CN106701807A - Recombinant expression vector, engineering bacteria, preparation method and application thereof f - Google Patents
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Abstract
The invention relates to the field of gene engineering, and in particular relates to a recombinant expression vector, engineering bacteria, a preparation method and application thereof. Human IL-18 gene engineering bacteria can be effectively expressed by a gene recombination technology, a simple and effective IL-18 purification technology is established, the problem of preparation of a large amount of IL-18 can be solved, and the clinical needs in the future can be met. A stable and simple inclusion body renaturation and IL-18 purification technology is established, and protein purity can reach more than 97%; and an IL-18 activity identification method can be established. The problems of complex IL-18 purification process, low recovery rate and low purity and the like can be mainly solved, and the process has the advantages of being simple, fast, high in recovery rate and high in purity (97%).
Description
Technical field
The present invention relates to genetic engineering field, more particularly to recombinant expression carrier, engineering bacteria, preparation method
And its application.
Background technology
Wet age-related macular degeneration (AMD) and diabetic retinopathy (DR) lack preferably to be controlled
Treatment measure, the anti-VEGF treatment for clinically using is related to regular intraocular injection anti-VEGF monoclonal antibody (such as
Lucentis), it is not only with certain nocuity and expensive and just for confirmed cases, additionally,
Although incidence is very low, per injection can bring the serious of intraocular infection, retinal hemorrhage and stripping
Risk, and this no terminal for the treatment of.Global diabetic retinopathy (DR) patient 9300 in 2013
Ten thousand, wherein proliferative DR (PDR) patient 17,000,000, diabetic macular edema 21,000,000, prestige
Coerce the DR patient 28,000,000 of eyesight.Onset diabetes after 5 years diabetic retinopathy incidence be 25%,
60% is increased to after 10 years, 75%--80% is may be up to after 15 years.It is so visible, accelerate development of new treatment eye
Bottom vascular lesion preparation, not only with social benefit, and will produce huge economic benefit.At present
Specific effective treatment method is also lacked to AMD and DR, it is clinically main to use symptomatic treatment or auxiliary
Helping property treatment method is controlled or delays advancing of disease, and conventional treatment method is as follows：Hypoglycemic medicine,
Anti-inflammatory drug, part are short of money using TNF-α antagonist, Fenofibrate, VEGF (VEGF)
Anti-agent, RAS system inhibitors, non-glycosylation inhibitor, composite treatment and laser therapy.
Interleukin-18 (Interleukin-18, IL-18) be immune-regulating factor important in human body it
One, with antitumor, suppress the effects such as new vessels generation, with very big clinical value, GSK
Company has carried out antineoplastic II clinical trial phases.Because IL-18 is the presence of the few cells factor in human body
(pg grades) is so the source of IL-18 can only solve by gene recombination technology.But current IL-18 preparation sides
There is complex process, purity low (90%) in method, renaturation yield is low and yield is more low to a certain extent,
Not only increase its cost, and industrialization is difficult.
Due to the potential application values of IL-18, related preparation research is more.(J Immunol. in 1996
156,4274-4279) " human il-18 and variant are expressed in Escherichia coli and mammalian cell " is reported,
IL-18 is expressed in the form of inclusion body, and its purification process once uses hydrophobic chromatography (pheny-sepharose),
Anion-exchange chromatography (DEAE-Sepharose), gel chromatography (Superdex75), reverse hydrophobic layer
Analysis (C4) etc. method, it is purified after IL-18 SDS-PAGE detections purity reach 90%.2006 (in
State's Journal of Immunology, 22,141-144) report " expression of the recombined human IL-18 in Escherichia coli and antitumor
Effect ", human il-18 is expressed with IL-18 in the form of inclusion body, and expression product is centrifuged after ice bath, forgives
Body precipitation is dissolved after fully washing with 8mol/L urea, through renaturation, is dialysed and G50 gel chromatographies, pure
IL-18 SDS-PAGE detections purity after change reaches 90% or so.Chinese patent (10169340A) in 2010
Disclose " the preparation method of human interleukin-18；It is classified using recombinant yeast pichia pastoris fermented and cultured supernatant
It is concentrated by ultrafiltration, hydrophobic chromatography is once used after (NH4) 2SO4 precipitations of more than 50% saturation degree
(pheny-sepharose), anion-exchange chromatography (Q-Sepharose HP) purifying, it is purified after
IL-18 SDS-PAGE detections purity reaches 97%.There is technique to a certain extent and answer in these preparation methods
Miscellaneous, purity is low, renaturation yield is low and low some problems of yield, increases its cost.
