CN1534044A - Recombination human interferon alpha 8 polypeptide coding cDNA sequence preparation method and application - Google Patents
Recombination human interferon alpha 8 polypeptide coding cDNA sequence preparation method and application Download PDFInfo
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Abstract
A recombinant human alpha-interferon 8 polypeptide for treating tumor and HBV, HCV and HIV infections, its expression in colibacillus, and its application are disclosed. It is prepared through optimizing its coding cDMA sequence by mutation, configuring colibacillus expression carrier pBV220/INF alpha 8, engineered bacteria screening, protein separation, purifying and freeze drying.
Description
Technical field
The present invention relates to biological technical field.Be specifically related to recombinant human interferon-alpha-8 (rhIFN α 8) peptide coding cDNA sequence and expression in intestinal bacteria and medicinal application.
Background technology
(Interferon is that a class by emiocytosis has multiple bioactive protein such as antiviral IFN) to Interferon, rabbit.It expresses multiple effect protein by directly or indirectly acting on cell Interferon, rabbit effector, thereby brings into play multifarious biological function, regulates material as a kind of cell function, and Interferon, rabbit belongs to cytokine.
The Interferon, rabbit kind of formally determining title through international Interferon, rabbit NK has: IFN α, IFN β, IFN γ, IFN ω, IFN τ etc. are several, and the Interferon, rabbit of same type is divided into some hypotypes again, and wherein IFN α research at most.Up to now, an IFN α gene surplus Chinese scholars has cloned 20 from the people's gene group.
IFN α 8 genes are successively by Yelverton E, Leung D, Weck P, et al., (NucleicAcids Res 1981,9 (3): 731-741) Goeddel, D.V., Leung, D, W., Dull, t, J.., etal., (Nature 1981,290 (5801): 20-26) Bowden DW, Mao J, GiU T, etal., (Gene 1984,27 (1): 87-99) wait the author to obtain in clone in 1981,1981,1984.Studies show that IFN α 8 assignments of genes gene mapping are in karyomit(e) 9P
22On, the coded protein product is 189 aa, and wherein preceding 23 aa are signal peptide, and sophisticated coded polypeptide is 166 aa.It is raw material that the inventor adopts normal Chinese's peripheral blood and tire hepatic tissue sample, through total RNA extracting, IFN α universal primer reverse transcription PCR (RT-PCR), the clone has obtained Chinese IFN α 8 gene (see figure 1)s, relatively more in full accord with the sequence that the outer author of predecessor State reports through sequential analysis, this is the good basis of having established of the present invention.
The research of relevant IFN α 8 biological activity assay also is seen in report, and that representative is wherein arranged is Foster GR et al., and the report that waits studies show that, people IFN α 8 Interferon, rabbit have the highest antiviral activity in each hypotype of IFN α, and can induce U
1Mutant clone produces antiviral state.(Foster?GR?et?al.，Different?relative?activities?of?humancell-derived?interferon-alpha?subtypes：IFN-alpha?8?has?very?highantiviral?potency.J.Interferon?Cytokine?Res，1996(12)：1027-1033).
FinterNB et al., Deng adopting WISH (people), Hela (people), MDBK (ox), L929 (mouse), RK13 (rabbit), Vero cell strains such as (monkeys) to compare the antiviral activity of IFN α 1, α 2, α 8, α C, multiple type Interferon, rabbit such as α F, Le, which system no matter IFN α 8 adopt as a result, and its antiviral activity all belongs to higher.Discover that different recombinant interferon have 90 times difference to the promoter action of NK cell, wherein α 8 (B), α 2 (A), α 1 (D), α C are higher, and α 6 (K), α F are lower, and α 7 (J) is minimum.(FinterNB?et?al.，J.Interferon?Cytokine?Res，1991(s)：185.)
Up to now, domestic and international business development for IFN α 8 does not appear in the newspapers yet.
Summary of the invention
But technical problem to be solved by this invention is to provide a kind of great expression, purifying, through sudden change, IFN α 8 coded cDNA sequences optimized.
RhIFN α 8cDNA sequence disclosed by the invention has gene order and the encoding amino acid sequence shown in the sequence 1,2.This sequence is on the basis of natural hIFN α 8cDNA sequence, according to the preferences of e. coli codon, is guaranteeing under the constant situation of encoding amino acid sequence acquisition through suddenling change after.Described rhIFN α 8 polypeptide molecular weights size is 20kda, and the pI value is 4.9.
Another technical problem to be solved by this invention is to disclose the expression method of above-mentioned rhIFN α 8 in intestinal bacteria.
