CN104561022B - Construction of domestic porcine tumor necrosis factor mutant and protein expression purification method - Google Patents
Construction of domestic porcine tumor necrosis factor mutant and protein expression purification method Download PDFInfo
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Abstract
The invention relates to site-directed mutagenesis of a domestic porcine tumor necrosis factor (TNF-alpha) gene, and a protein preparation method and application. One mutant gene of the porcine tumor necrosis factor (TNF-alpha) has the sequence disclosed as SEQ ID NO.2. The mutant gene translation protein amino acid sequence is disclosed as SEQ ID NO.3; the mutant can be connected into a pET28a prokaryotic expression plasmid and transformed into Escherichia coli BL21 (DE3) to induce expression, and Ni＜+＞-/NTA column purification is performed to obtain the recombinant His6-PmTNF-alpha; and the CCK-8, LDH detection and other experiments prove that the mutant protein can obviously enhance the cytotoxicity for L929 cells and lower the toxic or side effects on normal cells L02 as compared with the TNF-alpha wild type protein.
Description
Technical field
The present invention relates to biological gene engineering field, and in particular to the structure of hog tnf mutein body gene,
Expression, protein purification and activity identification technology.
Background technology
Tumor necrosis factor (tumor necrosis factor, TNF-α) is that the one kind for finding for 1975 has antitumor
The cytokine of effect, is the cytokine best to tumor cytotoxicity for finding so far, belongs to tumor necrosis factor and surpasses
Family member.TNF-α in addition to it there are direct Inhibit proliferaton and necrocytosiss to tumor cell and act on, to other cells (including
Myocardial cell) Growth and Differentiation also have an impact, while also antiviral and bacterium infection；Stimulate T cell, B cell, monokaryon macrophage
Cell etc. so as to which function strengthens；Induction inflammatory reaction, promotes the generation and secretion of IL1, IL2, IL6 etc.；Promote EGFR and main
Expression of Histocempatibilitygens classⅡ antigen (Ag of MHC II) etc. etc., plays an important role in host defense.TNF-α master
If by tying with Tumor Necrosis Factor Receptors on target cell membrane (tumor necrosis factor receptor, TNFR)
Close, the biological functions such as its cytotoxicity, antiviral bacterium infection, immunomodulating are realized, with extensive biologic activity.
People can be by kidney rapid drainage and by various proteases for decomposing with TNF-α, half-life very short (15- in vivo
30min), and playing a role needs 12-36h.If frequent large bolus injection, can cause serious untoward reaction, such as generate heat, dislike
Heart vomiting, headache, hypotension etc., or even shock, death can be caused.TNF-α is only the 1/ of its effective dose in the dosis tolerata of human body
10~1/50.Therefore, it is limited to be administered in tumor tissues with TNF-α treating cancer, to obtain higher local concentration.In order to
TNF-α is preferably utilized, its biologic activity is improved, its system toxic and side effects is reduced, in recent years, domestic and international scientist is by fixed
Point mutation Technique on T NF- α genes carry out rite-directed mutagenesises, so as to reach the purpose for increasing TNF-α activity and reducing its toxicity.
Professor Zhang Yingqi of The Fourth Military Medical University's biotechnology center in 2003 by protein engineering to TNF-α modified recombinant it
The hypotoxic new mutants of high activity, the tumor necrosis factor after mutation and Native recombinant human's tumor necrosis factor phase are obtained afterwards
Than toxicity reduces 100 to 1000 times, but the killing activity and tumor-inhibiting action to tumor cell reaches more than 60%.The research
First class national new drug Tetramune certificate is obtained, this is current first recombinant mutant human tumour for going through to produce in the world
Necrosin medicine, is also a genoid engineering antitumor drug of first listing of China.
Tumor necrosis factor (TNF-α) as the best cytokine of the antitumous effect for finding so far, always
The focus studied both at home and abroad, but almost all of research is all, around humanTNF-α, to be seldom related to the neoplasm necrosises of domestic animal
The factor.Pig is China's meat important sources, is the important component part of China's domestic animal aquaculture, but in recent years, pig some
The impact that commonly encountered diseases (such as heating, dysentery even swine fever, reproductive and respiratory syndrome, foot and mouth disease) are cultivated to it is increasingly serious.This problem is from state
From the aspect of the present situation of inside and outside research and the immunity and antiviral bacterium infection of enhancing pig, propose to pig TNF-α gene
Carry out mutation construction, RT-PCR expression and function to be studied, for hypoimmunity and the pig of inflammation infection disease, have
The immunostimulant and vaccine adjuvant of correlation is developed in prestige.