The existing IL-18 preparation methods with Escherichia coli as Hosts have 2 major defects：1. technique is answered
It is miscellaneous, it is unfavorable for industrialized production, and also the technique of complexity not only increases cost and process cycle is long,
The rate of recovery is low；2. purity of protein is low (90%), it is impossible to meet as the requirement of biological products.It is above-mentioned
The major defect of the IL-18 preparation methods with Pichia pastoris as Hosts is complex process, expression productivity
It is low, make large-scale production difficult.
The content of the invention
In view of this, the present invention provides a kind of recombinant expression carrier, engineering bacteria, preparation method and applications.
The present invention constructs the genetic engineering bacterium of high efficient expression human il-18 using gene recombination technology, establishes simple and direct
Effective purifying IL-18 technical matters, solves the problems, such as largely to prepare IL-18, disclosure satisfy that future clinical
Need.Establish and stablize simple and direct renaturing inclusion bodies, the process route of IL-18 purifying, purity of protein is reachable
More than 97%；Establish identification IL-18 activity methods.
It is complicated present invention mainly solves IL-18 purifying process, reclaim the low and low problem of purity.This technique is special
Simple and direct, the quick, rate of recovery of point is high and purity is (97%) high.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of recombinant vector, contain IL-18 genetic fragments；The IL-18 genetic fragments
Sequence as shown in SEQ ID No.1.
In some embodiments of the present invention, the plasmid that the recombinant vector that the present invention is provided is used is carried
Body is pBV220.
Present invention also offers a kind of engineering bacteria, described recombinant vector is integrated with.
In some embodiments of the present invention, the Host Strains of engineering bacteria are e.colistraindh5α.
Present invention also offers the method that the recombinant vector described in utilization prepares IL-18, comprise the following steps：
Step 1：Build and obtain engineering bacteria；The engineering bacteria is integrated with described recombinant vector；
Step 2：The engineering bacteria is taken, activated, fermentation, Fiber differentiation obtain inclusion body；
Step 3：The inclusion body is taken, after scrubbed, dissolving, renaturation, purified, determination of activity is obtained
Obtain IL-18.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
Purifying described in 3 is specially through hydroxyapatite chromatography column chromatography, collects purpose peak albumen, then pass through
Sephacryl S-200-HR gel chromatography column chromatographies, the protein peak occurred between collecting 100min~120min.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
The flow velocity of the sample solution of hydroxyapatite chromatography column chromatography described in 3 is 10-20ml/min；It is abundant with equilibrium liquid
Stream washes foreign protein to after baseline, respectively with containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1.0mol/L
The equilibrium liquid wash-out of NaCl, collects eluting peak, is identified through SDS protein electrophoresises, and destination protein is present in
In 0.1mol/L NaCl eluting peaks；
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
The flow velocity of Sephacryl S-200-HR gel chromatographies column chromatography sample solution is 2-5ml/min in 3.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
The construction method of recombinant vector described in 1 is the EcoR of the gene containing IL-18 I, the double digestions of BamH I,
Connected with the carrier pBV220 of EcoR I, the double digestions of BamH I, build the restructuring containing IL-18 genes
Carrier pBV220-IL-18.
In some embodiments of the present invention, institute in the method for IL-18 is prepared using described recombinant vector
The construction method of engineering bacteria is stated to take the recombinant vector conversion bacillus coli DH 5 alpha, is built and is obtained engineering bacteria
Strain DH5 α-pBV220IL-18.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
The activation in 2 is specially：Take the engineering bacteria and be inoculated into the nutrient agar plate medium containing Amp
On, 37 DEG C of 12~16h of culture；Picking single bacterium colony is inoculated into the LB fluid nutrient mediums containing Amp,
37 DEG C of 12~16h of shaking table culture.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
Fermentation described in 2 is specially：The bacterium solution for taking the activation acquisition is inoculated into the ratio of 1~10% (v/v)
In fermentation medium containing Amp, 28~32 DEG C of 12~16h of shaking table culture；Bacterium solution is pressed into 5~10% (v/v)
Ratio be inoculated into fermentation tank, cultivated under the conditions of 28~32 DEG C to bacterial concentration and reach OD600 for 2.0-3.0.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
Fiber differentiation is specially described in 2：Bacterium solution that the fermentation obtains is taken in 42 DEG C of Fiber differentiation 3-6h, together
Shi Chixu adds filler liquid, and the bacterium solution volume ratio that described filler liquid is obtained with the fermentation is 5%~10%；
Ammoniacal liquor automatically adjusts pH7.0；
Described filler liquid presses following proportions in units of 1000ml：
Peptone 100g
Yeast extract 100g
Glucose 200g (the independent high pressure of 700ml water dissolves, 8 pounds).
