agarose/TBE gel electrophoresises hole, carries out electrophoresis；
（11）DNA dyeing, viewed under ultraviolet radiation 934bpDNA bands are carried out with EB；
（12）Remaining PCR product adds 100ul H2O, then adds 150ul phenol/chloroforms/isoamyl alcohol（25/24/1）, mixing；
（13）Centrifugation, 13000rpm, 5 minutes；
（14）Supernatant is collected, adds 15ul 5M NaCl, 800ul ethyl alcohol, is mixed, -20 DEG C, overnight；
（15）Under the conditions of 4 DEG C, centrifugation, 13000rpm, 20 minutes；
（16) supernatant is removed, adds 70% ethyl alcohol of 800ul, under the conditions of 4 DEG C, centrifugation, 13000rpm, 10 minutes；
（17) supernatant is removed, dry microcentrifugation bottom of the tube DNA adds 50ul H20 dissolving DNAs.
Present invention structure FastBac-Flag-sST2 plasmids：
（1）With NdeI/XhoI restriction endonucleases（New England Biolabs）Cutting DNA
PCR DNA products 48ul
Cutsmart buffer 6ul
NdeI 3ul
XhoI3ul
PFastBac-Flag carrier DNAs 3ul（3ug）
Cutsmartbuffer 6ul
NdeI 3ul
XhoI3ul
H20 45ul
It is mixed, when 37 DEG C of reactions 2 are small；
（2）It is placed in 1% Agarose/TBE gel electrophoresis hole, carries out electrophoresis
（3）EB carries out DNA dyeing, viewed under ultraviolet radiation 934bp（PCR products）And 4.6kb（Carrier）DNA bands, respectively The lower DNA bands of cutting, are put into respectively in 1.5ml microcentrifugal tubes
（4）With QIAquick Gel Extraction Kit (QIAGEN), DNA in Agarose bands is extracted
Step is as follows：
Add in 700ul BufferQG to Agarose containing DNA band microcentrifugal tubes, be placed on 50 DEG C, 10 minutes, shake within every 2 minutes It swings once, dissolves Agarose bands
QIAquick centrifugal columns are put in 2ml collecting pipes, in addition stating in lysate 700ul to QIAquick centrifugal columns, place 2 Minute
Centrifugation, 3000g 1 minute remove liquid in collecting pipe
It centrifuges again once, 3000g 1 minute
Add 700ul Buffer PE to QIAquick centrifugal columns
Centrifugation, 12000g 1 minute remove liquid in collecting pipe
It centrifuges again once, 12000g 1 minute,
QIAquick centrifugal columns are put in new microcentrifugal tube, at room temperature, are placed 5 minutes
Add in 30ul H2O to QIAquick centrifugal columns, at room temperature, place 3 minutes
Centrifugation, 15000g 1 minute
Contain DNA in 1.5ml microcentrifugal tube collection liquids,
DNA concentration is measured with Nanodrop Spectrophotometer；
（5）With T4DNA ligase (New England Biolabs.), PCR DNA fragmentation pFastBac-Flag carriers are connected In,
Following substance is added in 1.5ml microcentrifugal tubes；
PCR DNA（NdeI/XhoI） 50ng
Carrier DNA（NdeI/XhoI） 50ng
10X T4 DNA Ligase Buffer 1 ul
T4 DNA Ligase 0.5ul
H2O to 10ul
It is mixed, 25 DEG C, when reaction 4 is small；
（6）Microcentrifugal tube is put on ice cube, adds 100ul competent bacterias（XL10 Gold bacterial strains）, it is gently mixed, ice cube It is upper to place 30 minutes
（7）Microcentrifugal tube is put under 42 DEG C of water bath conditions, 50 seconds, then be put on ice cube 3 minutes
（8）Add 700ul LB culture solutions, 37 DEG C, shake 1 it is small when
（9）Centrifugation, 10000g 5 minutes, remove supernatant, collect microcentrifugation bottom of the tube bacterium
（10）Kind is applied on 100ug/ml Ampicillin LB- agar plates, 37 DEG C, overnight incubation
（11）Single bacterium colony is selected, is inoculated into 3ml100ug/ml Ampicillin LB culture solutions, 37 DEG C, is shaken, overnight
（12）Bacteria plasmid DNA is extracted, with PureLink Quick Plasmid Miniprep Kits (Life Technologies Inc.)
Add 1.5ml bacterium liquid into microcentrifugal tube, centrifuge, 13000rpm3 minutes, remove supernatant,
Add 250ul suspensions BufferR3 into microcentrifugal tube, even suspension bacterium
250ul is added to dissolve BufferL7 into microcentrifugal tube, turned upside down microcentrifugal tube places microcentrifugal tube, room temperature Under, 5 minutes
Add 350ul precipitate Bs ufferN4 into microcentrifugal tube, turned upside down microcentrifugal tube places microcentrifugal tube in ice In block, 10 minutes
Centrifugation, 13000rpm, 10 minutes
Centrifugal column is put in 2ml collecting pipes, in addition stating centrifuged supernatant into centrifugal column, is placed 1 minute, centrifugation, 12000g, 1 minute, remove liquid in collecting pipe
Washing 500ul Buffer W10 is added to place 1 minute, centrifugation, 12000g, 1 minute into centrifugal column, remove collecting pipe Middle liquid
Washing 700ul Buffer W9 is added to place 1 minute, centrifugation, 12000g, 1 minute into centrifugal column, remove collecting pipe Middle liquid
Centrifugation, 12000g, 1 minute
Centrifugal column is put in new microcentrifugal tube, at room temperature, is placed 3 minutes
Add 75ul TE buffer into centrifugal column, at room temperature, place 3 minutes
Centrifugation, 12000g 2 minutes
Contain DNA in 1.5ml microcentrifugal tube collection liquids
DNA concentration is measured with Nanodrop Spectrophotometer；
（13）PstI restriction endonucleases（New England Biolabs）, cutting pFastBac-Flag-sST2 DNA
DNA 0.8ug
Cutsmartbuffer 1ul
PstI 1ul
H2O to 10ul
It is mixed, 37 DEG C, when reaction 2 is small,
（14）It is placed in 1% Agarose/TBE gel electrophoresis hole, carries out electrophoresis
（15）EB carries out DNA dyeing, and viewed under ultraviolet radiation forms DNA bands；
（16）665bp DNA band Plasmid DNA is selected, carries out determined dna sequence, sST2 DNA sequencing primers
(5’- tttttcatatggggttttggatcttagcaattc-3’;5’-tttttcatatgaagtttagtaaacaatc atggg-3’)
（17）Select the pFastBac-Flag-sST2 carriers of correct sST2 DNA sequences.
The present invention expresses sST2 using insect cell system, and insect cell is similar to human body cell, but yield is high, cost It is low, can mass production, be purified into sST2 with albumen natural structure and antigenicity, sST2 in similar serum, as detection sST2 The calibration object of concentration immune reagent kit.The present invention, as calibration object, it is dense to establish sST2 in measure serum with purifying Flag-sST2 ELISA kit is spent, available for diagnosis of heart failure.
