CN110079514A - A method of preparing protease 3 recombinant protein - Google Patents
A method of preparing protease 3 recombinant protein Download PDFInfo
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- CN110079514A CN110079514A CN201910294751.1A CN201910294751A CN110079514A CN 110079514 A CN110079514 A CN 110079514A CN 201910294751 A CN201910294751 A CN 201910294751A CN 110079514 A CN110079514 A CN 110079514A
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Abstract
The present invention relates to genetic engineerings and field of biotechnology, and in particular to a method of prepare protease 3 recombinant protein.The present invention realizes extensive, low cost using five instar larvaes of silkworm as bioreactor, by silkworm-baculovirus expression system, expeditiously prepares the immunocompetent recombined human PR3 of tool；By means of the present invention, recombinate shape virus infection five ages silkworm, after infection, PR3 fusion protein is expressed and is released in hemolymph in silkworm, and after isolating and purifying, every silkworm can prepare up to 2 micrograms or so destination protein enzyme 3, with very high expression quantity, production cost is effectively reduced, is convenient for large-scale industrial production, there is good economic benefit.
Description
Technical field
The present invention relates to genetic engineerings and field of biotechnology, and in particular to a kind of side for preparing protease 3 recombinant protein
Method.
Background technique
Protease 3 (Proteinase 3, PR3) is one of neutrophil leucocyte endochylema azurophilic granule serine stretch protein
Enzyme, the glycoprotein that molecular weight is about 29KDa.It is anti-neutrophil leucocyte and monocyte endochylema antibody (Anti-Neutrophil
Cytoplasmic Antibodies, ANCA) main target antigen, account for about 80% -90%.By ANCA, it is immunized using indirect
Two kinds of fluorescence modes (endochylema type cANCA and core week type pANCA) of fluorescence method, to self immune system vasculitis and inflammatory
Etc. diseases carry out diagnosis have become set tool.For example, diagnosing Wei lattice by PR3-cANCA since cANCA mainly identifies PR3
The specificity of Na Shi granuloma (WG) is greater than 95%, and usually it is also used for the detections of other a variety of primary angiitis.PR3-
Clinically another significant application value is that the antibody titer is consistent with state of an illness activity to cANCA, in primary angiitis such as WG
Patient is often by as judgment criteria.
For the physiopathologic research of deep progress PR3-cANCA and clinical diagnostic applications, obtain largely with immune anti-
Active PR3 albumen is answered to become the target that the whole world is made joint efforts as cANCA target antigen.Clinically it is used for self immune system
The PR3 of related disease in-vitro diagnosis take naive albumen (such as neutrocyte source) as best, but the separation of native protein
Purification difficult, yield is low, and purifying products obtained therefrom purity is lower, carries a certain amount of foreign protein secretly；The source of product is different, batch it
Between it is inconsistent and expensive, seriously constrain the development of self immune system related disease vitro diagnostic techniques.
Summary of the invention
In view of this, the main purpose of the present invention is to provide a kind of method of external preparation protease 3 recombinant protein, root
According to the embodiment of the present invention, can realize in expression system in vitro efficiently, cheap, large-scale preparation there is high immune response
Active protease 3 recombinant protein.
For achieving the above object, the invention provides the following technical scheme:
A method of protease 3 recombinant protein is prepared, the described method comprises the following steps:
(1) the genetic fragment PR3-His of human protease 3 is cloned into the transferring plasmid containing insect secretory signal peptide Mel
On pMelBacA, synthesis has the recombinant plasmid pMelBacA-PR3-His of insect secretory signal peptide Mel and people's PR3 sequence；.
(2) contain insect secretory signal by template building of the recombinant plasmid pMelBacA-PR3-His of step (1) synthesis
The recombination donor vehicle pFastBac1-Mel-PR3-His of peptide Mel:
(3) the recombination donor vehicle pFastBac1-Mel-PR3-His of step (2) building and baculoviral are transfected into silkworm jointly
Gonad cell BmN, preparation and reorganization virus rBm-pFastBac1-Mel-PR3-His；
(4) the recombinant virus rBm-pFastBac1-Mel-PR3-His inoculation transfection silkworm that will be prepared in step (3), in silkworm
Middle expression protease 3 recombinant protein；
(5) hemolymph of silkworm after recycling transfects；
(6) the protease 3 recombinant protein in hemolymph is isolated and purified.