In some embodiments of the present invention, institute in the method for IL-18 is prepared using described recombinant vector
Acquisition inclusion body is stated to be specially：The medium centrifugal collects thalline after the Fiber differentiation is taken, is buffered with TE
Liquid suspends, and under condition of ice bath, high-pressure homogenization crushes thalline, 4 times repeatedly.Thalline after centrifugal breaking,
Collect precipitation, as inclusion body.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
Dissolving described in 3 is specially：Ratio according to 2-10% (v/v) adds solubilization of inclusion bodies liquid, 4 DEG C of conditions
Under, magnetic agitation dissolving 5-10h；4 DEG C, 9000rpm is centrifuged 10min, collects supernatant；
The solubilization of inclusion bodies liquid presses following proportions in units of 1000ml：
8mol urea 480g
10mM DTT 1.65g
With 20mmolPB buffer solutions (pH7.0-8.0) constant volume to 100.
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
Renaturation is specially described in 3：Ratio according to 10-20% is slowly added to renaturing inclusion bodies liquid, makes protein concentration
Drop to 0.2%, 4 DEG C of renaturation 12-20h；
The renaturing inclusion bodies liquid presses following proportions in units of 1000ml：
20mmol PB buffer solution (pH7.0-8.0) 1000ml
DTT(20mM) 3.1g。
In some embodiments of the present invention, the method and step of IL-18 is prepared using described recombinant vector
Determination of activity described in 3 is specially：
Human leukemia cell's KG1 passage cultures：With the RPMI1640 culture mediums containing 10%FCS,
5%CO2, 37 DEG C of culture U937 cells；When cell density reaches 2 × 106Passed on during individual cell/ml；Pass
It is 4 × 10 for cell density5Cell/ml；
The RPMI1640 culture mediums without serum are used to be configured to 50nmol hrIL-18 standby；Take 96 holes
Tissue Culture Plate, in preceding 5 row per hole add the culture mediums of 100 μ l 20%FCS 1640 prepare cell density be
2×106Cell/ml, A, B, C, D row plus ConA, final concentration of 0.5mg/L；E、F、G、H
Hole is not added with ConA；100 μ l, 10 times of hrIL-18 of dilution are separately added into 1-5 row are per hole, are mixed,
The final concentration of 25nmol of first row；(each hole of dilution factor 4)；After being mixed, Tissue Culture Plate is put 37 DEG C,
5%CO2Incubator culture 24h；
IFN-γ potency is surveyed with ELISA method and collect culture supernatant, obtain IL-18 activity.
The medicine for the treatment of Fundus oculi lesion is being prepared present invention also offers IL-18 obtained in methods described
In application.
The present invention provides a kind of recombinant expression carrier, engineering bacteria, preparation method and applications.Profit of the invention
The genetic engineering bacterium of high efficient expression human il-18 is constructed with gene recombination technology, is established simple and direct effective pure
Change IL-18 technical matters, solve the problems, such as largely to prepare IL-18, disclosure satisfy that future clinical needs.Build
Stand and stablized simple and direct renaturing inclusion bodies, the process route of IL-18 purifying, purity of protein is up to more than 97%；
Establish identification IL-18 activity methods.
It is complicated present invention mainly solves IL-18 purifying process, reclaim the low and low problem of purity.This technique is special
Simple and direct, the quick, rate of recovery of point is high and purity is (97%) high.
Present invention also offers IL-18 activity test methods.The activity identification of interleukin-18 is generally adopted
Use PBMC cells：Stimulate PBMC with IL-18, according to the how much judgement IL-18's for producing INF γ
Activity；Second method is to stimulate NK cell proliferating numbers amount to judge the activity of IL-18, two kinds with IL-18
Method is all relatively cumbersome, and it is thin that we substitute PBMC using KG-1 cells (a kind of leukemia cell line)
Born of the same parents, because KG-1 cells are passage cells, simplify experimental arrangement.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to reality
The accompanying drawing to be used needed for example or description of the prior art is applied to be briefly described.
