CN109022382A - Suspend the method for cultivating insect cell preparation and reorganization TPO albumen - Google Patents
Suspend the method for cultivating insect cell preparation and reorganization TPO albumen Download PDFInfo
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Abstract
The invention discloses a kind of methods of culture insect cell preparation and reorganization TPO albumen that suspends, and first transfect into logarithmic phase insect cell recombinant baculovirus, supernatant is taken after 96h, harvest P0 for recombinant baculovirus liquid；Primary recombinant baculovirus cell secondary culture, triangular flask is accessed with the inoculum concentration for passing on inoculating cell density >=3-5 × 10^5,2-3 × 10^6 are grown to cell density, secondary culture again, when density is up to 2 × 10^6cells/mL, it is that 0.1-0.5 connect malicious P0 for virus liquid with MOI, harvests cell culture supernatant, it is virus liquid P1 generation after centrifugal treating, masking foil wraps up 4 DEG C and is kept in dark place；Above method is repeated, P2 is made for virus liquid；Continue to pass on, when density reaches 2 × 10^6cells/mL, be that 1-5 connect malicious P2 for virus liquid with MOI, cell culture supernatant is harvested after 3-5 days, be virus liquid P3 generation after centrifugal treating, after bioactivity, obtains recombinating TPO albumen.The antigentic specificity of the method for the present invention preparation is good, and high sensitivity and potency height can greatly improve sensitivity, accuracy and the stability of kit.
Description
Technical field
The present invention relates to Measurements for Biotechnique, cultivate insect cell preparation and reorganization TPO albumen more particularly, to a kind of suspension
Method.
Background technique
Baculoviral is the carrier system for being widely used in high efficient expression foreign protein in recent years, the genome of baculoviral
For single closed hoop double chain DNA molecule, size is 80~160 kb, and genome in insect cell endoreduplication and can turn
Record.It is assembled in after DNA replication dna in the nucleocapsid of baculoviral, the latter has biggish flexibility, can accommodate larger segment
Exogenous DNA insertion, therefore be the ideal carrier for expressing large fragment DNA.Carrier is done with baculoviral, it can high efficient expression external source base
Up to the present cause has the several hundred genes including animals and plants, virus, bacterium, fungi and obtains in insect cell or larva body
To high efficient expression.This expression system be mainly characterized by obtain it is a large amount of antigenicity, immunogenicity it is preferable, with natural egg
White intimate soluble recombinant protein.This feature is better than bacterium, yeast and mammalian cell expression system.
After recombinate shape virus infection insect cell, the transcription post-processing of many eukaryocytes can be carried out to foreign protein
Effect, including glycosylation, phosphorylation, acylation, correct signal peptide cleavage, proteolysis and folding appropriate effect, and
And recombinant protein can also be assembled and be positioned on the same organelle of native protein, moreover it is possible to carry out oligomerization assembly appropriate.Cause
This is the ideal carrier for the protein that expression has bioactivity.
Thyroid peroxidase (thyroid peroxidase, TPO) is the important enzyme for being catalyzed thyroid hormone.TPO
It is synthesized by thyroid follicular cells, it is by 10% color being saccharified that the molecular weight that 933 amino acid residues form is 103kD
Plain sample protein.TPO is the key enzyme for being catalyzed thyroid hormone synthesis, it takes part in TG(thyroglobulin) tyrosine residue
Iodate and iodotyrosine coupled action, generation with autoimmune thyroid disease, development are closely related.TPOAb
(thyroid peroxidase antibody) can cause thyroid follicle to damage as the major autoantibody of autoimmune thyroid disease
Wound, leads to hypothyroidism.Hyperthyroidism is a kind of common autoimmune disease, TPOAb for hyperthyroidism pathogenesis,
Diagnoses and treatment and Index for diagnosis are of great importance.
It recombinates TPO albumen to use in thyroid peroxidase antibody detection kit as antigen, it is desirable that biology is living
Property high, high sensitivity.Recombination TPO albumen is prepared early period using spinner cultivation.When due to Spinner culture, body is cultivated
Product is the 1/2 of bottle body product, but bottle cap stuffiness again, therefore the DO of cell growth process is compared with Customers ' Legal Right, so as to cause imitating
The low phenomenon of valence；On the other hand, Spinner bottles are vials, and have 2 glass rotors, need when use especially careful, are easy broken
Damage, and price is more expensive, in addition each Spinner equipment is only capable of accommodating 4 bottle positions, and receiving bottle position is less, and amplification need to purchase again
Equipment, amplification are difficult.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of culture insect cell preparation and reorganization TPO albumen that suspends, and prepare
TPO protein biological activity it is high, be applied in thyroid peroxidase antibody detection kit, kit can be greatly improved
Sensitivity, accuracy and stability.