Natural IFN α 8cDNA sequence clone imports intestinal bacteria DH on the pBV220 carrier
5 α, after testing, gene is not expressed.Adopt the DNA software analysis to find, contain many intestinal bacteria rare codons in the natural IFN α 8cDNA sequence, infer that this may be exactly the major cause that IFN α 8 does not express.The present invention is according to the preferences of e. coli codon, guaranteeing under the constant situation of encoding amino acid sequence, synthetic one section encoding gene be the IFN α 8cDNA sequence (seeing sequence 1) of intestinal bacteria preference.In order further to improve the destination gene expression level, before IFN α 8cDNA coding region initiator codon atg, also increased by one section ribosome bind site sequence (seeing sequence 3).In addition, 5 ' end in IFN α 8cDNA sequence has been introduced EcoR I restriction enzyme site, has increased the SalI restriction enzyme site after 3 ' the end terminator codon (TAA), and then makes up coli expression carrier pBV220/IFN α 8, through the engineering bacteria screening, obtain target protein after albumen sepn, purifying, the freeze-drying.
The expression of rhIFN α 8 of the present invention in intestinal bacteria comprises the following steps:
One, the structure of intestinal bacteria recombinant expression vector pBV220/IFN α 8
1.IFN the subclone of α 8 genes and with being connected of carrier
At first with the IFN α 8cDNA sequence (sequence 1,2) of synthetic and ribosome bind site sequence (sequence 3) subclone on the pUC18 carrier, become pUC18/IFN α 8.With EcoRI/Sal I double digestion digestion pUC18/IFN α 8 and coli expression carrier pBV220, reclaim IFN α 8 and carrier segments.After purified, adopt Bao Bio-Engineering Company's dna ligation kit to connect (see figure 2), then transformed competence colibacillus intestinal bacteria DH
5 α, the dull and stereotyped incubated overnight of LB.
2. engineering bacteria screening
Positive bacteria on the picking LB flat board is dropped into the evaluation of cutting and check order of capable enzyme.Identify that the positive colony after confirming is inoculated in the 5ml liquid LB nutrient solution, 30 ℃, 250rpn/min cultivated 12-18 hour.Nutrient solution is changed in the 50ml liquid LB nutrient solution, continue to be cultured to the OD value for 0.4-0.6, improve temperature to 42 suddenly and ℃ induced 4-5 hour.Centrifugal collection thalline carries out the SDS-PAGE electrophoretic analysis, and the result has obtained the very high engineering strain of a strain expression amount.
Two, the engineering bacterium fermentation condition gropes and optimizes
1. the activation of bacterial classification
Get-70 ℃, the bacterial classification 100ul of 20% glycerine preservation, be inoculated in the test tube that contains 4ml LB substratum, adding penbritin, to make its final concentration be 100ug/ml; 32 ℃, 220r/min are cultivated about 10h, again by 2% inoculative proportion transferred species in the triangular flask that contains 50mlLB, adding penbritin, to make its final concentration be 100ug/ml; 32 ℃, 220r/min are cultivated 4h, become activated seed liquid, 4 ℃ of preservations.
2. fermentation seed liquid is cultivated
Get the activatory seed liquor, be inoculated in the 1000ml triangular flask that contains 500ml LB substratum with 1: 1000 inoculative proportion, it is 100ug/ml that the adding penbritin makes its final concentration.32 ℃, 220r/min are cultivated 10h, OD
600Be about 2.5.
3. fermentor cultivation
Use the BioFloIII type 7L of New Brunswick Scientific company to control fermentor tank automatically, fermentation parameter is adjusted according to fermented liquid multi-parameter monitoring result and experience and is optimized.
4. centrifugal receipts bacterium gets wet thallus
5. bacterial cell disruption
Three, the separation of recombinant protein IFN α 8, purifying
1. the washing of inclusion body
(1) with 20mM Tris.cl pH8.0,1mMEDTA washs broken thalline, and 9000rpm * 30min abandons supernatant.
(2) use 0.2%Triton X-100,20mM Tris.cl pH8.0,1mM EDTA washing, 9000rpm * 30min abandons supernatant.
Through (1), (2) washing, inclusion body purity can reach about 60%.
2. sex change, renaturation, ultrafiltration
(1) will wash good inclusion body and be dissolved in the 7M Guanidinium hydrochloride in weight in wet base by 1: 20 (v/w), 50mMTris.cl pH8.0,10mM beta-mercaptoethanol, in the 10mM edta buffer liquid, stir 4h under the room temperature, centrifugal 9000rpm * 30min gets transparent supernatant liquor, i.e. sex change liquid.
(2) adopt the method for dilution refolding to carry out renaturation sex change liquid, renaturation buffer is 2.5MVrea, 50mMtris.cl, 1mMETDA.Slowly drip sex change liquid during renaturation, making the renaturation solution final concentration of protein is 0.1-0.2mg/ml, renaturation time 36h.
(3) protein renaturation liquid is filtered earlier, ultrafiltration again (millipore) obtains ultrafiltration and concentration liquid.
3. column chromatography purification
(1) adopts Q-sepharose FF anion-exchange column
With 20mM Tris.cl pH8.0 balance Q post, go up sample more earlier, at first use 20mMTris.cl pH8.0 wash-out after last sample is intact, use 100mM NaCl stepwise elution again, use the 400mMNaCl stepwise elution at last, 280nm detects the eluted protein peak, through the SDS-PAGE electrophoresis detection, purity is about 85%.