Description of the drawings
Fig. 1 is protective effect figures of the sST2 antagonisms IL-33 to cardiac muscle；
Fig. 2 is in synthesis cDNA and PCR amplification sST2 DNA steps, and DNA dyeing, viewed under ultraviolet radiation are carried out with EB 934bpDNA histograms；
Fig. 3 is in structure FastBac-Flag-sST2 plasmid procedures, and EB carries out DNA dyeing, and viewed under ultraviolet radiation forms DNA Histogram；
Fig. 4 is in restructuring Flag-sST2 AcNPV Plasmid DNA steps, and DNA dyeing, viewed under ultraviolet radiation are carried out with EB 934bp DNA histograms；
Fig. 5 is that argentation measures Flag-sST2 purity figures；
Fig. 6 is to measure Westernblot results with ImageReader LAS-4000 (FUJIFILM)；
Fig. 7 is BAS concentration standard curves；
Fig. 8 is that Flag-sST2 calibration objects prepare canonical plotting.
Specific embodiment
SST2 concentration in patients serum is measured, as heart failure Novel marker, for judging the generation of heart failure More after.It needs to develop the immunological method kit for measuring sST2 concentration in serum, while needs to purify sST2 as examination The calibration object of agent box.
T47D cell RNAs are extracted, for synthesizing ability cDNA, go out sST2 DNA with PCR amplification（Nucleotide sequence 55-987）. Insect cell expression vector pFastBac-Flag is selected, promoter containing Polyhedrin is expressed exogenous in insect cell Albumen；This carrier is inserted into 8 amino acid residues（DYKDDDDK）Small peptide（Flag）DNA sequences, for expressing Flag fusion eggs In vain；This carrier carries part AcNPV DNA sequences, for generating genetic recombination insect viruses in HD10Bac bacteriums （AcNPV）Plasmid.SST2 DNA and pFastBac-Flag are cut with restriction endonuclease NdeI/XhoI, sST2 DNA are connected to PFastBac-FlagNdeI/XhoI positions are built into pFast-Flag-sST2 carriers；This carrier DNA is transduceed thin into HD10Bac In bacterium, restructuring Flag-sST22 AcNPV plasmids are generated；Restructuring Flag-sST2 AcNPV Plasmid DNA is transduceed into SF9 cells, Generate restructuring Flag-sST2 AcNPV；SF9 cells are infected with this AcNPV, give expression to Flag-sST2 albumen, sST2 albumen has 328 amino acid residues, sST2 N- ends 1-18 amino acid residues are secretory protein signal boot sequence, and sST2 in blood has been cut off Signal boot sequence 1-18 amino acid residues.19-328 amino acid residues are presented in present invention expression sST2, with sST2 phases in blood Together, sST2N- ends are expressed and marks small peptide with Flag, Flag- can be purified into Anti-Flag M2 affinity agarose sST2。
Purify Flag-sST2 and carry out SDS-PAGE electrophoresis, then do argentation, measure Flag-sST2 purity reach 99% with On, it is further confirmed with Western blot methods, anti-sST2 antibody combination Flag-sST2 albumen.Anti- sST2 antibody is bought, Use horseradish peroxidase（HRP）SST2 antibody is tagged to, with purifying Flag-sST2 as calibration object, establishes ELISA for surveying Determine sST2 concentration in serum.
The present invention, which prepares Flag-sST2, has albumen natural structure and antigenicity, is measured as ELISA in serum or liquid The calibration object of sST2 concentration.
With reference to specific experiment, the principle of the invention and result are described further, multistep of the present invention is listed below and tested Journey：
Step of the present invention is：
A, T47D RNA are extracted, T47D cDNA gene pools is synthesized, goes out sST2 DNA, nucleotide sequence 55-987, structure with PCR amplification Build pFastBac-Flag-sST2 carriers；
B, the transduction of pFastBac-Flag-sST2 carrier DNAs generates restructuring Flag-sST2AcNPV into HD10Bac bacteriums Plasmid；
C, recombinate the transduction of Flag-sST2 AcNPV Plasmid DNA and enter SF9 cells, generate restructuring Flag-sST2 AcNPV；
D, Flag-sST2 AcNPV infection SF9 cells are recombinated and are given expression to 8 amino acid residue --- -- DYKDDDDK, it is short The Flag-sST2 albumen of peptide Flag, sST2-----19-328 amino acid residue；
E, it is purified into Anti-Flag M2 affinity agarose with albumen natural structure and antigenicity Flag-sST2 Albumen.
Insect cell expression sST2 described in claim 1 is as the calibration object for measuring sST2 concentration in serum, feature It is：With Flag-sST2 as calibration object, double fastener heart enzyme-linked immunization is established ----ELISA is measured in serum or other body fluid SST2 concentration.
Insect cell expression sST2 described in claim 1 is as the calibration object for measuring sST2 concentration in serum, feature It is：
One, prepares Flag-sST2 albumen
（One）Extract T47D cell RNAs
With RNeasy Mini kit (QIAGEN), process is as follows：
1. DMEM culture solutions are used, containing 10% hyclone（FBS）, cultivate T47D breast cancer cells
2. fresh cell medium containing 1.0nmol/LHeregulin is used, when culture T47D cells 4 are small
3. collect 2X106Cell is added in 15ml centrifuge tubes, adds 10ml PBS, with suction pipe pressure-vaccum mixing, is centrifuged, 5000g, 5 Minute, remove supernatant
4. using 1ml PBS cell mixings, cell is gone to 1.5ml Eppendorf micro tubes, is centrifuged, 5000g 3 minutes, is gone Except PBS
5. add 300ul Buffer RLT, with micropipettor pressure-vaccum, mixing
6. with 20-gauge (diameter 0.9mm) needle applicator, aspirate 5 times
7. add 70% ethyl alcohol of 300ul into cytolysate, micropipettor pressure-vaccum, mixing
8.RNeasy micro-columns are placed in 2ml collecting pipes, in addition stating 700ul mixed dissolutions liquid into RNeasy micro-columns
9. centrifugation, 8000g 30 seconds, remove liquid in collecting pipe
10. add in 700ul Buffer RW1 to RNeasy micro-columns
11. centrifugation, 8000g 30 seconds, remove liquid in collecting pipe
12. putting RNeasy micro-columns in new 2ml collecting pipes, add in 500ul Buffer RPE to RNeasy micro-columns
13. centrifugation, 8000g 30 seconds remove liquid in collecting pipe
14. again plus in 500ul Buffer RPE to RNeasy micro-columns
15. centrifugation, 8000g 2 minutes, remove collecting pipe
16. RNeasy micro-columns are put in new 2ml collecting pipes, maximum velocity centrifugation (13000rpm), 1 minute
17. putting RNeasy micro-columns in 1.5ml Eppendorf micro tubes, add in 30ulH2O to RNeasy micro-columns
18. it places 3 minutes, centrifugation, 8000g, 1 minute
19.1.5ml liquid is RNA extracting solutions in Eppendorf micro tubes,
20. measure RNA concentration with Nanodrop Spectrophotometer.