Preferably, present invention recombinant plasmid as described in step (1) pMelBacA-PR3-His's the preparation method comprises the following steps: to people
PR3 cDNA is that template carries out first round pcr amplification reaction, the PCR amplification primer such as SEQ ID NO.1-2 institute in sequence table
Show, amplifies PR3-His segment, it is double to being carried out to PR3-His segment, transferring plasmid pMelBacA respectively with Xho I and Kpn I
Endonuclease reaction；Two kinds of double enzyme digestion products are purified, are connected, are obtained with insect secretory signal peptide Mel and people's PR3 sequence
Recombinant plasmid pMelBacA-PR3-His；
Preferably, the preparation of donor vehicle pFastBac1-Mel-PR3-His is recombinated described in step (2) of the present invention are as follows: with
The recombinant plasmid pMelBacA-PR3-His prepared in step (1) is that template progress the second wheel pcr amplification reaction obtains Mel-
PR3-His target fragment；Double enzymes are carried out to Mel-PR3-His target fragment, donor plasmid pFastBac1 with Xba I and Kpn I
Reaction is cut, two kinds of double enzyme digestion products are purified, are connected, it is thin that the product after connection is transformed into bacillus coli DH 5 alpha competence
In born of the same parents, Plasmid DNA is extracted after culture；Double digestion identification is carried out with Plasmid DNA of Xba I and the Kpn I to extracting, screening is contained
There is insect secretory signal peptide Mel to recombinate donor vehicle pFastBac1-Mel-PR3-His.
Preferably, the preparation method of recombinant virus rBm-pFastBac1-Mel-PR3-His described in step (3) of the present invention
For the recombination donor vehicle pFastBac1-Mel-PR3-His transfection Escherichia coli for constructing step (2), recombinate in donor vehicle
Swivel base occurs for the Tn7 transposable element and silkworm BmNPV baculovirus shuttle vector Bacmid contained, and the recombination of swivel base occurs for extracting
Shuttle vector Bacmid DNA simultaneously transfects silkworm ovary cells BmN, prepares recombinant virus rBm-pFastBac1-Mel-PR3-
His。
Preferably, the transfection of inoculation described in step (4) of the present invention is by recombinant virus rBm-pFastBac1-Mel-
PR3-His is subcutaneously injected to Silkworm, Bombyx mori.
Preferably, silkworm described in step (4) of the present invention is five age silkworms.
Preferably, hemolymph described in step (5) of the present invention is the 4-5 days hemolymphs generated after silkworm transfection.
Preferably, it is isolated and purified described in step (6) of the present invention to first pass through the affine layer of nickel column after diluting hemolymph
Analysis purifying, purifying successively obtain the purpose product protease 3 weight of high-purity after purification through ion exchange column Mono Q and Mono S
Histone；The buffer dilution hemolymph being diluted to two volumes.
Another aspect of the present invention is the protease 3 recombinant protein produced using the method for the present invention.
The beneficial effects of the present invention are:
The present invention using silkworm larva as bioreactor, by silkworm-baculovirus expression system realize it is extensive, low at
Originally, the immunocompetent recombined human PR3 of tool is expeditiously prepared.As the representative of eukaryotic expression system, silkworm-baculoviral table
Have many advantages, such as high expression level and powerful protein post-translational modification function up to system.Pass through silkworm-rhabdovirus system
The human recombination protein of expression has very high bioactivity, and antigenicity, immunogenicity are similar to the native protein of people, and silkworm
Raise it is simple and convenient, it is at low cost.