The content of the invention
The present invention is directed to the deficiencies in the prior art, there is provided a kind of hog tnf mutein body gene (PmTNF- α)
Construction method, and expression and purification process and the Activity determination of hog tumor necrosis factor (TNF-α) mutant protein are provided.
Pig tumor necrosis factor (TNF-α) mutant gene sequence of the present invention is mutated base as shown in SEQ ID NO.2
Compare with wild type because translating protein amino acid sequence, N-terminal lacks 7 aminoacid, the Ser of the 8th, the Asp of the 9th and the
The Ile of 156 sports respectively Lys, Arg and Phe, and its sequence is as shown in SEQ ID NO.3.
Hog tumor necrosis factor (TNF-α) mutant gene construction method is as follows：
(1) pig spleen total tissue RNA is extracted, and reverse transcription is the first chain cDNA；
(2) large fragment primer of the design packet containing mutational site：
F1：5’-AAGCGCAAGCCCGTCGCCCA-3’(SEQ ID NO.4)
R1：5’-TCAGAAGGCAATGATCCCAA-3’(SEQ ID NO.5)
(3) by the pcr amplification product of above-mentioned primer, pMD-19T carriers (TaKaRa) are cloned into, and carry out base sequence survey
It is fixed, obtain pig tumor necrosis factor (TNF-α) mutant gene sequence (PmTNF- α).
The invention also discloses a kind of construction method of recombinant expression carrier pET28-mTNF- α, is with pig the first chain cDNA
Masterplate, F2, R2 enter performing PCR for primer, and the sequence of F2, R2 obtains purpose base as shown in SEQ ID NO.6 and SEQ ID NO.6
Cause, i.e. band restriction enzyme site hog tnf mutein body gene PmTNF- α, and pET28a carriers BamH I and Hind III
Enzyme action, the carrier and genes of interest after T4 DNA Ligas connection enzyme action, obtains pET28-PmTNF- α recombiant plasmid.
A kind of expression and purification method of hog tnf mutein body protein, comprises the following steps：
(1) strain containing pET28-PmTNF- α recombiant plasmid is recovered, amplification culture and abduction delivering take -80 DEG C of jellies
Deposit the μ l of strain 50 and be inoculated in the 5ml LB culture medium added with 5 μ lKan and recover 12 hours, the bacterium solution after recovery is added to 500ml
In sterile LB medium, and add 500 μ lKan antibiotic, 37 DEG C, 200rpm shakes 2h；Plus the μ l of 1M IPTG 100, reach final concentration
To 0.2mM, 16 DEG C, 150rpm induces 20h.Receive bacterium, -70 DEG C, it is frozen overnight.
(2) ultrasonication：The resuspended frozen thalline of 30ml PBS, excusing from death is broken, 4 DEG C, 12000rpm, and 20min is centrifuged, and collects
Supernatant.
(3) nickel column chromatography：The each 200ml of elution buffer 20mM, 50mM, 100mM, 250mM imidazoles is prepared, 20mM miaows are first used
Azoles balances pillar, after end of the sample, starts eluting.20mM imidazoles eluting is first used, 50mM, 100mM imidazoles eluting is used respectively afterwards,
Last 250mM, now eluting peak is purpose albumen.
(4) dialyse and concentrate：By the 250mM imidazoles wash-out proteins collected, in being put into bag filter, in 4 DEG C, in 1L PBS
Dialysis 24 hours, is concentrated to suitable concn with PEG20000 afterwards.
The present invention further discloses the restructuring application of the hog tnf mutein body gene PmTNF- α, is
By gene engineering method, production restructuring His6- PmTNF- α albumen, as pig immunostimulant or the immunity assistant of relevant disease
Agent, or bio-pharmaceutical preparation.
