CN109880842A - A kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen - Google Patents
A kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen Download PDFInfo
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Abstract
The invention discloses a kind of preparation processes of genetic recombination high activity serum amyloid protein SAA antigen, belong to technical field of bioengineering.The preparation process of the genetic recombination high activity serum amyloid protein SAA antigen, comprising the following steps: 1) preparation optimization SAA genetic fragment;2) expression plasmid containing SAA and the expression bacterial strain containing SAA are constructed；3) fermentation of genetic recombination SAA；4) collection and cracking of cell；5) purifying of SAA, using the SAA of SUMO protease digestion fusion SUMO label, affinitive layer purification is recycled again, obtains genetic recombination high activity serum amyloid protein SAA antigen.The host strain Escherichia coli Growth period that the present invention uses is short, and production cost is low, high conversion rate and can be significantly larger than other expression systems with high-expression target proteins, expression, realizes that large-scale production lays the foundation for recombination high activity product.
Description
Technical field
The invention belongs to technical field of bioengineering more particularly to a kind of genetic recombination high activity serum amyloid proteins
The preparation process of SAA antigen.
Background technique
Serum amyloid protein SAA is the precursor substance of tissue amyloid A, similar to c reactive protein (CRP), is
A kind of Acute reaction protein generated by liver cell.SAA exists in the serum of normal person with constant, but in inflammation or
SAA can increase hundreds of to thousands of times in 48~72h of acute stage of infection, and decline rapidly in the convalescence of disease, be a kind of
The marker of bacterium or virus infection is more sensitively diagnosed than CRP.
The main acute phase protein that SAA is largely generated during being APR.Currently, the raising of SAA concentration has become inflammation
Clinical marker.SAA can be activated including a variety of receptors such as scavenger receptor SR-BI, ATP receptor P2X7.
In general, high-density lipoprotein (HDLs) is considered as antiatherosclerosis.In research HDL protein group
At it was found that, acute phase protein serum amyloid A protein (SAA) may be incorporated into HDL particle.Some researches show that
SAA is the strong marker of cardiovascular mortality.One perspective study discovery, Serum SA A be during rejection most
One of early raised index.SAA is also used as monitoring liver transfer operation early rejection, kidney transplantation exclusion reaction and acute grafing
Therefore the Sensitive mark of the anti-host response of object with the continuous intensification that people study SAA, causes it in cardiovascular disease
Diagnosis on can hold out broad prospects.
For Escherichia coli as excellent genes engineering bacteria, growth cycle is short, and production cost is low, high conversion rate and can high table
Up to destination protein, expression is significantly larger than other expression systems.Realize industrialization and the recombinant product of maturation of product
Purifying process is closely bound up, therefore realizes the combination of technique for gene engineering and protein Process, undoubtedly can be high living for recombination
Property product realize large-scale production lay the foundation.
Summary of the invention
In view of the above problems, the present invention provides a kind of genetic recombination high activity serum amyloid protein SAA antigen
Preparation process, this preparation process can greatly improve recombination serum amyloid protein SAA antigen yield and activity.
The technical solution adopted by the invention is as follows:
A kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen, comprising the following steps:
1) codon optimization is carried out to SAA genetic fragment, SAA genetic fragment must be optimized；
2) building of the expression plasmid containing SAA and the expression bacterial strain containing SAA: the optimization SAA genetic fragment is connected to
On expression plasmid, the expression plasmid containing SAA is obtained；Then the expression plasmid containing SAA is transferred in expression bacterial strain, obtains the table containing SAA
Up to bacterial strain, that is, genetic recombination SAA；
3) fermentation of genetic recombination SAA: the expression bacterial strain containing SAA is cultivated in 37 DEG C of fermentation medium to OD600
=2~3, IPTG Fiber differentiation is then added, obtains fermentation liquid；
4) collection and cracking of cell: the fermentation liquid is carried out thallus is collected after centrifugation, is then by mass volume ratio
Lysis buffer is added in 1:10~15, and cell cracking, centrifugation are collected supernatant at 4 DEG C followed by high pressure homogenizer；
5) purifying of SAA: the supernatant and affinity chromatography filler are incubated for 60~80min, then carry out affinity chromatography
Purifying obtains the SAA of fusion SUMO label, the SAA of SUMO label is then merged using the digestion of SUMO protease, again affine layer
Purification and recovery is analysed, genetic recombination high activity serum amyloid protein SAA antigen is obtained.