Fig. 1 shows the purification process flow chart of present invention offer；
Fig. 2 shows pBV220-IL-18 Prokaryotic expression vector construction schemes；
Fig. 3 is shown as the PCR of expression vector, and sign is respectively in figure：
M：Molecular weight standard, be followed successively by 10000 from big to small, 7000,4000,2000,1000,
500、250bp；
1 compares for carrier pBV220, and 2 is Expression Vectors pBV220-IL-18PCR results；
Fig. 4 shows hydroxyapatite chromatography post result after purification；Sign is respectively in figure：
M：Molecular weight standard, be followed successively by 97.2 from big to small, 66.7,44.3,29.8kD；
1：0.1M NaCl eluting peaks；2：0.2M NaCl eluting peaks；3：0.5M NaCl eluting peaks；
Fig. 5 shows Sephacryl S-200-HR gel chromatography column purification results, and sign is respectively in figure：
M：Molecular weight standard, is followed successively by 97.2,66.7,44.3,29.8,20.0,14.0kD from big to small；
1-5：The foreign protein 6 of removal：Sample 8 after purification：Sample before purification；
Fig. 6 shows that hrIL-18 induction KG1 cells produce INF γ；
Fig. 7 shows the result of control group 1 in embodiment 6；
Fig. 8 shows the result of control group 2 in embodiment 6.
Specific embodiment
The invention discloses a kind of recombinant expression carrier, engineering bacteria, preparation method and applications, this area
Technical staff can use for reference present disclosure, be suitably modified technological parameter realization.In particular,
All similar replacements and change are apparent to those skilled in the art, and they are considered as
It is included in the present invention.The method of the present invention and application are described by preferred embodiment, related
Personnel substantially can not depart from present invention, spirit and scope to enter method described herein and application
Row is changed or suitably changed with combining and realizes and apply the technology of the present invention.
The technical scheme is that (Fig. 1)：Construction of expression vector pBV220-IL-18, the expression vector
The gene for containing EcoR I, BamH I double digestion that will be obtained by PCR, then with EcoR I,
The carrier pBV220 connections of the double digestions of BamH I；Build engineered strain DH5 α-pBV220-IL-18；In temperature
Under degree induction, engineered strain DH5 α-pBV220-IL-18 high density fermentations, high efficient expression IL-18 albumen；
The hrIL-18 dissolvings of expression, renaturation and purifying.
Technical scheme is comprised the concrete steps that：The engineered strain for taking -70 DEG C of preservations is inoculated into the nutrition fine jade containing Amp
On fat plating medium, 37 DEG C of cultures；Picking single bacterium colony is inoculated into the LB fluid nutrient mediums containing Amp
In, 37 DEG C of shaking table cultures；Bacterium solution is inoculated into the fermentation medium containing Amp in proportion, 28-32 DEG C is shaken
Bed culture；Bacterium solution is inoculated into fermentation tank in proportion, is cultivated under the conditions of 28-32 DEG C；Treat that bacterial concentration reaches
To OD600After about 2.0-3.0,42 DEG C of Fiber differentiation 3-6h are warmed up to, while persistently adding filler in a small amount
Liquid, ammoniacal liquor automatically adjusts pH7.0, and thalline is collected by centrifugation；Thalline is suspended with TE buffer solutions, condition of ice bath
Under, high-pressure homogenization crushes thalline, and precipitation, as inclusion body is collected by centrifugation；Appropriate inclusion body is weighed, plus
After entering the washing of inclusion body cleaning solution, solubilization of inclusion bodies liquid is added, under the conditions of 4 DEG C, magnetic agitation dissolving 5-10h
More than；Supernatant is collected by centrifugation；Proportionally it is slowly added to renaturation solution, room temperature magnetic agitation dissolving 4h；4℃
More than renaturation 12-20h；By the hydroxyapatite chromatography post balanced with equilibrium liquid, purpose peak is collected；Again
The gel chromatography column balanced with equilibrium liquid is gone up after purpose peak is concentrated by ultrafiltration again, purpose peak is collected, as
Destination protein, hydro-acupuncture preparation or lyophilized formulations are distributed into by the purifying protein.
Each liquid formulations scope and preparation are as follows：
LB fluid nutrient mediums：Following proportions, autoclaving after dissolving are pressed in units of 1000ml.
Tryptone 10g
Yeast extract 5g
NaCl 10g
Fermentation medium：Following proportions, autoclaving after dissolving are pressed in units of 1000ml.
20mmol PB buffer solutions (pH7.0-8.0) press following proportions in units of 1000ml：
Sodium dihydrogen phosphate .2H2O 3.12g
Disodium hydrogen phosphate .12H2O 7.16g
EDTA 0.37g。
Solubilization of inclusion bodies liquid presses following proportions in units of 1000ml：
8mol urea) 480g
10mM DTT 1.65g
With 20mmol PB buffer solutions (pH7.0-8.0) constant volume to 100
Renaturation solution presses following proportions in units of 1000ml：
20mmol PB buffer solution (pH7.0-8.0) 1000ml
DTT(20mM) 3.1g。
Eluent 1
20mmol PB buffer solution (pH7.2) 1000ml
0.1mol NaCL 5.8g。
Eluent 2
20mmol PB buffer solution (pH7.2) 1000ml
0.2mol NaCL 11.6g。
Eluent 3
20mmol PB buffer solution (pH7.2) 1000ml
0.5mol NaCL 29.0g
Feed supplement liquid is by following proportions in units of 1000ml：
Peptone 100g
Yeast extract 100g
Glucose 200g (the independent high pressure of 700ml water dissolves, 8 pounds).