By means of the present invention, recombinate shape virus infection five ages silkworm, after infection 4-5 days, PR3 fusion protein exists
It expresses and is released in hemolymph in silkworm, after isolating and purifying, every silkworm can prepare up to 2 micrograms or so destination protein
Enzyme 3 has very high expression quantity；Meanwhile being verified by embodiment, the protease 3 and people PR3 protein 10 0% of this method preparation are same
Source, it is enough to be identified by PR3-ANCA positive serum.Silkworm can be commercialized sterile raising on a large scale, and silkworm is as biological respinse
Device prepare PR3 avoid culture medium expensive needed for cell culture in AcMNPV shape virus-insect cell expressioning system and
Production cost is effectively reduced in fetal calf serum, is convenient for large-scale industrial production, has good economic benefit.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis for the genetic fragment PR3-His that first round pcr amplification reaction obtains in embodiment 1
Figure；M is DNA molecular amount standard；Number 1 represents genetic fragment PR3-His；
Fig. 2 is the knot that agarose gel electrophoresis is carried out to transferring plasmid pMelBacA and the PR3-His segment after double enzyme digestion reaction
Fruit figure；M is DNA molecular amount standard；Number C represents the recombination transfer of the MPO of myeloperoxidase containing people prepared in the same way
The gel electrophoresis figure of carrier double enzyme digestion reaction；Number 1 represents transferring plasmid pMelBacA and PR3-His segment；
Fig. 3 is the agarose gel electrophoresis figure of genetic fragment Mel-PR3-His in embodiment 1；M is DNA molecular amount standard；Number
1 represents genetic fragment Mel-PR3-His；
Fig. 4 is the recombination donor vehicle pFastBac1-Mel-PR3- after 2 double enzyme digestion reactions selected at random in embodiment 1
The agarose gel electrophoresis figure of His；M is DNA molecular amount standard；Number 1 ~ 2 represents genetic fragment carrier pFastBac1-Mel-
PR3-His；
Fig. 5 is the PCR amplification result figure of recombinant shuttle vector Bacmid DNA in embodiment 2；M is DNA molecular amount standard；Number
1 ~ 2 represents the hickie recombinant shuttle vector Bacmid DNA that swivel base has occurred, and number 3 represents locus coeruleus non-recombinant Bacmid DNA；
Fig. 6 is the expression analysis figure of recombined human PR3 different time in hemolymph after silkworm virus inoculation in embodiment 3；
Fig. 7 is the PAGE gel electrophoretic analysis figure of affinity chromatography Purification of Human PR3 albumen in embodiment 4；M is standard egg
White Marker, unit kDa；Number Bc is Haemolymph Supernatant；Number Ft is that the foreign protein not being combined after nickel column is added
Fraction；Number W1 ~ W4 is to wash down the fraction containing foreign protein；Number E1 ~ E9 is the fraction eluted；
Fig. 8 is the PAGE gel electrophoretic analysis figure of Mono Q ion exchange column purification people's PR3 albumen in embodiment 4, and Bc is
Nickel-affinity column purifies latter incorporated fraction, and 8-27 is the fraction that gradient elution gets off；M is standard protein Marker；
Fig. 9 is the PAGE gel electrophoretic analysis figure of Mono S ion exchange column purification people's PR3 albumen in embodiment 4, and Bc is
Sample of latter incorporated 10th fraction of Mono Q column purification after dialysing before pre- upper Mono S column；W is the foreign protein fraction of cleaning；
7-20 is the fraction that gradient elution gets off；M is standard protein Marker；
Figure 10 is the Western Blot qualification figure of destination protein PR3 in embodiment 4；Wherein, primary antibody is anti-His- mark in left figure
The mouse monoclonal antibody of label, primary antibody is anti-human PR3 mouse monoclonal antibody in right figure, and swimming lane 1 indicates recombination after purification in figure
PR3 protein immunization response sample, swimming lane M indicate standard protein Marker；
Figure 11 is the MALDI-TOF identification sequential covering result figure of PR3-His albumen in embodiment 5.
Specific embodiment
With reference to the accompanying drawing and specific embodiment the present invention is further described, but protection scope of the present invention and unlimited
In this.With reference to the accompanying drawing and specific embodiment the present invention is further described, but protection scope of the present invention is not limited to
This.Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can be bought by commercial channel
It obtains.