Further, the codon optimization software that codon optimization described in the step 1) is developed using the safe biology of moral
MaxCodonTM Optimization Program V13。
Further, expression plasmid described in the step 2) is pSUMO.
Further, expression bacterial strain described in the step 2) is E. coli BL21 (DE3).
Further, fermentation medium described in the step 3) is TB culture medium, the condition of the culture are as follows: revolving speed
200~300rpm, tank pressure are 0.03~0.05Mpa, and initial ventilatory capacity is 3L/min, maintain DO 25% by adjusting ventilatory capacity
~40%；Final concentration of 0.1~the 0.5mmol/L of IPTG.
Further, lysis buffer described in the step 4) includes the Triton X- of 1 × PBS, 0.5%~2%
100,1~2mmol/L DTT, 0.2~0.6mmol/L PMSF and 20~30mmol/L Imidazole.
Further, the homogenization pressure of high pressure homogenizer described in the step 4) is 700~900Mpa.
Further, filler used in affinity chromatography described in the step 5) is NI-IDA, is put down used in the affinity chromatography
Weighing apparatus buffer is 1 × PBS and 20~50mmol/L Imidazole.
Further, the temperature of digestion described in the step 5) is 4~30 DEG C, and the time of digestion is 1~3h；It is described to melt
The mass ratio for closing SAA and the SUMO protease of SUMO label is 1:400~800.
Further, recycling filler used described in the step 5) is NI-IDA, the incubation time 30 of the recycling~
60min, recycling equilibration buffer used is 1 × PBS.
The beneficial effects of the present invention are: the host strain Escherichia coli Growth period that the present invention uses is short, production cost is low, turns
Rate is high and can be significantly larger than other expression systems with high-expression target proteins, expression, to recombinate high activity product
Realize that large-scale production lays the foundation.
(1) the codon optimization software MaxCodonTM Optimization Program of the safe biology exploitation of moral is utilized
V13 optimizes SAA protein gene, so that it is conducive to escherichia coli high-level expression, genetic recombination SAA is obtained, by genetic recombination SAA
By high-temperature cultivation and induction, expresses that SAA albumen in the form of active supernatant, avoid and deposited with inactive inclusion bodies
?；Due to selecting special restriction enzyme site to improve final SAA by affinitive layer purification fusion protein in purification phase
The purity and fermentation product yield of albumen are easy to subsequent digestion and close reclaimer operation；Later period merges by the digestion of SUMO protease
The SAA of SUMO label obtains the recombination SAA albumen of high activity.
(2) present invention is to reduce the temperature to 25 DEG C to start to induce using cultivating at 37 DEG C of high temperature to logarithmic growth phase, in this way
Protein expression can be allowed in supernatant, ensure that the activity and yield of albumen while not losing expression quantity, selected in shattering process
High-pressure homogeneous crusher machine is selected, since it can be controlled with low temperature, keeps protein stabilized structure, and percentage of damage is good, treating capacity is big.
Therefore entire simple production process, efficiently quickly, production cost is low.
Detailed description of the invention
Fig. 1 PCR product agarose gel electrophoresis figure.
Fig. 2 SAA albumen is in BL21 (DE3) expression figure.
Fig. 3 SAA protein purification figure.
Fig. 4 SAA proteolytic cleavage recycling figure.
Fig. 5 SAA albumen quality inspection figure.
The standard curve of Fig. 6 measurement SAA protein concentration.
Specific embodiment
Embodiments of the present invention are as follows:
A kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen, comprising the following steps:
1) codon optimization is carried out to SAA genetic fragment, SAA genetic fragment must be optimized；
2) building of the expression plasmid containing SAA and the expression bacterial strain containing SAA: the optimization SAA genetic fragment is connected to
On expression plasmid, the expression plasmid containing SAA is obtained；Then the expression plasmid containing SAA is transferred in expression bacterial strain, obtains the table containing SAA
Up to bacterial strain, that is, genetic recombination SAA；
3) fermentation of genetic recombination SAA: the expression bacterial strain containing SAA is cultivated in 37 DEG C of fermentation medium to OD600
=2~3, IPTG Fiber differentiation is then added, obtains fermentation liquid；
4) collection and cracking of cell: the fermentation liquid is carried out thallus is collected after centrifugation, is then by mass volume ratio
Lysis buffer is added in 1:10~15, and cell cracking, centrifugation are collected supernatant at 4 DEG C followed by high pressure homogenizer；
5) purifying of SAA: the supernatant and affinity chromatography filler are incubated for 60~80min, then carry out affinity chromatography
Purifying obtains the SAA of fusion SUMO label, the SAA of SUMO label is then merged using the digestion of SUMO protease, again affine layer
Purification and recovery is analysed, genetic recombination high activity serum amyloid protein SAA antigen is obtained.