To achieve the above object, the present invention can take following technical proposals:
The method of the present invention for suspending culture insect cell preparation and reorganization TPO albumen includes the following steps:
Recombinant baculovirus is conventionally transfected in logarithmic phase insect cell, supernatant is taken after 96h by the first step,
P0 is harvested for recombinant baculovirus liquid；
Second step, primary recombinant baculovirus cell is after static gas wave refrigerator is recovered, through secondary culture, to pass on inoculating cell density
≥3-5In 10^5 inoculum concentration access triangular flasks, until cell density grows to 2-3At 10^6, passed on again
Culture, until cell is in logarithmic phase, density reaches 2It is that 0.1-0.5 carries out connecing malicious P0 generation virus with MOI when 10^6cells/mL
Liquid harvests cell culture supernatant, collects supernatant after centrifugal treating after 3-5 days, be virus liquid P1 generation, masking foil package after 4 DEG C
It is kept in dark place；
Third step repeats the second one step process, and P2 is made for virus liquid；
4th step continues to pass on, and when cell is in logarithmic phase, density reaches 2It is 1-5 progress with MOI when 10^6cells/mL
Malicious P2 is met for virus liquid, cell culture supernatant is harvested after 3-5 days, collects supernatant after centrifugal treating, is virus liquid P3 generation, potency
After detection, recombination TPO albumen is obtained.
The condition of secondary culture is 27 DEG C of temperature in the second step, shaking speed 100rpm/min.
The condition of centrifugal treating is revolving speed 1500rpm/min, time 3-5 minute in the second step, the 4th step.
The advantage of the invention is that antigenic structure and native protein are more using the antigen that baculovirus insect cell is expressed
It is close, and without being denaturalized purifying, the activity of antigen is preferably；It is special that recombinant baculovirus P1-P3 generation disease is carried out using shaking table culture
The preparation and amplification of venom, virus titer prepare virus liquid compared with static gas wave refrigerator and promote 10 times, the antigen effect of triangular flask culture preparation
Valence promotes 5 times compared with the antigen with spinner culture preparation, and is more conducive to amplify.Testing inspection, the method for the present invention preparation resist
Former specificity is good, and high sensitivity and potency height are used for thyroid peroxidase antibody detection kit, can greatly improve reagent
Sensitivity, accuracy and the stability of box.
Detailed description of the invention
Fig. 1 is that the antigen of the method for the present invention preparation is compared with antigen clinic coincidence rate is compareed.
Specific embodiment
More detailed explanation is made to the method for the present invention below, the understanding in order to those skilled in the art to the application.
Unless otherwise specified, reagent used in the method for the present invention and equipment are commercial product, and the method used is also normal for the industry
Advise the method used.
Embodiment 1 recombinates the preparation of TPO albumen (antigen)
The first step will recombinate bar according to the method for II kit specification of Cellfectin of work Invitrogen company production
Shape virus transfection takes supernatant into logarithmic phase insect cell after 96h, harvest P0 for recombinant baculovirus liquid, concrete operations are such as
Under:
1, by cell with 1-1.2x106The quantity of a cells/well spreads six orifice plates, and rear cell can cover with board bottom 85% or so overnight；
2, it prepares solution A -- plasmid/culture medium: being that 2ug recombinates Bacmid plasmid and is added to 100ul serum-free, unparalleled by quality
It is mixed gently in anti-III SFM of SF900；
3, it prepares B solution-liposome/culture medium: the Cellfectin II of volume 8uL is added to 100ul serum-free, unparalleled
In anti-III SFM culture medium of SF900, mix gently；
4, it prepares C solution: solution A and B solution being mixed, are incubated at room temperature 20min after mixing gently；
5, it takes out step 1 and spreads six orifice plates, abandon supernatant, it is primary with serum-free, III SFM culture medium board-washing of SF900 without double antibody,
C solution is added dropwise in six orifice plates, light shake makes its mixing, 27 DEG C of culture 4h；
6, supernatant is sucked out, 1.5ml SF-900III culture medium, 27 degree of culture 4d(96h is added), whether observation fluorescence occurs:
If fluorescence rate is greater than 85% or more, supernatant is collected, 1500rpm/min is centrifuged 3-5min, collects supernatant, is virus liquid P0 generation, tinfoil paper
It is kept in dark place for 4 DEG C after paper bag is wrapped up in.