（Two）Synthesize cDNA and PCR amplification sST2 DNA：
（1）Synthesize cDNA random primings（cDNA synthesis kit, Life Technologies, Inc.）
T47D cell RNAs 2ul（600ng）
10mM dNTP1ul
Random primer (50ng/ul) 2ul
H2O 5ul
It is placed in 0.5ml microcentrifugal tubes, mixing；It is placed under the conditions of 65 DEG C, 5 minutes, is placed in ice cube 5 minutes；
（2）After centrifugation, add following substance into above-mentioned 0.5ml microcentrifugal tubes
5XSSIV buffer 4ul
100mM DTT 1ul
RNase out 1ul
RT 1ul
H2O 4ul；
（3）Mixing after centrifugation, is placed under the conditions of 50 DEG C, is reacted 10 minutes；
（4）It is placed under the conditions of 80 DEG C, inactivates 10 minutes；
（5）Add 1ul E.coilRNaseH, under the conditions of 37 DEG C, react 20 minutes, obtain cDNA；
（6）Synthetic primer, primer -1,5 '-tttttcatatgaagtttagtaaacaatcatggg-3 '
Primer -2,5 '-tttttctcgagtcagaaacactccttacttggatt-3 '；
（7）Carry out PCR
Reaction buffer 10ul
High GC enhancer10ul
10mMdNTP1ul
25pm/ul primers -11ul
25pm/ul primers -11ul
cDNA2ul
Q5 HF-DNA Polymerase 0.5ul
H2O 25ul；
（8）PCR conditions are as follows：
A. 95 DEG C 2 minutes
B. 95 DEG C 1 minute
C. 56 DEG C 1 minute
D. 72 DEG C 1 minute
Repeat B-D steps 30 time
E. 95 DEG C 30 seconds 1 minute
F. 56 DEG C 30 seconds 1 minute
G. 72 DEG C 6 minutes
25 DEG C are cooled to, completes PCR；
（9）5ul PCR products are drawn, add 2ul 5X DNA loading buffer；
（10）It is placed in 1% Agarose/TBE gel electrophoresises hole, carries out electrophoresis；
（11）DNA dyeing, viewed under ultraviolet radiation 934bpDNA bands are carried out with EB；See Fig. 2；
（12）Remaining PCR product adds 100ul H2O, then adds 150ul phenol/chloroforms/isoamyl alcohol（25/24/1）, mixing；
（13）Centrifugation, 13000rpm, 5 minutes；
（14）Supernatant is collected, adds 15ul 5M NaCl, 800ul ethyl alcohol, is mixed, -20 DEG C, overnight；
（15）Under the conditions of 4 DEG C, centrifugation, 13000rpm, 20 minutes；
（16) supernatant is removed, adds 70% ethyl alcohol of 800ul, under the conditions of 4 DEG C, centrifugation, 13000rpm, 10 minutes；
（17) supernatant is removed, dry microcentrifugation bottom of the tube DNA adds 50ul H20 dissolving DNAs.
（Three）Build FastBac-Flag-sST2 plasmids：
（1）With NdeI/XhoI restriction endonucleases（New England Biolabs）Cutting DNA
PCR DNA products 48ul
Cutsmart buffer 6ul
NdeI 3ul
XhoI3ul
PFastBac-Flag carrier DNAs 3ul（3ug）
Cutsmartbuffer 6ul
NdeI 3ul
XhoI3ul
H20 45ul
It is mixed, when 37 DEG C of reactions 2 are small；
（2）It is placed in 1% Agarose/TBE gel electrophoresis hole, carries out electrophoresis
（3）EB carries out DNA dyeing, viewed under ultraviolet radiation 934bp（PCR products）And 4.6kb（Carrier）DNA bands, respectively The lower DNA bands of cutting, are put into respectively in 1.5ml microcentrifugal tubes
（4）With QIAquick Gel Extraction Kit (QIAGEN), DNA in Agarose bands is extracted
Step is as follows：
Add in 700ul BufferQG to Agarose containing DNA band microcentrifugal tubes, be placed on 50 DEG C, 10 minutes, shake within every 2 minutes It swings once, dissolves Agarose bands
QIAquick centrifugal columns are put in 2ml collecting pipes, in addition stating in lysate 700ul to QIAquick centrifugal columns, place 2 Minute
Centrifugation, 3000g 1 minute remove liquid in collecting pipe
It centrifuges again once, 3000g 1 minute
Add 700ul Buffer PE to QIAquick centrifugal columns
Centrifugation, 12000g 1 minute remove liquid in collecting pipe
It centrifuges again once, 12000g 1 minute,
QIAquick centrifugal columns are put in new microcentrifugal tube, at room temperature, are placed 5 minutes
Add in 30ul H2O to QIAquick centrifugal columns, at room temperature, place 3 minutes
Centrifugation, 15000g 1 minute
Contain DNA in 1.5ml microcentrifugal tube collection liquids,
DNA concentration is measured with Nanodrop Spectrophotometer；
（5）With T4DNA ligase (New England Biolabs.), PCR DNA fragmentation pFastBac-Flag carriers are connected In,
Following substance is added in 1.5ml microcentrifugal tubes；
PCR DNA（NdeI/XhoI） 50ng
Carrier DNA（NdeI/XhoI） 50ng
10X T4 DNA Ligase Buffer 1 ul
T4 DNA Ligase 0.5ul
H2O to 10ul
It is mixed, 25 DEG C, when reaction 4 is small；
（6）Microcentrifugal tube is put on ice cube, adds 100ul competent bacterias（XL10 Gold bacterial strains）, it is gently mixed, ice cube It is upper to place 30 minutes
（7）Microcentrifugal tube is put under 42 DEG C of water bath conditions, 50 seconds, then be put on ice cube 3 minutes
（8）Add 700ul LB culture solutions, 37 DEG C, shake 1 it is small when
（9）Centrifugation, 10000g 5 minutes, remove supernatant, collect microcentrifugation bottom of the tube bacterium
（10）Kind is applied on 100ug/ml Ampicillin LB- agar plates, 37 DEG C, overnight incubation
（11）Single bacterium colony is selected, is inoculated into 3ml100ug/ml Ampicillin LB culture solutions, 37 DEG C, is shaken, overnight
（12）Bacteria plasmid DNA is extracted, with PureLink Quick Plasmid Miniprep Kits (Life Technologies Inc.)