(2) adopt Interferon, rabbit monoclonal antibody chromatography column
Earlier, go up sample again with PBS balance anti-interferon monoclonal antibody chromatography column, after the PBS wash-out is collected protein peak, through SDS-PAGE electrophoresis detection target protein purity greater than 99%.
A technical problem more to be solved by this invention is to disclose the application of above-mentioned rhIFN α 8 in preparation treatment human or animal's tumour and disease of viral infection medicine.
RhIFN α 8 of the present invention can be used as the active ingredient of said medicine, prepares various liquid preparations of acceptable or freeze-dried preparation on the various physiology.
Tumour of the present invention comprises various malignant tumours, as solid carcinoma and serous tumor etc.
Disease of viral infection of the present invention comprises the disease that infection such as HBV, HCV, HIV, HSV, HPV or influenza virus cause.
Carry out extracorporeal antivirus effect activity and animal experiment with rhIFN α 8 of the present invention:
I. the foundation of extracorporeal antivirus effect activity test method and check and analysis
One, principle
Cytopathic-effect inhibition assay is adopted in rhIFN-α 8 titrations.The rhIFN-α 8 of different concns attacks with the VSV vesicular stomatitis virus after adding people's amnion wish cell cultures,, calculates product biological activity to be checked and tires the pathology inhibition degree of wish cell by standard of comparison product and product to be checked.
Two, material
Human alpha type Interferon, rabbit national standard product (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute)
RhIFN-α 8 product to be checked
The wish cell strain
MEM substratum (GIBCO company)
Staining fluid: 50mg Viola crystallina adds in the 20ml ethanol, adds water to 100ml
Destainer: 50% ethanol, 50% distilled water, 0.1% acetate
96 well culture plates (NUNC company)
Microplate reader (model 550, Bio-rad company)
Three, method
1. adjusting the wish cell concn with the MEM nutrient solution that contains 10% bovine serum is 3.0 * 10
5/ mL is inoculated in 96 well culture plates, every hole 100 μ l; Cultivate 4-6h under 37 ℃, 5%CO2 condition.
2. standard substance and product to be checked all are diluted to 10 with the MEM that contains 7% bovine serum
3IU/ml is as last sample initial concentration, and after carrying out 4 times of doubling dilutions on the dilution plate, (8 extent of dilution) adds 96 orifice plates respectively, every hole 50ul, and (each extent of dilution is established multiple hole) sets up cell, virus control simultaneously; Cultivate 18-24h under 37 ℃, 5%CO2 condition.
3. cell plate are abandoned supernatant, and adding dosage is 100TCID
50VSV virus liquid, every hole 100ul; Cultivate 24h under 37 ℃, 5%CO2 condition.
4. abandon supernatant, every hole adds 50ul violet staining liquid, and room temperature is placed 30min.
5. flowing water carefully washes away staining fluid, blots residual moisture.Every hole adds the 100ul destainer, and room temperature is measured the O.D value in microplate reader 570,630nm place after placing 3-5min.
6. obtain product to be tested activity unit by reference substance and product to be tested curve ratio.
Four, result
Three batches of rhIFN-α 8 purification of samples in this laboratory are carried out titration, and specific activity is all 10 as a result
7-10
8In the IU/mg scope, show that purification of samples tentatively reaches the requirement of biological products rules.
The therapeutic action of 8 couples of HBV DNA of II.rhIFN α transgenic mice
1. experiment material
Recombinant human IFN α 8 trial targets are by Chengdu company of Diao Pharmaceutical Group preparation (purity is greater than 99%).
HBV DNA transgenosis Balb/c mouse is provided male and female half and half by air force's Guangzhou Hospitals.Blood detects HBsAg, HBeAg through the ELISA method before dispatching from the factory, and quantitative PCR detection HBVDNA copy number, hepatic tissue adopt immunohistochemical methods to detect HBsAg, HBeAg and express, and confirms that These parameters is all positive.
HBsAg, HBeAg ELISA detection reagent are provided by Huamei Bio-Engrg Co..
That HBsAg, HBcAg immunohistochemical methods detect is one anti-, two anti-ly provide by Gibco company.
HBV DNA quantitatively adopts Zhongshan Medical Univ. to reach peace gene diagnosis center HBV DNA detection by quantitative test kit to carry out on PE7700 type PCR instrument.
Positive control drug recombinant human IFN α 1b is provided by emerging biological products company of Shenzhen section.
2. experimental technique
HBV DNA transgenosis Balb/c mouse is raised in the SPF environment.Be divided into administration group (IFN α 8 at random, 100,000 IU/ only, calculate by body weight/kilogram and to be equivalent to 100 times of human dosage), administration group (IFN α 8,10,000 IU/ only, calculate 10 times that are equivalent to human dosage by body weight/kilogram), positive controls (IFN α 1b, 10,000 IU/ are only), 4 groups of negative control group, 6 every group.
That medication begins is preceding, medication begins back 2 weeks, 4 weeks, takes each 0.4ml of mouse socket of the eye venous blood 8 weeks respectively, and that medication begins is preceding, 4 weeks, performed the operation respectively in 8 weeks and take the murine liver tissue sample.