The extracellular functional areas clone of the gene is connected in pET28a prokaryotic plasrnids simultaneously by existing gene engineering method
Expression in e. coli bl21 (DE3) is gone to, the present invention has found out suitable expression condition and (adjusted by repeatedly pre- expression experiment
Saving concentration, temperature and the rotating speed of IPTG makes fusion protein as much as possible with soluble form expression), it is collected by centrifugation after ultrasonication
Supernatant crosses Ni+- NTA column purifications, we have obtained hog tumor necrosis factor (TNF-α) mutant protein after gradient elution
(His6-PmTNF-α).And carry out Western-blot identifications.External WST-8 dyestuffs biological activity assay and LDH testing results
Show, the mutein compares the cytotoxic activity not only increased to tumor cell L929 with wild-type protein, and drops
The low cytotoxicity to normal cell (L02).
Description of the drawings
Fig. 1 recombiant plasmid figures (A, wild type TNF-α, B, saltant type TNF-α)
Fig. 2 nickel column chromatography elution profiles (A, wild type recombiant protein, B, saltant type recombiant protein)
Fig. 3 hog tumor necrosis factor (TNF-α) maturation zone fragment expressions and purification, Western blot qualification figures (M,
Albumen Maker, 1, do not induce, 2, induction, 3, albumen after purification, 4, Western blot results.A, wild type recombiant protein, B,
Saltant type recombiant protein)
Cytotoxic activity of Fig. 4 CCK-8 methods analysis variable concentrations hog tumor necrosis factor (TNF-α) to L929 cells.
(PwTNF- α are wild type recombiant protein, and PmTNF- α are saltant type recombiant protein)
L929 cells are shot under 10 × 10 times of inverted microscopes of Fig. 5
Toxic and side effects of Fig. 6 microplate reader method analysis pig tumor necrosis factor (TNF-α) to L02 cells.
Specific embodiment
The term for being used in the present invention, unless otherwise specified, typically has those of ordinary skill in the art usual
The implication of understanding.
Embodiment and Application Example are prepared with reference to specific, and this is described in further detail with reference to data
It is bright.It should be understood that these embodiments are of the invention solely for the purpose of illustration, rather than the scope of the present invention is limited by any way.
Below in an example, the various processes and method not described in detail are conventional methods as known in the art.
Used primer, indicates when occurring first, thereafter same primers used, identical with the content indicated first.
Embodiment 1
The structure of pig tnf mutein body (PmTNF- α) gene, pig spleen is taken from Nanjing Qixia Yao's metaplasia pig and is slaughtered
Slaughterhouse, is Landrace：
(1) total serum IgE is extracted：It is total pig spleen tissue to be extracted using RNA extraction agent boxes (TIANGEN) according to its workbook
RNA, and its quality and purity are identified by denaturing formaldehyde agarose gel electrophoresiies, ultraviolet spectrophotometer determines its concentration.
SMARTTMRACE test kits (TaKaRa) reverse transcription is the first chain cDNA.
(2) large fragment primer of the design packet containing mutational site, by PCR method tnf mutein body base is built
Cause.Primer sequence such as SEQ ID NO.4 and SEQ ID NO.5, using reverse transcription gained cDNA templates performing PCR is entered：
1. reaction system is 50 μ l, 10 μm of ol/L F1, R1 each 2 μ l, 2.5mmol/L dNTP 8 μ l, 2 × GC buffer
II 25 μ l, the μ l of cDNA templates 3, DreamTaq enzymes 0.5 μ l, ddH2O 9.5μl.Response procedures are：94 DEG C of 5min, (94 DEG C of 30s,