The present invention provides a kind of recombinant expression carrier, engineering bacteria, preparation method and applications.Profit of the invention
The genetic engineering bacterium of high efficient expression human il-18 is constructed with gene recombination technology, is established simple and direct effective pure
Change IL-18 technical matters, solve the problems, such as largely to prepare IL-18, disclosure satisfy that future clinical needs.Build
Stand and stablized simple and direct renaturing inclusion bodies, the process route of IL-18 purifying, purity of protein is up to more than 97%；
Establish identification IL-18 activity methods.
It is complicated present invention mainly solves IL-18 purifying process, reclaim the low and low problem of purity.This technique is special
Simple and direct, the quick, rate of recovery of point is high and purity is (97%) high.
Present invention also offers IL-18 activity test methods.The activity identification of interleukin-18 is generally adopted
Use PBMC cells：Stimulate PBMC with IL-18, according to the how much judgement IL-18's for producing INF γ
Activity；Second method is to stimulate NK cell proliferating numbers amount to judge the activity of IL-18, two kinds with IL-18
Method is all relatively cumbersome, and it is thin that we substitute PBMC using KG-1 cells (a kind of leukemia cell line)
Born of the same parents, because KG-1 cells are passage cells, simplify experimental arrangement.
Raw material needed for recombinant expression carrier, engineering bacteria, preparation method and applications and reagent Jun Keyou cities
Field is bought.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1 builds the prokaryotic expression carrier pBV220-IL-18 of IL-18
By RT-PCR obtain EcoR I, BamH I double digestion of the gene containing IL-18, then with
The carrier pBV220 connections of the double digestion of EcoR I, BamH I；The prokaryotic expression containing IL-18 genes is built to carry
Body pBV220-IL-18, converts bacillus coli DH 5 alpha, builds engineered strain DH5 α-pBV220IL-18；
(referring to Fig. 2,3).
The fermentation of the hrIL-18 of embodiment 2 and high efficient expression
Activation, fermentation, induction and the recovery of inclusion body of engineering bacteria：The engineered strain for taking -70 DEG C of preservations connects
Plant onto the nutrient agar plate medium containing Amp, 37 DEG C of culture more than 12h；Picking single bacterium colony connects
Plant in the LB fluid nutrient mediums containing Amp, 37 DEG C of more than shaking table culture 12h；Bacterium solution is pressed into 1-10%
Ratio is inoculated into the fermentation medium containing Amp, more than 28-32 DEG C of shaking table culture 12h；Bacterium solution is pressed
5-10% ratios are inoculated into fermentation tank, are cultivated under the conditions of 28-32 DEG C；Treat that bacterial concentration reaches OD600About
After 2.0-3.0,42 DEG C of Fiber differentiation 3-6h are warmed up to, while persistently adding filler liquid in a small amount, ammoniacal liquor is automatic
Regulation pH7.0.After culture terminates, thalline is collected by centrifugation, thalline is suspended with TE buffer solutions, condition of ice bath
Under, high-pressure homogenization crushes thalline, 4 times repeatedly.Thalline after centrifugal breaking, collects precipitation, as wraps
Contain body.
Fermentation volume：15-25L；Obtain inclusion body：150-300g；It is concentrated by ultrafiltration 10-20 times.
The renaturation of the hrIL-18 of embodiment 3 and purifying
The washing of 3.1 inclusion bodys, dissolving and renaturation：Inclusion body obtained in appropriate embodiment 2 is weighed, addition is forgiven
After body cleaning solution washs 2 times, solubilization of inclusion bodies liquid, under the conditions of 4 DEG C, magnetic force are added according to the ratio of 2-10%
More than stirring and dissolving 5-10h；4 DEG C, 9000rpm is centrifuged 10min, collects supernatant；According to 10-20%
Ratio be slowly added to renaturation solution, protein concentration is dropped to 0.2%, 4 DEG C of more than renaturation 12-20h；Immediately
Purifying.