Key instrument and material used in following embodiment:
The conventional reagents such as PCR related reagent, endonuclease reaction, connection kit are purchased from PROMEGA company；LB culture medium, insect
The chemical reagent such as cell culture related reagent, CELLFECTIN Reagent are GIBCO BRL Products；Plastic recovery kit
Purchased from QIAGEN company；PCR product QIAquick Gel Extraction Kit is purchased from OMEGA company；The cDNA of source of people protease 3 (PR3) is from
Disclosed DNA sequence dna (GenBank:BC096183.1) is purchased from PROTEINTECH GROUP company；Believe containing insect secretory
Baculovirus transfer plasmids pMelBacA, silkworm ovary cells BmN, donor plasmid pFastBac1 and the large intestine bar of number peptide Mel
Bacterium BmDH10Bac is INVITROGEN Products；Bacillus coli DH 5 alpha is Takara Products；Silkworm is kind 306, is come
Derived from sericulture research institute, the Chinese Academy of Agricultural Sciences；His label protein purifies dedicated buffering liquid and is purchased from Pierce company；Ion exchange column
Mono Q and Mono S are purchased from GE HEALTHCARE company；The mouse monoclonal antibody (27E8) of anti-His- label is purchased from Cell
Signaling Technology company；Anti-human PR3 mouse monoclonal antibody (D-1) is SANTACRUZ BIOTECHNOLOGY public
Take charge of product；People's PR3 protein ELISA detection kit is Ka Maishu (Shanghai) Biotechnology Co., Ltd product (Cat No.
KMS-EL9531c).
Do not make the Examination on experimental operation illustrated in following embodiment referring to " Molecular Cloning:A Laboratory guide "
(Sambrook etc. writes, Science Press, version in 1992) and product description carry out.
Embodiment one: the building containing insect secretory signal peptide Mel recombination donor vehicle pFastBac1-Mel-PR3-His
Using the cDNA of people PR3 (GenBankNo:X55668.1) as stencil design PCR amplification primer, in people's PR3 gene order
The end N- and the end C- introduce Xho I and Kpn I restriction enzyme site respectively, so that the segment is connected to equally after double digestion
In plasmid vector；Its end C- introduces the base of 6 histidines of coding, and in favor of the protein purification of expression: primer is as follows:
Forward primer -1(SEQ ID NO.1): AGGAGACTCGAGATCGTGGGCGGGCACGAGGCG；Reverse primer -1(SEQ
ID NO.2): TACGGTACCCTAGTGATGGTGATGGTGATGACGGCGCAGCGTGGAACG.
Using the above primer, first round pcr amplification reaction, reaction system and condition are carried out by template of the cDNA of people PR3
It is as shown in table 1:
1. pcr amplification reaction system of table and condition item
| Component | Volume/μ L(50 μ L) |
| 5 × Pfu buffer | 10 |
| 2.5mM dNTPs | 3 |
| Pfu enzyme | 0.5 |
| Forward primer (primer F) | 1 |
| Reverse primer (primer R) | 1 |
| cDNA | 0.3 |
| ddH2O | 34.2 |
PCR reaction condition: 94 DEG C of 4 min of initial denaturation, 94 DEG C of 30 s of denaturation, 55 DEG C of 30 s of annealing, 72 DEG C extend 1.5
Min re-extends 10 min for 72 DEG C after recycling 30 times.By the PR3 gene piece of the label containing His- of acquisition after first round PCR amplification
Section PR3-His carries out 1% agarose gel electrophoresis.
Fig. 1 is the agarose gel electrophoresis figure of genetic fragment PR3-His, as seen from Figure 1, obtains PR3- in 708 bp or so
The target fragment of His.The gel of the segment containing PR3-His is cut down with scalpel, is amplified with plastic recovery kit recycling
PR3-His segment carries out double enzyme digestion reaction with PR3-His segment of Xho I and the Kpn I to recycling；Meanwhile with Xho I and Kpn
I is shown in sequence table SEQ ID NO.3:ATGAA to containing insect secretory signal peptide Mel(insect secretory signal peptide Mel gene order
ATTCTTAGTCAACGTTGCCCTTGTTTTTATGGTCGTATACATTTCTTACATCTATG CG) transferring plasmid pMelBacA
Double enzyme digestion reaction is carried out, then is purified respectively to above two double enzyme digestion reaction product with PCR product purification kit, it will be pure
PCR product after change is connected on transferring plasmid by connecting kit, is obtained and is had insect secretory signal peptide Mel and people PR3
The recombinant plasmid pMelBacA-PR3-His of sequence.