56 DEG C of 30s, 72 DEG C of 1min), 35 circulations, last 72 DEG C of extensions 7min.
2. react after the completion of by product in 1% agarose gel electrophoresis detection, with rubber tapping QIAquick Gel Extraction Kit reclaim obtain
PCR primer, is cloned into pMD19-T carriers (TaKaRa), the agarose plate screening conversion of Jing antibiotic containing Amp (50mg/ml)
Son, choosing positive colony and being sent to Shanghai English fine horse sequencing company carries out base sequence measure.After the sequencing of many experiments result, pass through
Alignment determines constructed pig tnf mutein body gene order.Such as conception, by comprising mutational site
Primer (sequence such as SEQ ID NO.4 and SEQ ID NO.5) caused specific mutation, mutant gene sequence such as SEQ ID
NO.2。
Embodiment 2：
The structure of hog tumor necrosis factor (TNF-α) mutant recombinant expression carrier and its induction in escherichia coli
Expression and Purification；
According to TNF-α mutant sequence (gene order such as SEQ ID NO.2) is had been built up, primers F 2, R2 is designed.Wherein
5 ' the ends that the 5 ' ends of F2 have the restriction enzyme sites of BamH I, R2 have the restriction enzyme sites of Hind III, with pig the first chain cDNA as masterplate, F2,
R2 be primer (F25 '-CGCGGATCCAAGCGCAAGCCCGTCGCCCACGTTGT-3 ' (SEQ ID NO.6), R25 '-
CCCAAGCTTTCAGAAGGCAATGATCCCAAAATAGT-3 ' (SEQ ID NO.7), PCR expands the gene (10 × pfu
The μ l of Buffer 5, dNTP (2.5mM) 4 μ l, F2 (10 μm of ol/L) 2 μ l, R2 (10 μm of ol/L) 2 μ l, H2The μ l of O 33.5, the μ of cDNA 3
μ l of l, pfu Taq 0.5) reaction condition is as follows：94 DEG C of 5min, (94 DEG C of 30s, 59 DEG C of 30s, 72 DEG C of 1min), 35 circulations, most
Afterwards 72 DEG C of extension 7min, afterwards by PCR primer rubber tapping recovery, recovery product and pET28a carriers (Invitrogen, USA), use
BamH I and the enzyme action of Hind III, T4 DNA Ligase (TaKaRa) connections, obtain recombiant plasmid and (are named as pET28a-PmTNF-
α) (Figure 1B), in connection product Transformed E .coli DH5a, obtains the bacterial strain containing recombiant plasmid pET28a-PmTNF- α.Select
Positive restructuring bacterial strain is sequenced, and correct bacterial strain amplification culture is sequenced and extracts pET28a-PmTNF- α recombiant plasmid, is transformed into large intestine
In bacillus BL21 (DE3), positive strain abduction delivering is selected.
Hog tumor necrosis factor (TNF-α) wild-type protein prokaryotic expression carrier is in kind built, is named as
PET28a-PwTNF- α (Figure 1A).Primers F 3, R3, wherein F3 are designed according to hog tumor necrosis factor (TNF-α) gene order
5 ' end have the restriction enzyme sites of BamH I, R3 5 ' end have the restriction enzyme sites of Hind III.With pig the first chain cDNA as masterplate, F3,
R3 be primer (F35 '-CGCGGATCCCTCAGATCATCGTCTCAAACCTCAGATA-3 ' (SEQ ID NO.8), R35 '-
CCCAAGCTTTCACAGGGCAATGATCCCAAAATAGA-3 ' (SEQ ID NO.9) constructs hog tumor necrosis factor
(TNF-α) wild type expressing gene, sequence as shown in SEQ ID NO.1,
Through multiple pre- expression experiment, when IPTG concentration is 0.2mM, 16 DEG C of inducing temperature, rotating speed 150rpm induces 20h,
There is more destination protein with soluble form expression.16 DEG C, bacterium, after -70 DEG C of frozen nights, 30ml PBS are received in 6000rpm centrifugations
It is resuspended, ultrasonication on ice (200W, work 4s, time-out 8s, totally 150 times) expression thalline, 4 DEG C, 12000rpm, 20min centrifugation
After ultrasonication liquid, precipitation is abandoned, supernatant crosses Ni+- NTA posts.Simplified process is：Binding Buffer(20mmol/L
Imidazole, 0.5mol/L NaCl, 20mmol/L Tris HCl, pH 8.0) balance Ni+After-NTA posts, solubility will be contained
The slow loading of supernatant of recombiant protein, after end of the sample first with Binding buffer (20mmol/L Imidazole,
0.5mol/L NaCl, 20mmol/L Tris-HCl, pH 8.0) foreign protein is washed away, then washed with the imidazoles Tis-HCl of variable concentrations
De-, destination protein is mainly 250mmol/L Imidazole, 0.5mol/L NaCl, 20mmol/L Tris in wash-out concentration
HCl, pH 8.0 is eluted (elution curve is as shown in Figure 2).It is in charge of collection eluting destination protein, PBS overnight desalination.