The purifying of 3.2 hrIL-18：Hydroxyapatite chromatography post is balanced with equilibrium liquid, by the renaturation solution after above-mentioned dilution
Whole loadings, the equilibrium liquid used by balance hydroxyapatite chromatography post is 20mmol PB buffer solutions
(pH7.0-8.0), flow velocity is 10-20ml/min；Foreign protein is washed to after baseline with equilibrium liquid fully stream, first
Eluted with the equilibrium liquid containing 0.1,0.2,0.5 and 1.0M NaCl afterwards, collect eluting peak, through SDS albumen electricity
Swimming identification, destination protein is present in 0.1NaCl eluting peaks (Fig. 4)；By above-mentioned 0.1M NaCl eluting peaks
Sample is gone up the Sephacryl balanced with equilibrium liquid in bed volume 2-8% ratios again after being concentrated by ultrafiltration
S-200-HR gel chromatography columns, flow velocity is 2-5ml/min；Occur between Fraction collection 100min~120min
Protein peak, -20 DEG C of preservations are (Fig. 5) to be identified.
The prourokinase identification of 3.3 purifying uses SDS protein electrophoresises, and purity is more than 97%；Using ultraviolet
Spectrophotometric determination protein content, about 0.5mg/ml.Purifying IL-18 volumes 2-6L.
The hrIL-18 biological activity determinations of embodiment 4
4.1 human leukemia cell's KG1 passage cultures：With the RPMI1640 culture mediums containing 10%FCS,
5%CO2, 37 DEG C of culture U937 cells；When cell density reaches 2 × 106Passed on during individual cell/ml；Pass
It is 4 × 10 for cell density5Cell/ml.
4.2 by hrIL-18, with the RPMI1640 culture mediums without serum, to be configured to 50nmol standby；Take 96 hole cell trainings
Plate is supported, adds the cell density that the culture mediums of 100ul 20%FCS 1640 are prepared to be 2 × 10 per hole in preceding 5 row6Cell
/ ml, A, B, C, D row plus ConA, final concentration of 0.5mg/L；E, F, G, H hole are not added with ConA.
The hrIL-18 that the embodiment 3 of 10 times of dilutions of 100ul is prepared is separately added into 1-5 row are per hole, is mixed
It is even, the final concentration of 25nmol of first row；(each hole of dilution factor 4).After mixing, Tissue Culture Plate is put
37 DEG C, 5%CO2Incubator culture 24h；
4.3 survey IFN-γ potency with ELISA method collects culture supernatant, obtains the activity (Fig. 6) of IL-18.
The contrast test of embodiment 5
Control group：It is divided into control group 1, control group 2, control group 3；
Wherein, (the application number of control group 1：200820054133.1)；
(the application number of control group 2：98103307.5)；
Control group 3 (J Immunol.156 (1996), 4274-4279)
Experimental group：Zymotic fluid, inclusion body and IL-18 that the embodiment of the present invention 2,3 is prepared.
The results are shown in Table 1.
The result of the test of table 1
As seen from Table 1, the IL-18 yield that the present invention is obtained is apparently higher than control group (P ＜ 0.05)；It is pure
Degree is better than control group.
The IL-18 activity test method comparative examples of embodiment 6
Comparative example 1：RhIL-18 stimulates PBMCs to produce IFN-γ experiment
Test method：
(1) preparation of PBMCs：The physiological saline for taking appropriate anticoagulated whole blood plus equivalent is mixed, and equivalent is added and contained
The 50ml centrifuge tubes (or 10ml) of lymphocyte separation medium, centrifugation 2000rpm (horizontal rotor),
10min, takes buffy coat, and with brine 2 times, 1500rpm, 10min, regulation is thin
Born of the same parents' concentration is 1 × 106/ml；
(2) above-mentioned cell suspension is added into IL-2 or IL-12 (100u/ml), adds 96 well culture plates；100μl/
Hole；
(3) IL-18 (control IL-18 and self-control IL-18) concentration is diluted with 1640-10%FCS to be respectively：
1ng/ml,10ng/ml,100ng/ml,1μg/ml,10μg/ml。
(4) above-mentioned 96 orifice plates containing PBMCs are added, each titre adds 4 holes.
(5) 37 DEG C of 5%CO2 incubator culture 48h, collect cells and supernatant；
(6) IFN-γ content is determined with ELISA kit.
(7) with standard IFN-γ as abscissa, A values are ordinate, change standard curve；Calculate each IL-18 groups
IFN-γ content, convert specific activity.
Expected results：
IL-18 can stimulate PBMCs cell secretion of gamma-IFN under the collaboration of IL-2 or IL-12, and
The secretory volume of IFN-γ is proportionate with the consumption of IL-18 and activity.Result is shown in Fig. 7.