Add 1.5ml bacterium liquid into microcentrifugal tube, centrifuge, 13000rpm3 minutes, remove supernatant,
Add 250ul suspensions BufferR3 into microcentrifugal tube, even suspension bacterium
250ul is added to dissolve BufferL7 into microcentrifugal tube, turned upside down microcentrifugal tube places microcentrifugal tube, room temperature Under, 5 minutes
Add 350ul precipitate Bs ufferN4 into microcentrifugal tube, turned upside down microcentrifugal tube places microcentrifugal tube in ice In block, 10 minutes
Centrifugation, 13000rpm, 10 minutes
Centrifugal column is put in 2ml collecting pipes, in addition stating centrifuged supernatant into centrifugal column, is placed 1 minute, centrifugation, 12000g, 1 minute, remove liquid in collecting pipe
Washing 500ul Buffer W10 is added to place 1 minute, centrifugation, 12000g, 1 minute into centrifugal column, remove collecting pipe Middle liquid
Washing 700ul Buffer W9 is added to place 1 minute, centrifugation, 12000g, 1 minute into centrifugal column, remove collecting pipe Middle liquid
Centrifugation, 12000g, 1 minute
Centrifugal column is put in new microcentrifugal tube, at room temperature, is placed 3 minutes
Add 75ul TE buffer into centrifugal column, at room temperature, place 3 minutes
Centrifugation, 12000g 2 minutes
Contain DNA in 1.5ml microcentrifugal tube collection liquids
DNA concentration is measured with Nanodrop Spectrophotometer；
（13）PstI restriction endonucleases（New England Biolabs）, cutting pFastBac-Flag-sST2 DNA
DNA 0.8ug
Cutsmartbuffer 1ul
PstI 1ul
H2O to 10ul
It is mixed, 37 DEG C, when reaction 2 is small,
（14）It is placed in 1% Agarose/TBE gel electrophoresis hole, carries out electrophoresis
（15）EB carries out DNA dyeing, and viewed under ultraviolet radiation forms DNA bands；
（16）665bp DNA band Plasmid DNA is selected, carries out determined dna sequence, sST2 DNA sequencing primers
(5’- tttttcatatggggttttggatcttagcaattc-3’;5’-tttttcatatgaagtttagtaaacaatc atggg-3’)
（17）Select the pFastBac-Flag-sST2 carriers of correct sST2 DNA sequences.
（Four）Prepare restructuring Flag-sST2 AcNPV Plasmid DNA
1. taking 50ng, pFastBac-Flag-sST2 DNA are put into microcentrifugal tube, then are placed on ice cube, and 100ul is added to experience State bacterium（HD10Bac bacterial strains）, it is gently mixed, on ice cube, places 30 minutes
2. microcentrifugal tube is put under 42 DEG C of water bath conditions, 50 seconds, then be put on ice cube 3 minutes
3. add 700ul LB culture solutions, when 37 DEG C of shakes 4 are small
4. taking 20ul bacterium liquid, kind is applied in 50ug/ml Kanamycin, 7ug/ml Gentamicin, 10ug/ml On Tetracycline, 100ug/ml X-gal, 40ug/ml IPTGLB- agar plates, 37 DEG C, overnight incubation
5. selecting single white colony, 3ml 50ug/ml Kanamycin, 7ug/ml Gentamicin, 10ug/ml are inoculated into In TetracyclineLB culture solutions, 37 DEG C, shake, overnight incubation
6. extract bacteria plasmid DNA
A. plus 1.5ml bacterium liquid is into microcentrifugal tube, centrifugation, 13000rpm3 minutes, removes supernatant,
Plus 300ulBufferI b.（15mM Tris, pH8.0,10mM EDTA,100ug/ml RNaseA）To microcentrifugal tube In, even suspension bacterium
C. plus 300ulBufferII (0.2N NaOH, 1%SDS) is into microcentrifugal tube, turned upside down microcentrifugal tube 8 times, It places at room temperature, 5 minutes
Plus 300ul Buffer III (KCH d.3COO, pH5.5) into microcentrifugal tube, turned upside down microcentrifugal tube 8 times, Microcentrifugal tube is placed in ice cube, 10 minutes
E. centrifuge, 13000rpm, 10 minutes
F. 700ul centrifuged supernatants are shifted into microcentrifugal tube, adds 700ul Isopropanol into microcentrifugal tube, mixes , under the conditions of placing -20 °C, overnight
G. centrifuge, 14000g, 20 minutes, remove supernatant,
H. plus 70% Ethanol of 1.0ml are into microcentrifugal tube, washing, 14000g, 10 minutes, remove supernatant
I. microcentrifugation bottom of the tube DNA is dried
J. plus 40ul TE buffer are into microcentrifugal tube, dissolving DNA
7. with sST2 primer -1, primer -2 carries out PCR
Reaction buffer 10ul
High GC enhancer 10ul
10mMdNTP 1ul
25pm/ul primers -11ul
25pm/ul primers -21ul
Recombinate Flag-sST2 AcNPV Plasmid DNA 1ul
Q5 HF-DNA Polymerase 0.5ul
H2O 25ul
8.PCR conditions are as follows：
A. 95 DEG C 2 minutes
B. 95 DEG C 1 minute
C. 56 DEG C 1 minute
D. 72 DEG C 1 minute
Repeat B-D steps 30 time
E. 95 DEG C 30 seconds 1 minute
F. 56 DEG C 30 seconds 1 minute
G. 72 DEG C 6 minutes
25 DEG C are cooled to, completes PCR
9. drawing 5ul PCR products, add 2ul 5X DNA loading buffer
10. being placed in 1% Agarose/TBE gel electrophoresises hole, electrophoresis is carried out
11. carrying out DNA dyeing, viewed under ultraviolet radiation 934bp DNA bands with EB, 1,2,5,6 plasmid is positive restructuring Flag-sST2 AcNPV plasmids.