Fig. 2 is to carry out agarose gel electrophoresis to transferring plasmid pMelBacA and the PR3-His segment after double enzyme digestion reaction
Result figure；M is DNA molecular amount standard；Number C represents the recombination of the MPO of myeloperoxidase containing people prepared in the same way
The gel electrophoresis figure of transfer vector double enzyme digestion reaction；Number 1 represents transferring plasmid pMelBacA and the PR3-His piece after double digestion
Section obtains the target fragment of PR3-His in 708 bp or so, the transferring plasmid after 2277 bp or so obtain digestion
The segment of pMelBacA.It is identified through double digestion, successfully PR3-His segment is cloned into and is turned containing insect secretory signal peptide Mel
It moves on plasmid pMelBacA.
Design forward primer -2(SEQ ID NO.4): 5 '-CCGTCTAGAATGAAATTCTTAGTCAAC-3 '；Using just
To primer -2 and reverse primer 1, second is carried out as template using the recombinant plasmid pMelBacA-PR3-His prepared in above-mentioned steps
Pcr amplification reaction is taken turns, reaction system and condition are the same as first round PCR amplification.
Fig. 3 is the agarose gel electrophoresis figure of genetic fragment Mel-PR3-His, as it can be seen that obtaining in 792 bp or so in figure
The target fragment of Mel-PR3-His.Contain target fragment Mel-PR3- under cutting from gel with PCR QIAquick Gel Extraction Kit
The amplified production of His is recycled；Double enzyme digestion reaction is carried out to recovery product with Xba I and Kpn I, meanwhile, with Xba I and
Kpn I also carries out double enzyme digestion reaction to donor plasmid pFastBac1, then double to two kinds respectively by PCR product purification kit
Endonuclease reaction product is purified；After PCR product Mel-PR3-His after purification is connected to digestion by connection kit
On pFastBac1, the product after connection is then transformed into bacillus coli DH 5 alpha competent cell, and coated plate is in containing 100 μ g/
The LB culture medium flat plate of mL ampicillin；The single bacterium colonies for selecting 37 DEG C of inversion overnight incubations, through LB liquid medium culture,
Extract Plasmid DNA；Double digestion identification is carried out with Plasmid DNA of Xba I and the Kpn I to extracting, filters out and has been inserted into Mel-PR3-
The recombination donor plasmid of His genetic fragment, is detected by DNA sequence dna, and comparison result is obtained containing insect secretory signal peptide Mel weight
Group donor vehicle pFastBac1-Mel-PR3-His.
Fig. 4 is the solidifying of the recombination donor vehicle pFastBac1-Mel-PR3-His after 2 double enzyme digestion reactions selected at random
Gel electrophoresis figure is plasmid in 4776 bp or so as it can be seen that being the target fragment of Mel-PR3-His in 792bp or so in figure
The segment of pFastBac1.So far, it is identified by sequencing and double digestion, obtains and recombinate donor containing insect secretory signal peptide Mel
Carrier pFastBac1-Mel-PR3-His.
Embodiment two: the preparation of recombinant Bombyx mori baculovirus rBm-pFastBac1-Mel-PR3-His
The insect secretory signal peptide Mel that contains constructed in embodiment one is recombinated into donor vehicle pFastBac1-Mel-PR3-
The Tn7 transposable element contained in His transfection Escherichia coli BmDH10Bac, carrier pFastBac1-Mel-PR3-His and silkworm
Swivel base occurs for BmNPV baculovirus shuttle vector Bacmid, reflects after the screening of blue hickie to the recombinant shuttle vector of acquisition
It is fixed.2 hickie recombinant shuttle vector Bacmid DNA are selected at random, and using locus coeruleus non-recombinant Bacmid DNA as control, are answered
With the general pUC/M13 forward primer (SEQ ID NO.5) of specificity: 5 '-CCCAGTCACGACGTTGTAAAACG-3 ' and reversely
Primer (SEQ ID NO.6): 5 '-AGCGGATAACAATTTCACACAGG-3 ' carry out recombinant shuttle vector Bacmid DNA
PCR reaction verifying.