Second step, preparation of the P1 for virus liquid: after the recovery of primary cell static gas wave refrigerator, by secondary culture, with passage
Inoculating cell density is not less than 3-5In 10^5 inoculum concentration access 250mL triangular flasks, triangular flask liquid amount is total volume
1/5(50mL), cell density grows to 2-3At 10^6, progress secondary culture, 27 DEG C of cultivation temperature, shaking speed
100rpm/min.After continuous passage 2-3 times, cell state is preferable, and survival rate is greater than 95%.It takes in logarithmic phase cell, with 8 ×
10^5cells/mL is inoculated with 250mL triangular flask, and density reaches 2It is that 0.1-0.5 carries out meeting malicious P0 with MOI when 10^6cells/mL
For virus liquid, cell culture supernatant is harvested after 3-5 days, and cell culture supernatant is centrifuged 3- in revolving speed 1500rpm/min
5min collects supernatant, is virus liquid P1 generation, is kept in dark place for 4 DEG C after masking foil wraps up.
Third step, preparation of the P2 for virus liquid: by primary cell after static gas wave refrigerator is recovered, by secondary culture, with
It passes on inoculating cell density and is not less than 3-5In 10^5 inoculum concentration access 500mL triangular flasks, triangular flask liquid amount is overall
Long-pending 1/5(100mL), cell density grows to 2-3At 10^6, progress secondary culture, 27 DEG C of cultivation temperature, shaking speed
100rpm/min.After continuous passage 2-3 times, cell state is preferable, and survival rate is greater than 95%.It takes in logarithmic phase cell, with 810
^5cells/mL is inoculated with 250mL triangular flask, and density reaches 2It is that 0.1-0.5 carries out connecing malicious P1 generation with MOI when 10^6cells/mL
Virus liquid harvests cell culture supernatant after 3-5 days, cell culture supernatant is centrifuged 3-5min in revolving speed 1500rpm/min,
Supernatant is collected, is virus liquid P2 generation, is kept in dark place for 4 DEG C after masking foil wraps up.
4th step, preparation of the P3 for virus liquid: by primary cell after static gas wave refrigerator is recovered, by secondary culture, with
It passes on inoculating cell density and is not less than 3-5In 10^5 inoculum concentration access 1000mL triangular flasks, triangular flask liquid amount is overall
Long-pending 1/5(250mL), cell density grows to 2-3At 10^6, progress secondary culture, 27 DEG C of cultivation temperature, shaking speed
100rpm/min.After continuous passage 2-3 times, cell state is preferable, and survival rate is greater than 95%.It takes in logarithmic phase cell, with 810
^5cells/mL is inoculated with 250mL triangular flask, and density reaches 2It is that 1-5 carries out connecing malicious P2 generation virus with MOI when 10^6cells/mL
Liquid harvests cell culture supernatant after 3-5 days, cell culture supernatant is centrifuged 3-5min in revolving speed 1500rpm/min, is collected
Supernatant is virus liquid P3 generation, and 4 DEG C are kept in dark place.
The P3 of harvest is carried out bioactivity for virus liquid, is concentrated with 30KD hollow fiber column, according to effect by the 5th step
Valence is concentrated 2-5 times, and 0.22 μm of bacterial filter filtering after glycerol adding, obtained recombination TPO protein-20 DEG C saves.
The application of the recombination TPO albumen of 2 embodiment 1 of embodiment preparation
By thyroid peroxidase antibody detection kit coating technique, it is coated with after being diluted according to respective concentration with compareing
Antigen is coated with simultaneously, Feng Bao, drying, corresponding index test results in the following table:
1, bioactivity: detecting the potency of antigen prepared by the present invention, after being diluted according to different concentration gradients, and compares
Envelope antigen is coated with simultaneously, Feng Bao, drying, and antigen valence prepared by the present invention is promoted to compared with promoting 5 times producing antigen by 1/50
1/250, it meets the requirements.
2, sensitivity: 10 holes are measured in parallel the luminous value of B0 internal control product, calculate the average (`X) and standard of measurement result
Poor (SD) calculates sensitivity with (`X ± 2SD), and testing result should be not higher than 2IU/ml, and the present invention prepares the sensitivity of antigen
There is promotion compared in production antigen, minimum detected value is promoted to 0.28IU/ml from 0.35IU/ml, meets the requirements.
3, clinical comparison: clinical sample requires randomness/gradient, 100 parts of comparison or more, with control batch yin-yang value base
This is consistent, as shown in Figure 1, clinical coincidence rate R2Reach 0.9952, meets the requirements.