（Five）Prepare Flag-sST2 AcNPV
1. solution A：6ul recombinates Flag-sST2 AcNPVDNA+100ul TNM-FH liquid（No FBS, No antibiotics）
2. solution B：6ul Cellfectin (Invitrogen)+100ul TNM-FH liquid (No FBS, No antibiotics）
3. mixed solution A and solution B with micropipettor gently pressure-vaccum 3 times, at room temperature, after placing 30 minutes, add 800ul TNM-FH liquid, mixing become Plasmid DNA cell transfecting liquid
4. suctioning out SF9 cell TNM-FH culture solutions in 6 hole culture dishes, add 2ml TNM-FH liquid, washing SF9 cells are once
5. add Plasmid DNA cell transfecting liquid into SF9 Tissue Culture Dish, be placed on shaking table and slowly shake, at room temperature, 5 it is small when
6. suctioning out DNA cell transfecting liquid from SF9 Tissue Culture Dish, 2.5ml is added to contain 10%FBS TNM-FH culture solutions, 28 DEG C, it cultivates 3 days
7. drawing cell culture fluid into centrifuge tube, 3000rpm is centrifuged 10 minutes, collected supernatant, as Flag-sST22 AcNPV liquid
8. add 2X106SF9 cells add TNM-FH culture solutions of the 5ml containing 10%FBS into 100mm Tissue Culture Dish, 28 DEG C, Place 2 it is small when
9. suctioning out old TNM-FH culture solutions, add TNM-FH culture solutions of the 5ml containing 10%FBS, 100ul Flag-sST2 AcNPV Liquid is into SF9 Tissue Culture Dish
10. at room temperature, gently shake SF9 Tissue Culture Dish, 1 it is small when
11. adding TNM-FH culture solutions of the 5ml containing 10%FBS into SF9 Tissue Culture Dish, 28 DEG C, cultivate 3 days
12. drawing cell culture fluid into centrifuge tube, 3000rpm is centrifuged 10 minutes, collected supernatant, to expand Flag- SST2 AcNPV liquid
（Six）Prepare Flag-sST2 albumen
1. add 8X106SF9 cells add 20ml TNM-FH containing 10%FBS culture solutions into 150mm Tissue Culture Dish, 28 DEG C, Place 2 it is small when
2. suctioning out old TNM-FH culture solutions, add TNM-FH culture solutions of the 10ml containing 10%FBS, add 300ul Flag-sST2 In AcNPV to SF9 Tissue Culture Dish
3. at room temperature, gently shake SF9 Tissue Culture Dish, 1 it is small when
4. adding 20ml TNM-FH containing 10%FBS culture solutions into SF9 Tissue Culture Dish, 28 DEG C, cultivate 3 days
5. SF9 cells are collected into 50ml centrifuge tubes, centrifugation, 3000rpm, 5 minutes
6. removing supernatant, add 20ml PBS into centrifuge tube, centrifuge, 3000rpm, 5 minutes,
7. adding 1ml PBS into centrifuge tube, transfer SF9 cells are into 1.5ml microcentrifugal tubes
8. centrifugation, 3000rpm 5 minutes, remove supernatant
9. add 1.0ml lysing solutions (10mM Hepes. pH7.5,100mM NaCl, 0.5% NP-40,1mM EDTA, 1mM DTT, 1mM PMSF, 10ug/ml leupetine, 10ug/ml Aprotinin) into microcentrifugal tube, With micropipettor gently pressure-vaccum, it is mixed, is placed in ice cube 15 minutes
10. under the conditions of 4 DEG C, centrifugation, 5000rpm, 5 minutes, transfer supernatant was into new 1.5ml microcentrifugal tubes
11. under the conditions of 4 DEG C, centrifugation, 13000rpm, 20 minutes
12. shift supernatant（SF9 cytolysates）Into new 1.5ml microcentrifugal tubes
13. take 50ul Anti-Flag M2 affinity agarose（Sigma-Aldrich）Be put into 1.5ml it is micro from In heart pipe
14. add 1.2ml cleaning solutions (40mM Tris, pH8.0,300mM NaCl, 5% Glycerol, 0.01% NP-40, 1mM EDTA, 1mM DTT, 1mM PMSF, 10ug/ml Leupetine, 10ug/ml Aprotinin) arrive Anti-Flag In M2 affinity agarose microcentrifugal tubes,
15. under the conditions of 4 DEG C, centrifugation, 3000g 1 minute, suctions out supernatant, washes repeatedly 2 times
16. add SF9 cytolysates into Anti-Flag M2 affinity agarose microcentrifugal tubes
17. under the conditions of 4 DEG C, shake microcentrifugal tube, 1.5 it is small when
18. under the conditions of 4 DEG C, centrifugation, 3000rpm 1 minute, absorbs supernatant
19. add 1.2ml cleaning solutions into Anti-Flag M2 affinity agarose microcentrifugal tubes, in 4 DEG C of conditions Under, shake microcentrifugal tube, 20 minutes
20. under the conditions of 4 DEG C, centrifugation, 3000rpm 1 minute, absorbs cleaning solution, and the same terms wash repeatedly 3 times
21. add 40ul eluents（0.1M Glycine-HCl, pH3.0）To Anti-Flag M2 affinity agarose In microcentrifugal tube, with micropipettor gently pressure-vaccum, it is placed in ice cube 2 minutes
22. under the conditions of 4 DEG C, centrifugation, 6000rpm, 30 seconds, Aspirate supernatant, into new microcentrifugal tube, containing in 2ul It with liquid (2M Tris, pH8.0,3M NaCl), is mixed, the same terms, repeats elution 4 times,
23. combine 4 eluents, be transferred in bag filter (Spectra/Pro), be placed on dialyzate (20mM Tris, PH7.5,150mM NaCl, 5% Glycerol, 1mM DTT, 1mM EDTA) in, under the conditions of 4 DEG C, slowly stir, mistake Night
24. protein liquid in bag filter is collected to move on in microcentrifugal tube, under the conditions of 4 DEG C, centrifugation, 13000rpm, 10 points Clock,
25. dispensing protein liquid into different microcentrifugal tubes, 4 DEG C of short-term preservations are -20 DEG C or -80 DEG C long-term to preserve.
（Seven）Measure purifying sST2 protein concentrations and purity
A. argentation measures Flag-sST2 purity
1. freezing 10ul protein liquids to be put into 37 DEG C of water-baths, flash melt is put into ice cube,
2. add 3ul 4X albumen sample-adding buffer（50 mMTris, pH 6.8,2% SDS, 10% Glycerol, 1% β- mercaptoethanol, 12.5mM EDTA, 0.02% Bromophenol blue）, 100 DEG C, 3 minutes
3.10ulsST2 adds 3ul 4X albumen sample liquid, 100 DEG C, 3 minutes
4. 10ul protein liquids sample is added to carry out electrophoresis into the hole of 10% SDS-PAGE gels
5. after completing electrophoresis, obtaining SDS-PAGE gels, Silver stain is carried out, step is as follows：
A. plus 50% Methanol of 100ml are into the container of PAGE gel, at room temperature, slowly shake, 15 minutes,
Remove Methanol liquid
B. plus 5% Methanol of 100ml are into PAGE gel container, at room temperature, slowly shake, 10 minutes, removal Methanol liquid, is washed with water PAGE gel 1 time
C. plus in 10ul DTT to SDS-PAGE glue containers, there is 200ml water, at room temperature, slowly shake, 10 minutes, remove DTT Liquid, is washed with water PAGE gel 1 time
Plus 0.1% AgNO of 100ml d.3Into PAGE gel container, at room temperature, slowly shake, 15 minutes, removal AgNO3Liquid, is washed with water PAGE gel 2 times
Plus 3% Na of 100ml e.2CO30.05% Formaldehyde of ＆ react at room temperature into PAGE gel container, Until seeing clear protein band
F. plus Critic Acid are into PAGE gel container, terminate reaction
G. after washing with water SDS-PAGE glue, drying PAGE gel judges Flag-sST2 purity
Flag-sST2 purity is up to more than 99%.