Administration group and positive controls adopt tibialis anterior meat injection to give relative medicine, the next day once, totally 5 times, negative group adopts the 0.9%NS liquid injection of same method afford and positive group equivalent.
Institute's extracting blood sample is used for ELISA detection, HBV DNA detection by quantitative, and the hepatic tissue sample is used for HBsAg, the HBeAg immunohistochemical methods detects.
3. experimental result
(1) serum HBV DNA detection by quantitative result
Table 1 serum HBV DNA detection by quantitative result (copy number/ml)
Administration group administration group
The negative group of n n positive controls n n
(100,000 U/ only) (10,000 U/ only)
Treat preceding 3.51 * 10
76 8.02 * 10
66 4.42 * 10
76 7.88 * 10
76
2 weeks 3.90 * 10
46 7.62 * 10
36 7.88 * 10
46 4.38 * 10
66
4 weeks 1.25 * 10
35 4.56 * 10
35 6.42 * 10
35 5.76 * 10
76
8 weeks 6.50 * 10
65 3.78 * 10
65 5.35 * 10
65 9.36 * 10
76
(2) serum HBsAg, HBeAg changing conditions
Serum HBsAg is all positive before and after each group administration, the positive rate no change.HBeAg negative rate variation before and after administration sees Table 2.
Table 2 serum HBeAg positive rate changing conditions
(%) 8 weeks (%) 4 weeks in (%) 2 weeks (%) before the treatment
Administration group 100 100
6/6 6/6 4/5 (80％) 5/5 (100％)
(100,000 U/ only) % %
Administration group 100 100
6/6 6/6 4/5 (80％) 5/5 (100％)
(10,000 U/ only) % %
Positive group 6/6 100 6/6 100 6/6 (100%) 6/6 (100%)
Negative group 6/6 100 6/6 100 6/6 (100%) 6/6 (100%)
(3) hepatic tissue immunohistochemical methods changing conditions
Positive group and administration group see that all the HBsAg expression weakens, and HBcAg changes not obvious.
4. conclusion:
IFN α 8 and IFN α 1b all have antivirus action to HBV DNA transgenic mice, heavy dose of and low dose of IFN α 8 curative effect no significant differences.
Description of drawings
Fig. 1 IFN α 8 gene agarose gel electrophoresis figure
Wherein M is nucleic acid molecular weight Marker, be followed successively by from the bottom to top 200bp, 300bp,
400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1 is
IFN α gene, the about 500bp of length
Fig. 2 recombinant expression vector pBV220/IFN α 8 plasmid construction figure
Fig. 3 pBV220/IFN α 8 recombinant expression vector enzymes are cut the evaluation electrophorogram
Wherein M is nucleic acid molecular weight Marker, be followed successively by from the bottom to top 250bp, 500bp,
750bp、1000bp、2000bp、3000bp、3500bp、4000bp、5000bp、
6000bp, 8000bp, 9000bp, 10000bp,, 1,2,3,4 is pBV220/IFN α 8
Vector gene, the about 4500bp of length
Fig. 4 pBV220/IFN α 8 engineering bacterium fermentation product S DS-PAGE electrophorograms
Wherein M is protein molecular weight Marker, be followed successively by from the bottom to top 14200da,
20100da、24000da、29000da、36000da、45000da、66000da，1、
2,3 is pBV220/IFN α 8 vector expression products, and 1,2 is through optimization of fermentation conditions
Product, the destination gene expression product is about 22Kda
The purified engineering bacterium fermentation liquid of Fig. 5 product S DS-PAGE electrophorogram
Wherein M is protein molecular weight Marker, be followed successively by from the bottom to top 14200da,
20100da、24000da、29000da、36000da、45000da、66000da，1、
2,3 is pBV220/IFN α 8 vector expression products, and 1 is final purified product.Order
Gene expression product be about 22Kda
Embodiment
The structure of embodiment 1 intestinal bacteria recombinant expression vector pBV220/IFN α 8
1.IFN the sudden change and the optimization of α 8 genes
According to the preferences of e. coli codon, guaranteeing under the constant situation of encoding amino acid sequence, synthetic one section encoding gene be the IFN α 8 cDNA sequences (seeing sequence 1) of intestinal bacteria preference.In order further to improve the destination gene expression level, before IFN α 8cDNA coding region initiator codon atg, also increased by one section ribosome bind site sequence (seeing sequence 3).In addition, introduced the EcoRI restriction enzyme site, increased the SalI restriction enzyme site after 3 ' the end terminator codon (TAA) at 5 ' end of IFN α 8cDNA sequence.
2.IFN the subclone of α 8 genes and with being connected of carrier
The concrete grammar of gene clone and connection with reference to Sa nurse Brooker etc. show " molecular cloning experiment guide " (third edition).At first with the IFN α 8 cDNA sequences of synthetic and ribosome bind site sequence subclone on the pUC18 carrier, become pUC18/IFN α 8.With EcoRI/Sal I double digestion digestion pUC18/IFN α 8 and coli expression carrier pBV220, reclaim IF α 8 and carrier segments.After purified, adopt Bao Bio-Engineering Company's dna ligation kit to connect (see figure 2), then transformed competence colibacillus intestinal bacteria DH
5 α, the dull and stereotyped incubated overnight of LB.