SDS-PAGE detects each collecting pipe purity of protein, and ultraviolet spectrophotometer determines protein concentration.As shown in Fig. 3 (band 3), expression
The destination protein Jing Ni for obtaining+- NTA column purifications obtain the destination protein that purity is up to 97%.
The Western markings are analyzed：
Used one resists for the anti-His of Mus in Western-blot6Monoclonal antibody, two resist for the sheep of horseradish peroxidase (HRP) labelling
Anti- Mus IgG.Its brief step is：Sample is first carried out SDS-PAGE, with immersion method by albumen electrotransfer to celluloid
On film (NC films), with each bands of marking pen labelled protein marker, then successively the defatted milk powder room temperatures of Jing 5% close 1h, with one
Anti- incubation 1h, with two anti-igg of HRP labellings 1h is incubated, and after the completion of often walking film is strictly washed, and finally adds the colour developing of TMB lucifuges.As a result
Specific band is occurred in that as shown in Fig. 3 (band 4) in destination protein position.Preserve after the destination protein sucking filtration subpackage that will be obtained
In -70 DEG C.
Embodiment 3
CCK-8 methods analyze His6Cytotoxic activities of-PmTNF- the α to L929 cells；
7ml 1640 (10%FBS) culture medium culturing L929 cells, 24 hours.After removing culture medium, the digestion of 1ml pancreatin
30s, plus the termination digestion of 1ml culture medium, re-suspended cell, in being drawn to 15ml centrifuge tubes, 1000g, 20 DEG C, 3min.Go out supernatant
Afterwards again plus 2ml culture medium (1640,10%FBS), re-suspended cell, in drawing 1ml to 50ml centrifuge tubes, plus 10%FBS 1640
Culture medium dilutes, and counting chamber is counted, and makes born of the same parents' number be 3 × 105Individual/ml.The cell suspension kind for drawing above-mentioned dilution enters 96 orifice plates
In, per the μ l of hole 100,37 DEG C, 5%CO2, cultivate 24h.Remove culture fluid.Variable concentrations are configured with the culture medium of 10%FBS 1640
TNF-α diluent (is divided to two groups of wild type and saltant type), adds 100 μ l per hole afterwards, makes TNF-α final concentration reach 105、104、
103、102、10、100、10-1、10-2、10-4Ng/ml, adds Act.D (actinomycin D) to make final concentration reach 0.4 μ l/ml per hole, if
It is empty white, PBS, Act.D matched group.37 DEG C, 5%CO2, cultivate 24 hours, it is directly right by micro- bat under inverted microscope
Shot per hole cell growth condition, as a result as shown in figure 5, afterwards plus CCK-8 (10% ratio), place in incubator 1h it
Afterwards, 450nm absorbances are surveyed in microplate reader.After carrying out cytotoxic activity calculating, as a result as shown in Figure 4.
Embodiment 4
Enzyme linked immunosorbent assay analyzes His6Toxic and side effects of-PmTNF- the α to L02 cells
7ml 1640 (10%FBS) culture medium culturing L02 cells, 24h.After removing culture medium, 1ml pancreatin digestion 30s, plus
1ml culture medium terminates digesting, re-suspended cell, in being drawn to 15ml centrifuge tubes, 1000g, and 20 DEG C, 3min.Go out after supernatant again
Plus 2ml culture medium (1640,10%FBS), re-suspended cell, in drawing 1ml to 50ml centrifuge tubes, plus the culture medium of 10%FBS 1640
Dilution, counting chamber is counted, and makes born of the same parents' number be 3 × 105Individual/ml.The cell suspension kind for drawing above-mentioned dilution enters in 24 orifice plates, per hole
500 μ l, 37 DEG C, 5%CO2, cultivate 24h.Culture fluid is removed, it is dilute with the culture medium of 10%FBS 1640 configuration variable concentrations TNF-α
Liquid (being divided to two groups of wild type and saltant type) is released, adds 500 μ l per hole afterwards, make TNF-α final concentration reach 105、104、103、102、
10、100、10-1、10-2、10-4Ng/ml, arranges blank, PBS control group.37 DEG C, 5%CO2, cultivate 24 hours, afterwards according to south
Cell toxicant of Bioengineering Research Institute's lactic acid dehydrogenase (LDH) detection kit step detection TNF-α to L02 cells is built up in capital
Pair, exercising result is as shown in Figure 6.