B.Westernblotting measures purifying Flag-sST2
1. freezing Flag-sST2 protein liquids to be put into 37 DEG C of water-baths, flash melt is put into ice cube, with PBS 1:60 dilutions Protein liquid
2.10ul dilution Flag-sST2 protein liquids add 3ul 4X albumen sample adding liquids, 100 DEG C, 3 minutes
3. add 10ul diluted protein liquid samples be added in the hole of 10% SDS-PAGE gels, carry out electrophoresis, 100V, 2 it is small when
4. after completing electrophoresis, obtain PAGE gel
5. shift on PAGE gel albumen in nitrocellulose membrane with protein delivery Buffer, 30V, 2 it is small when
6. with 5% skim milk powder in TBS Buffer (20mM Tris, pH7.5,150mM NaCl, 0.05% Tween- 20), close nitrocellulose membrane, room temperature, 1 it is small when, wash nitrocellulose membrane once with TBS Buffer,
7. add 1：The 4000 anti-sST2 Antibodies Monoclonal antibodies of dilution（R & D systems）In 1% skim milk powder-TBS Buffer is into NC Nitroncellulose film container, under the conditions of 4 DEG C, slowly shakes, overnight
8. suctioning out antibody liquid, nitrocellulose membrane is washed 4 times with TBS Buffer, 20 minutes every time
9. add 1：5000 dilution anti-rabbit IgG antibody-HRP（Promega）It is fine to nitro in 1% skim milk powder-TBS Buffer In the plain film container of dimension, at room temperature, slowly shake, 1.5 it is small when
10. suctioning out antibody liquid, nitrocellulose membrane is washed 4 times with TBS Buffer, 20 minutes every time
11. add ECL Western blotting substrates（Pierce）Liquid reacts 2 minutes in nitrocellulose membrane
Westernblot results are measured with ImageReader LAS-4000 (FUJIFILM).
C.Bradford methods measure Flag-sST2 concentration
1. 1ml is taken to concentrate dye reagent（Bio-Rad Protein Assay Dye Reagent Concentrate）With 4mlH2O is uniformly mixed
2. preparing BSA titers, concentration is respectively 0,12.5,25,50,100,200ug/ml
3. adding 80ul PBS into micro titer plate well, add 5ul various concentration BSA titers, 5ul purifying Flag-sST2 is arrived In micro titer plate well, each sample adds 3 holes
4. 80ul is added to dilute dye reagent into micro titer plate well, react 5 minutes
5. putting microtiter plate to Victor3V 1420MultilabelCounter, wavelength 495nm is selected, measures OD values
6. according to BSA concentration standard curves, Flag-sST2 concentration is calculated as 57.46ug/ml.
。
Prepared by one, measures sST2 ELISA kits
（One）HRP marks anti-sST2 antibody
With activation HRP labelled antibodies kit (Thermo Scientific), step is as follows：
1. 300ul（300ug）SST2 antibody（NovusBiologicals）, it is transferred in bag filter (Spectra/Pro), It is placed in dialyzate (100mM NaHCO3), under the conditions of 4 DEG C, slowly stirs, overnight
2. pre-process protein concentration column（ThermoScientific）
A. plus 500ul 100mM NaHCO3 liquid is added in protein concentration column, 15000g, is centrifuged 20 minutes
B.400ul liquid suctions out remaining liq by protein concentration column film
C. protein concentration column is put into new collecting pipe, for use
3. collecting sST2 antibody liquids in bag filter, it is added in protein concentration column
4. 15000g is centrifuged 5 to 10 minutes, residue 100ul sST2 antibody liquids on protein concentration column film
5. a 100ulsST2 antibody liquids are transferred in new centrifuge tube
6. 100ul H2O are added in HRP activating enzymes pipes, HRP activating enzymes are dissolved, are mixed with 100ulsST2 antibody liquids, 4 DEG C, overnight
7. add in 40mg lysine to HRP activating enzymes pipes, dissolving, 25 °C, when reaction 2 is small
8. pre-process ProteinA/G columns
A. the capping of ProteinA/G columns top and bottom is opened, flows out storing liquid automatically
B. plus 5ml combinations liquid is into ProteinA/G columns, passes through ProteinA/G columns
9. 200ul combinations liquid is added to be gently mixed into HRP activating enzymes pipes, go to ProteinA/G columns, flow out mixed liquor automatically
10. 10ml combinations liquid is added to pass through ProteinA/G columns to ProteinA/G columns
11. 10ml eluents is added to collect eluent 1ml/ to ProteinA/G columns and often manage, 280nm wavelength measures OD values
12. selecting the collecting pipe of antibody containing HRP-sST2, eluent is transferred in bag filter (Spectra/Pro), is placed on dialysis In liquid (50mMTris, pH7.5,100mM NaCl), under the conditions of 4 DEG C, slowly stir, overnight
13. collecting HRP-sST2 antibody liquids in bag filter, add in 1% BSA to HRP-sST2 antibody liquids, 4 °C, it is long-term to preserve
（Two）Establish sST2 ELISA kit conditions
1.50ul（1, 2,4ug/ml）SST2 antibody（R&DSystems）In 50mM carbonate buffer solutions, pH9.6 is added to micro During titer plate is per hole, under the conditions of 4 DEG C, overnight
2. antibody response liquid is absorbed, with 200ul PBS washing lotions, washing reaction hole 2 times
3. add 200ul confining liquids into every hole, at ambient temperature, when closing 2 is small
4. confining liquid is absorbed, with 200ul PBS washing lotions, washing reaction hole 2 times
5. add 100ul Flag-sSt2 calibration objects（0,62.5,125,500,1000pg/ml）Or serum（1：50 times of dilutions）It arrives Kong Zhong, at room temperature, when reaction 1 is small
6. reaction solution is absorbed, with 200ul PBS washing lotions, washing reaction hole 5 times
7. add 100ul ST2 antibody-HRP (1000,2000,4000 times of dilutions), at room temperature, when reaction 1 is small
8. ST2 antibody-HRP liquid is absorbed, with 200ul PBS washing lotions, washing reaction hole 5 times
It 9. adding 50ul substrate solution A+50ul substrate solution B, at room temperature, is protected from light, reacts 20 minutes
10. adding 100ul terminate liquids (1N HCl), reaction is terminated
11. putting microtiter plate to Victor3V 1420MultilabelCounter, wavelength 450nm is selected, measures OD values.
12. use Flag-sST2 calibration objects（0,62.5,125,250,500,1000pg/ml）, make sST2 concentration with OD value affinity criterions curves, calculate sST2 concentration (ng/ml) in blood serum sample
13. according to experimental result, 2.0ug/mlsST2 antibody coating microtiter plate is selected, 1:2000 dilution HRP-sST2 resist Body combination sST2 albumen establishes ELISA, measures sST2 concentration in serum.