3. engineering bacteria screening
Positive bacteria on the picking LB flat board drops into that capable enzyme is cut and the evaluation of checking order (enzyme cut identify electrophoresis see Fig. 3).Identify that the positive colony after confirming is inoculated in the 5ml liquid LB nutrient solution, 30 ℃, 250rpm/min cultivated 12-18 hour.Nutrient solution is changed in the 50ml liquid LB nutrient solution, continue to be cultured to the OD value for 0.4-0.6, improve temperature to 42 suddenly and ℃ induced 4-5 hour.Centrifugal collection thalline carries out the SDS-PAGE electrophoretic analysis, and the result has obtained the very high engineering strain of a strain expression amount.
The separation purifying technique of embodiment 2 engineering bacterium fermentations and IFN α 8 target proteins
One, engineering bacterium fermentation
1. nutrient solution preparation
(1) seed liquor substratum 500ml
Tryptone 5g
Yeast?Extract2.5g
NaCl 5g
Add deionized water and be settled to 500ml, at 121 ℃, 1.034 * 10
5Pa autoclaving 20min.
(2) basic medium
Tryptone 44g
Yeast?Extract 22g
NaCl 44g
Add deionized water and be settled to 3800ml, with fermentor tank at 121 ℃, 1.034 * 10
5Pa autoclaving 20min.
(3) phosphate buffered saline buffer
K
2HPO
4·
3H
2O 28.5g
KH
2PO
4 17g
Add deionized water and be settled to 120ml, at 121 ℃, 1.034 * 10
5Pa autoclaving 20min.
(4) supplemented medium I
Glucose 40g
Add deionized water and be settled to 80ml
MgSO
4·7H
2O 4g
Add deionized water and be settled to 20ml
At 121 ℃, 1.034 * 10
5Pa autoclaving 20min.
(5) supplemented medium II
Tryptone 20g
Add deionized water and be settled to 100ml
Yeast?Extract 40g
Add deionized water and be settled to 200ml
At 121 ℃, 1.034 * 10
5Pa autoclaving 20min.
Vitamin solution 10ml
Trace metal solution 10ml
(6) supplemented medium III
Tryptone 30g
Add deionized water and be settled to 150ml
Yeast?Extract 10g
Add deionized water and be settled to 50ml
Glucose 60g
Add deionized water and be settled to 120ml at 121 ℃, 1.034 * 10
5Pa autoclaving 20min.
2. the activation of bacterial classification:
Get-70 ℃, the bacterial classification 100ul of 20% glycerine preservation, be inoculated in the test tube that contains the 4mlLB substratum, adding penbritin, to make its final concentration be 100ug/ml; 32 ℃, 220r/min are cultivated about 10h, again by 2% inoculative proportion transferred species in the triangular flask that contains 50mlLB, adding penbritin, to make its final concentration be 100ug/ml; 32 ℃, 220r/min are cultivated 4h, become activated seed liquid, 4 ℃ of preservations.
3. fermentation seed liquid is cultivated:
Get the activatory seed liquor, be inoculated in the 1000ml triangular flask that contains 500ml LB substratum with 1: 1000 inoculative proportion, it is 100ug/ml that the adding penbritin makes its final concentration.32 ℃, 220r/min are cultivated 10h, OD
600Be about 2.5.
4. fermentor cultivation:
Use the BioFloIII type 7L of New Brunswick Scientific company to control fermentor tank automatically, fermentation parameter is as follows:
time temp stir air pH PO
2 OD
600
00:00 30-32 ℃ of 250 0.4 7.3 100 inoculations
01:00 30-32℃ 250 0.4 7.3 96.3 0.32
02:00 30-32℃ 250 1.0 7.3 82.0 0.54
03:00 30-32℃ 250 1.0 7.3 60.5 0.96
04:00 30-32℃ 300 1.4 7.3 55.2 1.54
05:00 30-32℃ 350 2.0 7.3 65.4 2.52
06:00 30-32℃ 350 2.4 7.3 50.8 5.35
07:00 30-32℃ 400 2.4 7.3 45.0 8.20
08:00 30-32℃ 400 2.4 7.3 28.3 10.53
09:00 40-42℃ 450 3.0 6.7 52.1 12.16
10:00 40-42℃ 450 3.0 6.7 36.7 18.56
11:00 40-42℃ 450 3.0 6.7 30.2 21.12
12:00 40-42℃ 450 3.0 6.7 42.5 24.32
Annotate: regulate pH with HCl, NaOH, add phosphate buffered saline buffer during inoculation, 01:00 adds feed supplement I with pump; 04:00 adds feed supplement II with pump; 07:00 adds feed supplement III with pump.
5. centrifugal receipts bacterium gets wet thallus 230g.