Fig. 5 is recombination and the PCR amplification result figure of non-recombinant shuttle vector Bacmid DNA；M is DNA molecular amount standard；
Number 1 ~ 2 represent the PCR of hickie recombinant shuttle vector Bacmid DNA as a result, number 3 represent do not occur swivel base locus coeruleus it is non-heavy
The PCR result of group Bacmid DNA；As shown in figure 5, Tn7 transposon does not occur for control group locus coeruleus non-recombinant Bacmid DNA
PCR reaction product there is purpose band in 300 bp or so, and 2 hickie recombinant shuttle vector Bacmid selected at random
There is band in 3161 bp of desired location or so in DNA, shows that 2 selected hickies pass through Tn7 transposons and turned
Seat, Mel-PR3-His genetic fragment have successfully passed through homologous recombination and have been integrated on silkworm shuttle vector Bacmid DNA.
Recombinant shuttle vector Bacmid DNA is mediated to transfect silkworm ovary cells BmN, warp with CELLFECTIN Reagent
The amplification of two-wheeled virus obtains Mel containing insect secretory signal peptide and encoding human PR3 gene order and has the recombination of His- label
Silkworm baculovirus rBm-pFastBac1-Mel-PR3-His carries out virus titer measurement to it, and measurement result is up to 2 × 107
PFU/mL shows that the concentration of virus can be used for infecting five age silkworms.
Embodiment three: table of the recombinant Bombyx mori baculovirus rBm-pFastBac1-Mel-PR3-His in five age silkworms
Up to situation
By the inoculation of recombinant Bombyx mori baculovirus rBm-pFastBac1-Mel-PR3-His prepared by embodiment two transfection to five ages
5 μ L recombinant Bombyx mori baculovirus rBm-pFastBac1-Mel-PR3- are subcutaneously injected in second day silkworm after phase, every silkworm
His, it may be assumed that every boss silkworm inoculation 1.0 × 105PFU virus.Every the silkworm hemolymph of collection in 24 hours after virus inoculation, together
When using the silkworm of non-virus inoculation as negative control group, hemolymph is collected together with the silkworm after virus inoculation, using people PR3
Protein ELISA detection kit measures institute in hemolymph in the mixing hemolymph that different time collects 5 boss silkworms as sample
The enzyme linked immunoassay of the coated people PR3 antibody of institute is active on the albumen of PR3 containing people and microwell plate, recombinates after calculating virus inoculation
Expression quantity of the people PR3 in hemolymph.
Fig. 6 is the expression analysis figure of recombined human PR3 different time in hemolymph after silkworm virus inoculation；Abscissa table
Show the time that silkworm hemolymph is collected after virus infection, ordinate indicates that people PR3 and people PR3 protein ELISA detect in hemolymph
The concentration that standard items comparing calculation obtains；As it can be seen that being able to detect that within 48 hours the expression of people's PR3 albumen after virus inoculation in figure,
Peak is reached when to 96 and 120 hours, illustrate expressed recombined human PR3 via insect secretory signal peptide mediate and by successfully
It is discharged into hemolymph, and can be by the coated anti-human source PR3 antibody of institute on people's PR3 protein ELISA detection kit microwell plate
It is identified.
The recombined human PR3's that example IV: expressing in five age silkworms and is discharged into hemolymph isolates and purifies
According to expression of the recombined human PR3 albumen in embodiment three in five age silkworms it is found that expression quantity is the 4th after virus inoculation
With at the 5th day reach peak.Accordingly, 600 silkworms are inoculated with, the silkworm hemolymph for collecting inoculation 4-5 days freezes in -800C ice
Case is spare；Hemolymph sample collected by 1mL is taken, is diluted with the 50mM phosphate buffer of two volumes, takes blood after high speed centrifugation
Lymph supernatant, through nickel-column separating purification；Fig. 7 is nickel-column affinitive layer purification people's PR3 albumen PAGE gel electrophoresis
Analysis chart；M is standard protein Marker, unit kDa；Number Bc is Haemolymph Supernatant；Number Ft is not have after nickel column is added
There is the foreign protein fraction being combined；Number W1 ~ W4 is to wash down the fraction containing foreign protein；Number E1 ~ E9 is eluted
Fraction.As it can be seen that doubtful destination protein is eluted from the first fraction to the 4th fraction (E1-E4) in figure, but it is containing more multicomponent
Foreign protein；Merge E1-E4 fraction and carry out dialysis desalination, is then purified through ion exchange column Mono Q.Fig. 8 is that Mono Q ion is handed over
Change the PAGE gel electrophoretic analysis figure of column purification people's PR3 albumen, in figure, Bc is that nickel-column affinitive layer purification is latter incorporated
Fraction, i.e., the sample before Mono Q column is added in advance after dialysing；8-27 is the fraction that gradient elution gets off；M is standard protein
Marker；As shown in figure 8, purer destination protein is eluted in the 10th fraction, and contained foreign protein the 11st fraction start by
It separates；Merge multiple batches of the 10th fraction through Mono Q after purification, after desalination of being dialysed again by ion exchange column Mono S into
Row purifying.Fig. 9 is the PAGE gel electrophoretic analysis figure of Mono S ion exchange column purification people's PR3 albumen, such as Fig. 9 arrow institute
Show, the destination protein of high-purity is eluted in the 14th and 15 fractions, and molecular weight is about in 32 kDa, with insect cell Sf-9
Expressed 34kDa recombined human PR3 albumen it is in the same size.