Flag-sST2 calibration object OD values
ELISA measures sST2 concentration in serum（1：50 dilute serums）
ELISA experiment liquid：
Carbonate buffer solution 1L: 8.4g NaHCO3 + 3.56g Na2CO3, adjust pH to 9.5
PBS：137 mMNaCl, 2.7 mMKCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4,pH 7.4
PBS washing lotions：PBS + 0.05% Tween-20
Confining liquid： PBS + 1% BSA
Dilution： PBS + 1% BSA
Substrate solution A:Tetramethylbenzidine, TMB（3,3 ', 5,5 '-tetramethyl benzidine）
Substrate solution B: H2O2
Terminate liquid:1N HCl.
Sequence table
<110>Jilin University
<120>Insect cell expression sST2 is as the calibration object for measuring sST2 concentration in serum
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 933
<212> DNA
<213> Biologic insect
<400> 1
aagtttagta aacaatcatg gggcctggaa aatgaggctt taattgtaag atgtcctaga 60
caaggaaaac ctagttacac cgtggattgg tattactcac aaacaaacaa aagtattccc 120
actcaggaaa gaaatcgtgt gtttgcctca ggccaacttc tgaagtttct accagctgca 180
gttgctgatt ctggtattta tacctgtatt gtcagaagtc ccacattcaa taggactgga 240
tatgcgaatg tcaccatata taaaaaacaa tcagattgca atgttccaga ttatttgatg 300
tattcaacag tatctggatc agaaaaaaat tccaaaattt attgtcctac cattgacctc 360
tacaactgga cagcacctct tgagtggttt aagaattgtc aggctcttca aggatcaagg 420
tacagggcgc acaagtcatt tttggtcatt gataatgtga tgactgagga cgcaggtgat 480
tacacctgta aatttataca caatgaaaat ggagccaatt atagtgtgac ggcgaccagg 540
tccttcacgg tcaaggatga gcaaggcttt tctctgtttc cagtaatcgg agcccctgca 600
caaaatgaaa taaaggaagt ggaaattgga aaaaacgcaa acctaacttg ctctgcttgt 660
tttggaaaag gcactcagtt cttggctgcc gtcctgtggc agcttaatgg aacaaaaatt 720
acagactttg gtgaaccaag aattcaacaa gaggaagggc aaaatcaaag tttcagcaat 780
gggctggctt gtctagacat ggttttaaga atagctgacg tgaaggaaga ggatttattg 840
ctgcagtacg actgtctggc cctgaatttg catggcttga gaaggcacac cgtaagacta 900
agtaggaaaa atccaagtaa ggagtgtttc tga 933

Claims (4)

1. a kind of insect cell expression sST2 is as the calibration object for measuring sST2 concentration in serum, it is characterised in that：Its step is：
A, T47D RNA are extracted, T47D cDNA gene pools is synthesized, goes out sST2 DNA, nucleotide sequence 55-987, structure with PCR amplification Build pFastBac-Flag-sST2 carriers；
B, the transduction of pFastBac-Flag-sST2 carrier DNAs generates restructuring Flag-sST2AcNPV into HD10Bac bacteriums Plasmid；
C, recombinate the transduction of Flag-sST2 AcNPV Plasmid DNA and enter SF9 cells, generate restructuring Flag-sST2 AcNPV；
D, Flag-sST2 AcNPV infection SF9 cells are recombinated and are given expression to 8 amino acid residue --- -- DYKDDDDK, it is short The Flag-sST2 albumen of peptide Flag, sST2-----19-328 amino acid residue；
E, it is purified into Anti-Flag M2 affinity agarose with albumen natural structure and antigenicity Flag-sST2 Albumen.
2. insect cell expression sST2 described in claim 1 exists as the calibration object for measuring sST2 concentration in serum, feature In：With Flag-sST2 as calibration object, double fastener heart enzyme-linked immunization is established ----ELISA is measured in serum or other body fluid SST2 concentration.
3. insect cell expression sST2 described in claim 1 exists as the calibration object for measuring sST2 concentration in serum, feature In：Synthesize cDNA and PCR amplification sST2 DNA：
（1）Synthesize cDNA random primings
T47D cell RNAs 2ul（600ng）
10mM dNTP1ul
Random primer (50ng/ul) 2ul
H2O 5ul
It is placed in 0.5ml microcentrifugal tubes, mixing；It is placed under the conditions of 65 DEG C, 5 minutes, is placed in ice cube 5 minutes；
（2）After centrifugation, add following substance into above-mentioned 0.5ml microcentrifugal tubes
5XSSIV buffer 4ul
100mM DTT 1ul
RNase out 1ul
RT 1ul
H2O 4ul；
（3）Mixing after centrifugation, is placed under the conditions of 50 DEG C, is reacted 10 minutes；
（4）It is placed under the conditions of 80 DEG C, inactivates 10 minutes；
（5）Add 1ul E.coilRNaseH, under the conditions of 37 DEG C, react 20 minutes, obtain cDNA；
（6）Synthetic primer, primer -1,5 '-tttttcatatgaagtttagtaaacaatcatggg-3 '
Primer -2,5 '-tttttctcgagtcagaaacactccttacttggatt-3 '；
（7）Carry out PCR
Reaction buffer 10ul
High GC enhancer10ul
10mMdNTP1ul
25pm/ul primers -11ul
25pm/ul primers -11ul
cDNA2ul
Q5 HF-DNA Polymerase 0.5ul
H2O 25ul；
（8）PCR conditions are as follows：
A. 95 DEG C 2 minutes
B. 95 DEG C 1 minute
C. 56 DEG C 1 minute
D. 72 DEG C 1 minute
Repeat B-D steps 30 time
E. 95 DEG C 30 seconds 1 minute
F. 56 DEG C 30 seconds 1 minute
G. 72 DEG C 6 minutes
25 DEG C are cooled to, completes PCR；
（9）5ul PCR products are drawn, add 2ul 5X DNA loading buffer；
（10）It is placed in 1% Agarose/TBE gel electrophoresises hole, carries out electrophoresis；
（11）DNA dyeing, viewed under ultraviolet radiation 934bpDNA bands are carried out with EB；
（12）Remaining PCR product adds 100ul H2O, then adds 150ul phenol/chloroforms/isoamyl alcohol（25/24/1）, mixing；
（13）Centrifugation, 13000rpm, 5 minutes；
（14）Supernatant is collected, adds 15ul 5M NaCl, 800ul ethyl alcohol, is mixed, -20 DEG C, overnight；
（15）Under the conditions of 4 DEG C, centrifugation, 13000rpm, 20 minutes；
（16) supernatant is removed, adds 70% ethyl alcohol of 800ul, under the conditions of 4 DEG C, centrifugation, 13000rpm, 10 minutes；
（17) supernatant is removed, dry microcentrifugation bottom of the tube DNA adds 50ul H20 dissolving DNAs.