6. bacterial cell disruption and gel electrophoresis (the SDS-PAGE electrophoresis result is seen Fig. 4).
Two, protein separation, purifying
1. the washing of inclusion body
(1) with 20mM Tris.cl pH8.0, the 1mMEDTA washing, 9000rpm * 30min abandons supernatant.
(2) use 0.2%Triton X-100,20mM Tris.cl pH8.0,1mM EDTA washing, 9000rpm * 30min abandons supernatant.
Through (1), (2) washing, inclusion body purity has reached about 60%.
2. sex change, renaturation, ultrafiltration
(1) will wash good inclusion body and be dissolved in the 7M Guanidinium hydrochloride in weight in wet base by 1: 20 (v/w), 50mMTris.cl pH8.0,10mM beta-mercaptoethanol, in the 10mM edta buffer liquid, stir 4h under the room temperature, centrifugal 9000rpm * 30min gets transparent supernatant liquor, i.e. sex change liquid.
(2) adopt the method for dilution refolding to carry out renaturation sex change liquid, renaturation buffer is 2.5MVrea, 50mMtris.cl, 1mMETDA.Slowly drip sex change liquid during renaturation, making the renaturation solution final concentration of protein is 0.1-0.2mg/ml, renaturation time 36h.
(3) protein renaturation liquid is filtered earlier, ultrafiltration again (millipore) obtains ultrafiltration and concentration liquid.
3. column chromatography purification
(1) adopts Q-sepharose FF anion-exchange column
With 20mM Tris.cl pH8.0 balance Q post, go up sample more earlier, at first use 20mMTris.cl pH8.0 wash-out after last sample is intact, use 100mM NaCl stepwise elution again, use the 400mMNaCl stepwise elution at last, 280nm detects the eluted protein peak, through the SDS-PAGE electrophoresis detection, purity is about 85%.
(2) adopt Interferon, rabbit monoclonal antibody chromatography column
Earlier, go up sample again with PBS balance anti-interferon monoclonal antibody chromatography column, after the PBS wash-out is collected protein peak, through SDS-PAGE electrophoresis detection target protein purity greater than 99% (the target protein SDS-PAGE electrophoresis of purifying is seen Fig. 5).
RhIFN α 8cDNA aminopeptidase gene acid sequence
ATG?TGT?GAT?CTT?CCT?CAG?ACT?CAT?TCT?CTT?GGT?AAT?CGT?CGT?GCT 45
Met?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Asn?Arg?Arg?Ala
CTT?ATT?CTT?CTT?GCT?CAG?ATG?CGT?CGT?ATT?TCT?CCT?TTT?TCT?TGT 90
Leu?Ile?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Pro?Phe?Ser?Cys
CTT?AAG?GAT?CGT?CAT?GAT?TTT?GAG?TTT?CCT?CAG?GAG?GAG?TTT?GAT 135
Leu?Lys?Asp?Arg?His?Asp?Phe?Glu?Phe?Pro?Gln?Glu?Glu?Phe?Asp
GAT?AAG?CAG?TTT?CAG?AAG?GCT?GAG?GCT?ATT?TCT?GTT?CTT?CAT?GAG 180
Asp?Lys?Gln?Phe?Gln?Lys?Ala?Gln?Ala?Ile?Ser?Val?Leu?His?Glu
ATG?ATT?GAG?CAG?ACT?TTT?AAT?CTT?TTT?TCT?ACT?AAG?GAT?TCT?TCT 225
Met?Ile?Gln?Gln?Thr?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser
GCT?GCT?CTT?GAT?GAG?ACT?CTT?CTT?GAT?GAG?TTT?TAT?ATT?GAG?CTT 270
Ala?Ala?Leu?Asp?Glu?Thr?Leu?Leu?Asp?Glu?Phe?Tyr?Ile?Glu?Leu
GAT?CAG?CAG?CTT?AAT?GAT?CTT?GAG?TCT?TGT?GTT?ATG?CAG?GAG?GTT 315
Asp?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ser?Cys?Val?Met?Gln?Glu?Val
GGT?GTT?ATT?GAG?TCT?CCT?CTT?ATG?TAT?GAG?GAT?TCT?ATT?CTT?GCT 360
Gly?Val?Ile?Glu?Ser?Pro?Leu?Met?Tyr?Glu?Asp?Ser?Ile?Leu?Ala
GTT?CGT?AAG?TAT?TTT?CAG?CGT?ATT?ACT?CTT?TAT?CTT?ACT?GAG?AAG 405
Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Thr?Glu?Lys
AAG?TAT?TCT?TCT?TGT?GCT?TGG?GAG?GTT?GTT?CGT?GCT?GAG?ATT?ATG 450
Lys?Tyr?Ser?Ser?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met
CGT?TCT?TTT?TCT?CTT?TCT?ATT?AAT?CTT?CAG?AAG?CGT?CTT?AAG?TCT 495
Arg?Ser?Phe?Ser?Leu?Ser?Ile?Asn?Leu?Gln?Lys?Arg?Leu?Lys?Ser
AAG?GAG?TGA 504
Lys Glu Trm sequence 3 recombinant expression vector pBV220/IFN α 8 ribosome bind site sequence chart
GAA?TTC?aac?agg?aga?ctt?tct?g?atg
Claims (12)
1, recombinant human interferon-alpha-8 is characterized in that having following gene order:
ATG?TGT?GAT?CTT?CCT?CAG?ACT?CAT?TCT?CTT?GGT?AAT?CGT?CGT?GCT 45
Met?Cys?Asp?Leu?Pro?Gln?Thr?His?Ser?Leu?Gly?Asn?Arg?Arg?Ala
CTT?ATT?CTT?CTT?GCT?CAG?ATG?CGT?CGT?ATT?TCT?CCT?TTT?TCT?TGT 90
Leu?Ile?Leu?Leu?Ala?Gln?Met?Arg?Arg?Ile?Ser?Pro?Phe?Ser?Cys
CTT?AAG?GAT?CGT?CAT?GAT?TTT?GAG?TTT?CCT?CAG?GAG?GAG?TTT?GAT 135
Leu?Lys?Asp?Arg?His?Asp?Phe?Glu?Phe?Pro?Gln?Glu?Glu?Phe?Asp
GAT?AAG?CAG?TTT?CAG?AAG?GCT?CAG?GCT?ATT?TCT?GTT?CTT?CAT?GAG 180
Asp?Lys?Gln?Phe?Gln?Lys?Ala?Gln?Ala?Ile?Ser?Val?Leu?His?Glu
ATG?ATT?CAG?CAG?ACT?TTT?AAT?CTT?TTT?TCT?ACT?AAG?GAT?TCT?TCT 225
Met?Ile?Gln?Gln?Thr?Phe?Asn?Leu?Phe?Ser?Thr?Lys?Asp?Ser?Ser
GCT?GCT?CTT?GAT?GAG?ACT?CTT?CTT?GAT?GAG?TTT?TAT?ATT?GAG?CTT 270
Ala?Ala?Leu?Asp?Glu?Thr?Leu?Leu?Asp?Glu?Phe?Tyr?Ile?Glu?Leu
GAT?CAG?CAG?CTT?AAT?GAT?CTT?GAG?TCT?TGT?GTT?ATG?CAG?GAG?GTT 315
Asp?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ser?Cys?Val?Met?Gln?Glu?Val
GGT?GTT?ATT?GAG?TCT?CCT?CTT?ATG?TAT?GAG?GAT?TCT?ATT?CTT?GCT 360
Gly?Val?Ile?Glu?Ser?Pro?Leu?Met?Tyr?Glu?Asp?Ser?Ile?Leu?Ala
GTT?CGT?AAG?TAT?TTT?CAG?CGT?ATT?ACT?CTT?TAT?CTT?ACT?GAG?AAG 405
Val?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?Tyr?Leu?Thr?Glu?Lys
AAG?TAT?TCT?TCT?TGT?GCT?TGG?GAG?GTT?GTT?CGT?GCT?GAG?ATT?ATG 450
Lys?Tyr?Ser?Ser?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met
CGT?TCT?TTT?TCT?CTT?TCT?ATT?AAT?CTT?CAG?AAG?CGT?CTT?AAG?TCT 495
Arg?Ser?Phe?Ser?Leu?Ser?Ile?Asn?Leu?Gln?Lys?Arg?Leu?Lys?Ser
AAG?GAG?TGA 504
Lys?Glu?Trm
2, recombinant human interferon-alpha according to claim 1-8 is characterized in that described rhIFN α 8 polypeptide molecular weights size is 22kda, and the pI value is 4.9.
3, the expression method of recombinant human interferon-alpha according to claim 1-8 in intestinal bacteria, it is characterized in that before IFN α 8 cDNA coding region initiator codon atg, increasing by one section ribosome bind site sequence, 5 ' end in IFN α 8 cDNA sequences is introduced the EcoRI restriction enzyme site, increase the SalI restriction enzyme site behind 3 ' the end terminator codon TAA, make up coli expression carrier pBV220/IFN α 8, through the engineering bacteria screening, obtain target protein after albumen sepn, purifying, the freeze-drying then.
4, the expression method of recombinant human interferon-alpha according to claim 3-8 in intestinal bacteria is characterized in that the wherein said one section ribosome bind site that increases before IFN α 8 cDNA coding region initiator codon atg, its sequence is:
GAA?TTC?aac?agg?aga?ctt?tct?g?atg
5, the expression method of recombinant human interferon-alpha according to claim 3-8 in intestinal bacteria is characterized in that described coli expression carrier is pBV220.