Comparative example 2：RhIL-18 enhancing NK cytoactives experiment (direct fluorescence flow cytometry instrument method)
Test method：
(1) preparation of PBMCs：The physiological saline for taking appropriate anticoagulated whole blood plus equivalent is mixed, and equivalent is added and contained
The 50ml centrifuge tubes (or 10ml) of lymphocyte separation medium, centrifugation 2000rpm (horizontal rotor),
10min, takes buffy coat, and with brine 2 times, 1500rpm, 10min, regulation is thin
Born of the same parents' concentration is 1 × 106/ml；
(2) above-mentioned cell suspension is added into IL-2 or IL-12 (100u/ml), adds 25ml blake bottles, altogether
8 bottles；
(3) IL-18 (control IL-18 and self-control IL-18) concentration is diluted with 1640-10%FCS to be respectively：
0.1ng/ml,1ng/ml,10ng/ml,100ng/ml。
(4) add in the above-mentioned PBMCs cells containing PBMCs, each titre adds 1 bottle.
(5) 37 DEG C of 5%CO2 incubator culture 24h, collect cell culture；Cell is adjusted after being washed with culture medium dense
Spend is 2 × 106/ml。
(6) marked with anti-CD56, CD69 monoclonal antibody of fluorescence labeling respectively；37 DEG C of incubation 2h.
(7) percentage of CD56, CD69 cell is determined with flow cytometer.
The percentage of each IL-18 groups CD56, CD69 cell is calculated, specific activity curve is drawn.Result is shown in
Fig. 8.
Experimental group：The embodiment of the present invention 4.Test result indicate that experimental group IL18 energy significant stimulations KG1 is thin
Intracrine INF γ, and have obvious dose-effect relationship, there is pole significant difference (p ＜ 0.01) with control group ratio
(Fig. 6).
The above is only the preferred embodiment of the present invention, it is noted that general for the art
For logical technical staff, under the premise without departing from the principles of the invention, some improvement and profit can also be made
Decorations, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of recombinant vector, it is characterised in that contain IL-18 genetic fragments；The IL-18 genes
The sequence of fragment is as shown in SEQ ID No.1.
2. recombinant vector according to claim 1, it is characterised in that the plasmid vector is
pBV220。
3. a kind of engineering bacteria, it is characterised in that be integrated with recombinant vector as claimed in claim 1 or 2.
4. engineering bacteria according to claim 3, it is characterised in that Host Strains are bacillus coli DH 5 alpha
Bacterial strain.
5. the method for preparing IL-18 using recombinant vector as claimed in claim 1 or 2, its feature exists
In comprising the following steps：
Step 1：Build and obtain engineering bacteria；The engineering bacteria is integrated with weight as claimed in claim 1 or 2
Group carrier；
Step 2：The engineering bacteria is taken, activated, fermentation, Fiber differentiation obtain inclusion body；
Step 3：The inclusion body is taken, after scrubbed, dissolving, renaturation, purified, determination of activity is obtained
Obtain IL-18.
6. method according to claim 5, it is characterised in that purifying described in step 3 is specially
Through hydroxyapatite chromatography column chromatography, purpose peak albumen is collected, then through Sephacryl S-200-HR gel layers
Analysis column chromatography, the protein peak occurred between collecting 100min~120min.
7. method according to claim 6, it is characterised in that the hydroxyapatite chromatography post layer
The flow velocity for analysing sample solution is 10-20ml/min；Foreign protein is washed to after baseline with equilibrium liquid fully stream, is used respectively
Equilibrium liquid wash-out containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1.0mol/L NaCl, collects wash-out
Peak, identifies, destination protein is present in 0.1mol/L NaCl eluting peaks through SDS protein electrophoresises；
The flow velocity of Sephacryl S-200-HR gel chromatography column chromatography sample solutions is 2-5ml/min.