4. insect cell expression sST2 described in claim 1 exists as the calibration object for measuring sST2 concentration in serum, feature In：Build FastBac-Flag-sST2 plasmids：
（1）With NdeI/XhoI restriction endonucleases（New England Biolabs）Cutting DNA
PCR DNA products 48ul
Cutsmart buffer 6ul
NdeI 3ul
XhoI3ul
PFastBac-Flag carrier DNAs 3ul（3ug）
Cutsmartbuffer 6ul
NdeI 3ul
XhoI3ul
H20 45ul
It is mixed, when 37 DEG C of reactions 2 are small；
（2）It is placed in 1% Agarose/TBE gel electrophoresis hole, carries out electrophoresis
（3）EB carries out DNA dyeing, viewed under ultraviolet radiation 934bp（PCR products）And 4.6kb（Carrier）DNA bands, respectively The lower DNA bands of cutting, are put into respectively in 1.5ml microcentrifugal tubes
（4）With QIAquick Gel Extraction Kit (QIAGEN), DNA in Agarose bands is extracted
Step is as follows：
Add in 700ul BufferQG to Agarose containing DNA band microcentrifugal tubes, be placed on 50 DEG C, 10 minutes, shake within every 2 minutes It swings once, dissolves Agarose bands
QIAquick centrifugal columns are put in 2ml collecting pipes, in addition stating in lysate 700ul to QIAquick centrifugal columns, place 2 Minute
Centrifugation, 3000g 1 minute remove liquid in collecting pipe
It centrifuges again once, 3000g 1 minute
Add 700ul Buffer PE to QIAquick centrifugal columns
Centrifugation, 12000g 1 minute remove liquid in collecting pipe
It centrifuges again once, 12000g 1 minute,
QIAquick centrifugal columns are put in new microcentrifugal tube, at room temperature, are placed 5 minutes
Add in 30ul H2O to QIAquick centrifugal columns, at room temperature, place 3 minutes
Centrifugation, 15000g 1 minute
Contain DNA in 1.5ml microcentrifugal tube collection liquids,
DNA concentration is measured with Nanodrop Spectrophotometer；
（5）With T4DNA ligase (New England Biolabs.), PCR DNA fragmentation pFastBac-Flag carriers are connected In,
Following substance is added in 1.5ml microcentrifugal tubes；
PCR DNA（NdeI/XhoI） 50ng
Carrier DNA（NdeI/XhoI） 50ng
10X T4 DNA Ligase Buffer 1 ul
T4 DNA Ligase 0.5ul
H2O to 10ul
It is mixed, 25 DEG C, when reaction 4 is small；
（6）Microcentrifugal tube is put on ice cube, adds 100ul competent bacterias（XL10 Gold bacterial strains）, it is gently mixed, ice cube It is upper to place 30 minutes
（7）Microcentrifugal tube is put under 42 DEG C of water bath conditions, 50 seconds, then be put on ice cube 3 minutes
（8）Add 700ul LB culture solutions, 37 DEG C, shake 1 it is small when
（9）Centrifugation, 10000g 5 minutes, remove supernatant, collect microcentrifugation bottom of the tube bacterium
（10）Kind is applied on 100ug/ml Ampicillin LB- agar plates, 37 DEG C, overnight incubation
（11）Single bacterium colony is selected, is inoculated into 3ml100ug/ml Ampicillin LB culture solutions, 37 DEG C, is shaken, overnight
（12）Bacteria plasmid DNA is extracted, with PureLink Quick Plasmid Miniprep Kits (Life Technologies Inc.)
Add 1.5ml bacterium liquid into microcentrifugal tube, centrifuge, 13000rpm3 minutes, remove supernatant,
Add 250ul suspensions BufferR3 into microcentrifugal tube, even suspension bacterium
250ul is added to dissolve BufferL7 into microcentrifugal tube, turned upside down microcentrifugal tube places microcentrifugal tube, room temperature Under, 5 minutes
Add 350ul precipitate Bs ufferN4 into microcentrifugal tube, turned upside down microcentrifugal tube places microcentrifugal tube in ice In block, 10 minutes
Centrifugation, 13000rpm, 10 minutes
Centrifugal column is put in 2ml collecting pipes, in addition stating centrifuged supernatant into centrifugal column, is placed 1 minute, centrifugation, 12000g, 1 minute, remove liquid in collecting pipe
Washing 500ul Buffer W10 is added to place 1 minute, centrifugation, 12000g, 1 minute into centrifugal column, remove collecting pipe Middle liquid
Washing 700ul Buffer W9 is added to place 1 minute, centrifugation, 12000g, 1 minute into centrifugal column, remove collecting pipe Middle liquid
Centrifugation, 12000g, 1 minute
Centrifugal column is put in new microcentrifugal tube, at room temperature, is placed 3 minutes
Add 75ul TE buffer into centrifugal column, at room temperature, place 3 minutes
Centrifugation, 12000g 2 minutes
Contain DNA in 1.5ml microcentrifugal tube collection liquids
DNA concentration is measured with Nanodrop Spectrophotometer；
（13）PstI restriction endonucleases（New England Biolabs）, cutting pFastBac-Flag-sST2 DNA
DNA 0.8ug
Cutsmartbuffer 1ul
PstI 1ul
H2O to 10ul
It is mixed, 37 DEG C, when reaction 2 is small,
（14）It is placed in 1% Agarose/TBE gel electrophoresis hole, carries out electrophoresis
（15）EB carries out DNA dyeing, and viewed under ultraviolet radiation forms DNA bands；
（16）665bp DNA band Plasmid DNA is selected, carries out determined dna sequence, sST2 DNA sequencing primers
(5’- tttttcatatggggttttggatcttagcaattc-3’;5’-tttttcatatgaagtttagtaaacaatc atggg-3’)
（17）Select the pFastBac-Flag-sST2 carriers of correct sST2 DNA sequences.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104151416A (en) * 2013-05-15 2014-11-19 中国科学院上海巴斯德研究所 Anti-human sST2 monoclonal antibody and application thereof
CN105671081A (en) * 2015-12-02 2016-06-15 肇庆大华农生物药品有限公司 Method of preparing chicken Akirin2 protein through insect cell expression system
CN106344914A (en) * 2014-11-05 2017-01-25 南通大学 Simple and convenient method for preparing periodic brugia malayi M29 epitope gene protein vaccine
CN107446949A (en) * 2017-07-25 2017-12-08 国家纳米科学中心 PLS3 recombinant proteins eukaryon expression plasmid and its construction method and application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104151416A (en) * 2013-05-15 2014-11-19 中国科学院上海巴斯德研究所 Anti-human sST2 monoclonal antibody and application thereof
CN106344914A (en) * 2014-11-05 2017-01-25 南通大学 Simple and convenient method for preparing periodic brugia malayi M29 epitope gene protein vaccine
CN105671081A (en) * 2015-12-02 2016-06-15 肇庆大华农生物药品有限公司 Method of preparing chicken Akirin2 protein through insect cell expression system
CN107446949A (en) * 2017-07-25 2017-12-08 国家纳米科学中心 PLS3 recombinant proteins eukaryon expression plasmid and its construction method and application

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