Claims (6)
1. a kind of hog tumor necrosis factor TNF-alpha mutant gene, it is characterised in that its sequence such as SEQ ID NO.2 institutes
Show.
2. a kind of albumen by hog tumor necrosis factor TNF-alpha mutant gene code described in claim 1, it is characterised in that
Its sequence is as shown in SEQ ID NO.3.
3. a kind of hog tumor necrosis factor TNF-alpha site-directed point mutation method, it is characterised in that comprise the following steps：
(1) hog spleen tissue total serum IgE is extracted, test kit is transcribed into the first chain cDNA
(2) designing long segment primer carries out the structure of mutant gene
Sense oligonucleotides primers F 1：5’-AAGCGCAAGCCCGTCGCCCA-3’
Antisense oligonucleotide primer R1：5’-TCAGAAGGCAATGATCCCAA-3’
(3) with F1, R1 as primer, the cDNA obtained by reverse transcription is template, enters performing PCR amplification；The PCR primer of gained is cloned into
PMD-19T carriers, and carry out base sequence measure；Obtain hog tumor necrosis factor-TNF-α-mutant gene, its sequence
Shown in SEQ ID NO.2.
4. a kind of construction method of recombinant expression carrier pET28-mTNF- α, it is characterised in that：With pig the first chain cDNA as masterplate,
F2, R2 enter performing PCR for primer, and the sequence of F2, R2 obtains genes of interest, i.e., as shown in SEQ ID NO.6 and SEQ ID NO.7
Band restriction enzyme site hog tnf mutein body gene PmTNF- α, and pET28a carriers BamH I and the enzyme action of Hind III,
Carrier and genes of interest after T4DNA Ligas connection enzyme action, obtains pET28-PmTNF- α recombiant plasmid.
5. a kind of expression and purification method of hog tnf mutein body protein, it is characterised in that comprise the following steps：
(1) the recovery of the strain containing pET28-PmTNF- α recombiant plasmid, amplification culture and abduction delivering, -80 DEG C of frozen bacterium are taken
Plant 50 μ l and be inoculated in the 5ml LB culture medium added with 5 μ l Kan and recover 12 hours, the bacterium solution after recovery is added to 500ml sterilizings
In LB culture medium, and add 500 μ l Kan antibiotic, 37 DEG C, 200rpm shakes 2h；Plus the μ l of 1M IPTG 100, reach final concentration
0.2mM, 16 DEG C, 150rpm induces 20h；Receive bacterium, -70 DEG C, it is frozen overnight；The pET28-PmTNF- α recombiant plasmid is by power
Profit requires that 4 method builds and obtains；
(2) ultrasonication：The resuspended frozen thalline of 30ml PBS, excusing from death is broken, 4 DEG C, 12000rpm, 20min is centrifuged, in collection
Clearly；
(3) nickel column chromatography：The each 200ml of elution buffer 20mM, 50mM, 100mM, 250mM imidazoles is prepared, it is first flat with 20mM imidazoles
Weighing apparatus pillar, after end of the sample, starts eluting；20mM imidazoles eluting is first used, 50mM, 100mM imidazoles eluting is used respectively afterwards, finally
250mM imidazoles, now eluting peak is purpose albumen；
(4) dialyse and concentrate：By the 250mM imidazoles wash-out proteins collected, in being put into bag filter, in 4 DEG C, dialyse in 1L PBS
24 hours, it is concentrated to suitable concn with PEG20000 afterwards.
6. hog tumor necrosis factor TNF-alpha mutant gene described in a kind of claim 1 is preparing pig immunostimulant or phase
Application in the immunological adjuvant or bio-pharmaceutical preparation of related disorders.
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