4, general internal control product measured value: multiple holes measure Q1, Q2 internal control product, calculate measurement concentration, measurement knot with qualified S1-S6
Fruit within the allowable range, meets the requirements.
5, stability: coating plate is placed 10 days through 37 DEG C, and clinical sample detects the luminous value range of decrease and is not higher than 10%.The present invention
Compared with antigen is being produced, the luminous value range of decrease is reduced to 3% by 7%, is below 10%, meets the requirements the antigen of preparation.
Claims (3)
1. a kind of method for the culture insect cell preparation and reorganization TPO albumen that suspends, it is characterised in that: include the following steps:
Recombinant baculovirus is conventionally transfected in logarithmic phase insect cell, supernatant is taken after 96h by the first step,
P0 is harvested for recombinant baculovirus liquid；
Second step, primary recombinant baculovirus cell are close to pass on inoculating cell through secondary culture after static gas wave refrigerator is recovered
In degree >=3-5 × 10^5 inoculum concentration access triangular flasks, until being carried out again when cell density grows to 2-3 × 10^6
Secondary culture, until cell is in logarithmic phase, it is that 0.1-0.5 carries out meeting malicious P0 with MOI when density reaches 2 × 10^6cells/mL
For virus liquid, cell culture supernatant is harvested after 3-5 days, collects supernatant after centrifugal treating, be virus liquid P1 generation, masking foil package
It is kept in dark place for 4 DEG C afterwards；
Third step repeats the second one step process, and P2 is made for virus liquid；
4th step continues to pass on, and is 1-5 progress with MOI when density reaches 2 × 10^6cells/mL when cell is in logarithmic phase
Malicious P2 is met for virus liquid, cell culture supernatant is harvested after 3-5 days, collects supernatant after centrifugal treating, is virus liquid P3 generation, potency
After detection, recombination TPO albumen is obtained.
2. the method for the culture insect cell preparation and reorganization TPO albumen according to claim 1 that suspends, it is characterised in that: institute
The condition for stating secondary culture in second step is 27 DEG C of temperature, shaking speed 100rpm/min.
3. the method for the culture insect cell preparation and reorganization TPO albumen according to claim 1 that suspends, it is characterised in that: institute
State second step, the condition of centrifugal treating is revolving speed 1500rpm/min, time 3-5 minute in the 4th step.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196643A (en) * | 2021-12-24 | 2022-03-18 | 中元汇吉生物技术股份有限公司 | Process and method for purifying TPO |
Citations (4)
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| WO2008073620A2 (en) * | 2006-11-02 | 2008-06-19 | Neose Technologies, Inc. | Manufacturing process for the production of polypeptides expressed in insect cell-lines |
| CN105861458A (en) * | 2016-04-20 | 2016-08-17 | 南阳师范学院 | Thyroid peroxidase expression method |
| CN106282131A (en) * | 2016-09-30 | 2017-01-04 | 广东温氏大华农生物科技有限公司 | A kind of keratinase expression system and its preparation method and application |
| CN107841491A (en) * | 2017-11-03 | 2018-03-27 | 南阳师范学院 | A kind of expression for increasing thyroid peroxidase expression quantity |
-
2018
- 2018-09-05 CN CN201811031155.6A patent/CN109022382A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008073620A2 (en) * | 2006-11-02 | 2008-06-19 | Neose Technologies, Inc. | Manufacturing process for the production of polypeptides expressed in insect cell-lines |
| CN105861458A (en) * | 2016-04-20 | 2016-08-17 | 南阳师范学院 | Thyroid peroxidase expression method |
| CN106282131A (en) * | 2016-09-30 | 2017-01-04 | 广东温氏大华农生物科技有限公司 | A kind of keratinase expression system and its preparation method and application |
| CN107841491A (en) * | 2017-11-03 | 2018-03-27 | 南阳师范学院 | A kind of expression for increasing thyroid peroxidase expression quantity |
Non-Patent Citations (3)
| Title |
|---|
| YING LOU ET AL.: ""Secretory expression and scale-up production of recombinant human thyroid peroxidase via baculovirus/insect cell system in a wave-type bioreactor"", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| 刘金泉等: "hTPO膜外区基因的克隆及其杆状病毒表达载体的构建", 《天津医药》 * |
| 娄颖: ""重组人源甲状腺过氧化物酶的分泌表达及扩大培养"", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196643A (en) * | 2021-12-24 | 2022-03-18 | 中元汇吉生物技术股份有限公司 | Process and method for purifying TPO |
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Application publication date: 20181218 |