Respectively using the mouse monoclonal antibody (27E8) of anti-His- label and anti-human PR3 mouse monoclonal antibody (D-1) as one
Anti-, the anti-mouse IgG alkaline phosphatase is marked carries out No. 15 fraction after Mono S column purification as secondary antibody
Western Blot identification, carries out chromogenic reaction by Alkaline Phosphatase Kit.Figure 10 is the Western of destination protein PR3
Blot qualification figure, wherein left figure is the immune response using the mouse monoclonal antibody of anti-His- label as primary antibody, with α-His
It indicates；Right figure is using immune response of the anti-human PR3 mouse monoclonal antibody as primary antibody, with α-PR3(Human) it indicates；In figure
Swimming lane 1 indicates that recombination PR3 protein immunization response sample after purification, swimming lane M indicate standard protein Marker；As shown in Figure 10,
The destination protein that prepared molecular weight is about 32 kDa not only identifies by the antibody of anti-His- label, while also can be by quotient
The antibody of the anti-human source PR3 of productization identified, the purified albumen of preliminary identification is recombined human PR3.The recombined human PR3 of acquisition is pure
Degree is about 32 kDa close to 100%, molecular weight, is learnt by measurement, and average every silkworm can get the weight of about 2 micrograms after purification
Group people PR3 destination protein.
Embodiment five: recombined human PR3 destination protein mass spectrum identification
First mass spectrometric (MS) is carried out to recombined human PR3 destination protein obtained in example IV and second order ms (MS/MS) are comprehensive
Analysis is closed, surveyed albumen is identified by ncbi database using 2.2 software of Mascot.Mass spectral analysis condition: instrument
Model: 5800 MALDI-TOF/TOF(AB SCIEX companies)；Detection mode: cation, reflective-mode；Sample target: 384
Opti-TOF 123 mm ╳81 mm ss AB SCIEX；Matrix: CHCA；Analyze the peptide that result is as shown in figure 11, and mass spectrum obtains
Section is to deepen font representation, the coverage rate that people's PR3 amino acid sequence in the peptide fragment and ncbi database of mass spectrum acquisition matches
It is 25.68%.Recombinant protein score 405 is learnt using people's PR3 albumen comparison in Mascot software and database, according to software
Algorithm and define the albumen and people PR3 protein 10 0% is homologous.Therefore it by mass spectral analysis, has further confirmed that after isolating and purifying
Albumen behaviour PR3 obtained.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Sequence table
<110>Jiangsu University
<120>a kind of method for preparing protease 3 recombinant protein
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<170> SIPOSequenceListing 1.0
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<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aggagactcg agatcgtggg cgggcacgag gcg 33
<210> 2
<211> 48
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<213>artificial sequence (Artificial Sequence)
<400> 2
tacggtaccc tagtgatggt gatggtgatg acggcgcagc gtggaacg 48
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<211> 63
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgaaattct tagtcaacgt tgcccttgtt tttatggtcg tatacatttc ttacatctat 60
gcg 63
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<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ccgtctagaa tgaaattctt agtcaac 27
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<212> DNA
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cccagtcacg acgttgtaaa acg 23
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agcggataac aatttcacac agg 23
Claims (10)
1. a kind of method for preparing protease 3 recombinant protein, which comprises the following steps:
(1) the genetic fragment PR3-His of human protease 3 is cloned into the transferring plasmid containing insect secretory signal peptide Mel
On pMelBacA, synthesis has the recombinant plasmid pMelBacA-PR3-His of insect secretory signal peptide Mel and people's PR3 sequence；
(2) contain insect secretory signal peptide Mel by template building of the recombinant plasmid pMelBacA-PR3-His of step (1) synthesis
Recombination donor vehicle pFastBac1-Mel-PR3-His:
(3) the recombination donor vehicle pFastBac1-Mel-PR3-His of step (2) building and Escherichia coli are transfected into silkworm jointly
Gonad cell BmN, preparation and reorganization virus rBm-pFastBac1-Mel-PR3-His；
(4) the recombinant virus rBm-pFastBac1-Mel-PR3-His inoculation transfection silkworm that will be prepared in step (3), in silkworm
Middle expression protease 3 recombinant protein；
(5) hemolymph of silkworm after recycling transfects；
(6) the protease 3 recombinant protein in hemolymph is isolated and purified.