6, the expression method of recombinant human interferon-alpha according to claim 3-8 in intestinal bacteria is characterized in that the structure of wherein said intestinal bacteria recombinant expression vector pBV220/IFN α 8 comprises:
1) subclone of IFN α 8 genes and with being connected of carrier
IFN α 8 cDNA sequences and ribosome bind site sequence subclone on the pUC18 carrier, are become pUC18/IFN α 8; With EcoRI/SalI double digestion digestion pUC18/IFN α 8 and coli expression carrier pBV220, reclaim IFN α 8 and carrier segments, purified after, adopt dna ligation kit to connect, then transformed competence colibacillus intestinal bacteria DH
5 α, the dull and stereotyped incubated overnight of LB;
2) engineering bacteria screening
Positive bacteria on the picking LB flat board is dropped into the evaluation of cutting and check order of capable enzyme; Identify that the positive colony after confirming is inoculated in the 5ml liquid LB nutrient solution, 30 ℃, 250rpm/min cultivated 12-18 hour; Nutrient solution is changed in the 50ml liquid LB nutrient solution, continue to be cultured to the OD value for 0.4-0.6, improve temperature to 42 suddenly and ℃ induced 4-5 hour; Centrifugal collection thalline carries out the SDS-PAGE electrophoretic analysis, promptly.
7, the expression method of recombinant human interferon-alpha according to claim 3-8 in intestinal bacteria is characterized in that separation, the purifying of wherein said recombinant protein IFN α 8 comprises:
1) washing of inclusion body
(1) with 20mM Tris.cl pH8.0,1mMEDTA washs broken thalline, and 9000rpm * 30min abandons supernatant;
(2) use 0.2%Triton X-100,20mM Tris.cl pH8.0,1mM EDTA washing, 9000rpm * 30min abandons supernatant;
2) sex change, renaturation, ultrafiltration
(1) will wash good inclusion body and be dissolved in the 7M Guanidinium hydrochloride in weight in wet base by 1: 20 (v/w), 50mMTris.cl pH8.0,10mM beta-mercaptoethanol, in the 10mM edta buffer liquid, stir 4h under the room temperature, centrifugal 9000rpm * 30min gets transparent supernatant liquor, i.e. sex change liquid;
(2) adopt the method for dilution refolding to carry out renaturation sex change liquid, renaturation buffer is 2.5MVrea, 50mMtris.cl, 1mMETDA.Slowly drip sex change liquid during renaturation, making the renaturation solution final concentration of protein is 0.1-0.2mg/ml, renaturation time 36h;
(3) protein renaturation liquid is filtered earlier, ultrafiltration again (millipore) obtains ultrafiltration and concentration liquid;
3) column chromatography purification
(1) adopts Q-sepharose FF anion-exchange column
With 20mM Tris.cl pH8.0 balance Q post, go up sample more earlier, at first use 20mMTris.cl pH8.0 wash-out after last sample is intact, use 100mM NaCl stepwise elution again, use the 400mMNaCl stepwise elution at last, 280nm detects the eluted protein peak;
(2) adopt Interferon, rabbit monoclonal antibody chromatography column
With PBS balance anti-interferon monoclonal antibody chromatography column, go up sample again, earlier after the PBS wash-out is collected protein peak.
8, the expression method of recombinant human interferon-alpha according to claim 3-8 in intestinal bacteria is characterized in that the recombinant human interferon-alpha-8 that obtains with this method, through SDS-PAGE electrophoresis detection purity of protein greater than 99%.
9, the application of recombinant human interferon-alpha according to claim 1-8 in preparation treatment human or animal's tumour and disease of viral infection medicine.
10, the application of recombinant human interferon-alpha according to claim 9-8 is characterized in that rhIFN α 8 can be used as the active ingredient of said medicine, prepares various liquid preparations of acceptable or freeze-dried preparation on the various physiology.
11, the application of recombinant human interferon-alpha according to claim 9-8 is characterized in that described tumour, comprises various malignant tumours, as solid carcinoma and serous tumor etc.
12, the application of recombinant human interferon-alpha according to claim 9-8 is characterized in that described disease of viral infection comprises the disease that infection such as HBV, HCV, HIV, HSV, HPV or influenza virus cause.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1746306B (en) * | 2005-08-02 | 2010-08-25 | 成都地奥制药集团有限公司 | Recombinant human interferon alpha 4 coded cDNA sequence, production and use thereof |
| CN101191130B (en) * | 2007-07-12 | 2010-12-22 | 中国农业大学 | ChIFNGR1 gene and its coding protein and application |
| CN108822202A (en) * | 2018-02-07 | 2018-11-16 | 电子科技大学 | A kind of 21 recombinant protein of leucocytes of grass carp interleukin and preparation method thereof |
-
2003
- 2003-03-28 CN CN 03116053 patent/CN1235909C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1746306B (en) * | 2005-08-02 | 2010-08-25 | 成都地奥制药集团有限公司 | Recombinant human interferon alpha 4 coded cDNA sequence, production and use thereof |
| CN101191130B (en) * | 2007-07-12 | 2010-12-22 | 中国农业大学 | ChIFNGR1 gene and its coding protein and application |
| CN108822202A (en) * | 2018-02-07 | 2018-11-16 | 电子科技大学 | A kind of 21 recombinant protein of leucocytes of grass carp interleukin and preparation method thereof |
| CN108822202B (en) * | 2018-02-07 | 2021-11-30 | 电子科技大学 | Grass carp interleukin 21 recombinant protein and preparation method thereof |
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