8. the method according to any one of claim 5 to 7, it is characterised in that described in step 1
The construction method of recombinant vector is the EcoR of the gene containing IL-18 I, the double digestions of BamH I, and with EcoR
The carrier pBV220 connections of the double digestion of I, BamH I, build the recombinant vector containing IL-18 genes
pBV220-IL-18；
The construction method of the engineering bacteria builds and obtains to take the recombinant vector conversion bacillus coli DH 5 alpha
Engineered strain DH5 α-pBV220IL-18；
The activation in step 2 is specially：Take the engineering bacteria be inoculated into the nutrient agar containing Amp put down
On plate culture medium, 37 DEG C of 12~16h of culture；Picking single bacterium colony is inoculated into the LB liquid training containing Amp
In foster base, 37 DEG C of 12~16h of shaking table culture；
Fermentation described in step 2 is specially：The bacterium solution of the activation acquisition is taken by the ratio of 1~10% (v/v)
Example is inoculated into the fermentation medium containing Amp, 28~32 DEG C of 12~16h of shaking table culture；Bacterium solution is pressed
The ratio of 5~10% (v/v) is inoculated into fermentation tank, is cultivated to bacterial concentration under the conditions of 28~32 DEG C and reached
OD600It is 2.0-3.0；
Fiber differentiation is specially described in step 2：The bacterium solution of the fermentation acquisition is taken in 42 DEG C of Fiber differentiations
3-6h, while persistently adding filler liquid, the bacterium solution volume ratio that described filler liquid is obtained with the fermentation is
5%~10%；Ammoniacal liquor automatically adjusts pH7.0；
Described filler liquid presses following proportions in units of 1000ml：
Peptone 100g
Yeast extract 100g
Glucose 200g (the independent high pressure of 700ml water dissolves, 8 pounds)
The acquisition inclusion body is specially：The medium centrifugal collects thalline after the Fiber differentiation is taken, is used
TE buffer solutions suspend, and under condition of ice bath, high-pressure homogenization crushes thalline, 4 times repeatedly；After centrifugal breaking
Thalline, collects precipitation, as inclusion body；
Dissolving described in step 3 is specially：Ratio according to 2-10% (v/v) adds solubilization of inclusion bodies liquid,
Under the conditions of 4 DEG C, magnetic agitation dissolving 5-10h；4 DEG C, 9000rpm is centrifuged 10min, collects supernatant；
The solubilization of inclusion bodies liquid presses following proportions in units of 1000ml：
8mol urea 480g
10mM DTT 1.65g
With 20mmol PB buffer solutions (pH7.0-8.0) constant volume to 100
Renaturation is specially described in step 3：Ratio according to 10-20% is slowly added to renaturing inclusion bodies liquid,
Protein concentration is set to drop to 0.2%, 4 DEG C of renaturation 12-20h；
The renaturing inclusion bodies liquid presses following proportions in units of 1000ml：
20mmol PB buffer solution (pH7.0-8.0) 1000ml
DTT(20mM) 3.1g。
9. the method according to any one of claim 5 to 8, it is characterised in that living described in step 3
Property determine be specially：
Human leukemia cell's KG1 passage cultures：With the RPMI1640 culture mediums containing 10%FCS,
5%CO2, 37 DEG C of culture U937 cells；When cell density reaches 2 × 106Passed on during individual cell/ml；Pass
It is 4 × 10 for cell density5Cell/ml；
The RPMI1640 culture mediums without serum are used to be configured to 50nmol hrIL-18 standby；Take 96 holes
Tissue Culture Plate, in preceding 5 row per hole add the culture mediums of 100 μ l 20%FCS 1640 prepare cell density be
2×106Cell/ml, A, B, C, D row plus ConA, final concentration of 0.5mg/L；E、F、G、H
Hole is not added with ConA；100 μ l, 10 times of hrIL-18 of dilution are separately added into 1-5 row are per hole, are mixed,
The final concentration of 25nmol of first row；(each hole of dilution factor 4)；After being mixed, Tissue Culture Plate is put 37 DEG C,
5%CO2Incubator culture 24h；
IFN-γ potency is surveyed with ELISA method and collect culture supernatant, obtain IL-18 activity.
10. IL-18 is preparing treatment Fundus oculi lesion according to obtained in any one of claim 5 to 9
Application in medicine.
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| CN107190033A (en) * | 2017-07-11 | 2017-09-22 | 北京宝易生物技术有限公司 | A kind of fermentation process in high density of the genetic engineering bacterium of recombinant interferon |
| CN113501857A (en) * | 2021-08-17 | 2021-10-15 | 沈阳百发科技有限公司 | Preparation method of high-activity recombinant protein |
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| CN1326992A (en) * | 2000-06-07 | 2001-12-19 | 北京双鹭药业有限责任公司 | Gene cloning, product preparation and use of Chinese interleukin-18 subtype (53 Arg IL-18) |
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| CN1326992A (en) * | 2000-06-07 | 2001-12-19 | 北京双鹭药业有限责任公司 | Gene cloning, product preparation and use of Chinese interleukin-18 subtype (53 Arg IL-18) |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107190033A (en) * | 2017-07-11 | 2017-09-22 | 北京宝易生物技术有限公司 | A kind of fermentation process in high density of the genetic engineering bacterium of recombinant interferon |
| CN113501857A (en) * | 2021-08-17 | 2021-10-15 | 沈阳百发科技有限公司 | Preparation method of high-activity recombinant protein |
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