2. the method according to claim 1 for preparing protease 3 recombinant protein, which is characterized in that as described in step (1)
Recombinant plasmid pMelBacA-PR3-His's the preparation method comprises the following steps: to people PR3 cDNA be template first round PCR amplification obtain PR3-
His segment carries out double enzyme digestion reaction to PR3-His segment, transferring plasmid pMelBacA respectively with Xho I and Kpn I；To two
Kind double enzyme digestion product is purified, is connected, and the recombinant plasmid for having insect secretory signal peptide Mel and people's PR3 sequence is obtained
pMelBacA-PR3-His。
3. the method according to claim 1 for preparing protease 3 recombinant protein, which is characterized in that described in step (2)
Recombinate donor vehicle pFastBac1-Mel-PR3-His's the preparation method comprises the following steps: recombinant plasmid to prepare in step (1)
PMelBacA-PR3-His is that template progress the second wheel pcr amplification reaction obtains Mel-PR3-His target fragment；With Xba I and
Kpn I to Mel-PR3-His target fragment, donor plasmid pFastBac1 carry out double enzyme digestion reaction, to two kinds of double enzyme digestion products into
Product after connection is transformed into competent escherichia coli cell by row purifying, connection, and extracting Plasmid DNA carries out evaluation and screening and obtains
Donor vehicle pFastBac1-Mel-PR3-His is recombinated to containing insect secretory signal peptide Mel.
4. the method according to claim 1 for preparing protease 3 recombinant protein, which is characterized in that weight described in step (3)
Group virus rBm-pFastBac1-Mel-PR3-His's the preparation method comprises the following steps: by step (2) construct recombination donor vehicle
PFastBac1-Mel-PR3-His transfection Escherichia coli, recombinate donor vehicle in Tn7 transposable element and silkworm BmNPV it is rod-shaped
Swivel base occurs for viral shuttle vector Bacmid, and extracting occurs the recombinant shuttle vector Bacmid DNA of swivel base and transfects silkworm ovary
Cells BmN prepares recombinant virus rBm-pFastBac1-Mel-PR3-His.
5. the method according to claim 1 for preparing protease 3 recombinant protein, which is characterized in that described in step (4)
Inoculation transfection is that recombinant virus rBm-pFastBac1-Mel-PR3-His is subcutaneously injected to Silkworm, Bombyx mori.
6. the method according to claim 1 for preparing protease 3 recombinant protein, which is characterized in that described in step (4)
Silkworm is five age silkworms.
7. the method according to claim 1 for preparing protease 3 recombinant protein, which is characterized in that described in step (5)
Hemolymph is the 4-5 days hemolymphs generated after silkworm transfection.
8. the method according to claim 1 for preparing protease 3 recombinant protein, which is characterized in that described in step (6)
Isolate and purify to first pass through affinity chromatography purifying after diluting hemolymph, purifying successively through ion exchange column Mono Q and
Mono S obtains the purpose product protease 3 recombinant protein of high-purity after purification.
9. the method according to claim 8 for preparing protease 3 recombinant protein, which is characterized in that described is diluted to use
The buffer of two volumes dilutes hemolymph.
10. the protease 3 recombinant protein of any one of -9 the method productions according to claim 1.
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| CN113528494A (en) * | 2021-08-10 | 2021-10-22 | 河南省医药科学研究院 | Purification method of recombinant mouse mature PRTN3 protein |
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