Further, the codon optimization software that codon optimization described in the step 1) is developed using the safe biology of moral
MaxCodonTM Optimization Program V13。
Further, expression plasmid described in the step 2) is pSUMO.
Further, expression bacterial strain described in the step 2) is E. coli BL21 (DE3).
Further, fermentation medium described in the step 3) is TB culture medium, the condition of the culture are as follows: revolving speed
200~300rpm, tank pressure are 0.03~0.05Mpa, and initial ventilatory capacity is 3L/min, maintain DO 25% by adjusting ventilatory capacity
~40%；Final concentration of 0.1~the 0.5mmol/L of IPTG.
Further, lysis buffer described in the step 4) includes the Triton X- of 1 × PBS, 0.5%~2%
100,1~2mmol/L DTT, 0.2~0.6mmol/L PMSF and 20~30mmol/L Imidazole.
Further, the homogenization pressure of high pressure homogenizer described in the step 4) is 700~900Mpa.
Further, filler used in affinity chromatography described in the step 5) is NI-IDA, is put down used in the affinity chromatography
Weighing apparatus buffer is 1 × PBS and 20~50mmol/L Imidazole.
Further, the temperature of digestion described in the step 5) is 4~30 DEG C, and the time of digestion is 1~3h；It is described to melt
The mass ratio for closing SAA and the SUMO protease of SUMO label is 1:400~800.
Further, recycling filler used described in the step 5) is NI-IDA, the incubation time 30 of the recycling~
60min, recycling equilibration buffer used is 1 × PBS.
In technical proposal scope listed by the present invention, the present invention be can be implemented including endpoint value, can generate nothing in this way
Number embodiment, details are not described herein, and set forth below is preferred embodiment of the invention, however it is not limited to protection model of the invention
It encloses.
Embodiment 1
1, the building of genetic recombination SAA
With the SAA amino acid sequence of people, using the codon optimization software MaxCodonTM of the safe biological recent development of moral
Optimization Program V13 carries out codon optimization to SAA genetic fragment and obtains cDNA sequence, according to target cDNA
Sequent synthesis primer merges to obtain full length sequence using round pcr；
Primer is specifically:
Primer1
AACTTTAAGAAGGAGATATACATATGCATCATCATCACCACCACCGCAGCTTTTTCAGCTTTCTGGGCG
AAG
Primer2
TGGTGCTCGAGTGCGGCCGCAAGCTTTCATTAATATTTTTCCGGCAGACCAGCCGGACGAAAGTGATTC
GGATC
2, using the cDNA genetic fragment of SAA as template, PCR amplification target gene fragment, reaction condition are as follows: 95 DEG C, 5min；
(95 DEG C of 30s, 60 DEG C of 60s, 72 DEG C of 45s, 25 circulations)；72 DEG C, 10min.Purify the SAA gene and expression plasmid amplified
PSUMO uses StuI and Hind III double digestion, connection respectively and converts to BL21 (DE3) competent cell, will be weighed by thermal shock
Group plasmid imports in e. coli bl21 (DE3), obtains the expression bacterial strain containing SAA.
3,300ul LB culture medium is added in the expression bacterial strain by step 2 containing SAA, takes 80 μ L equal after 37 DEG C of constant temperature incubation 4h
It is even to be applied on the kanamycins plating medium that 50ug/ml is added, it is cultivated 12~24 hours at 37 DEG C, can be obtained recombination
SAA Escherichia coli.
Embodiment 2
Recombinate the fermentation control of SAA Escherichia coli
Picking monoclonal on the plate for the recombinant bacterial strain that embodiment 1 obtains is seeded in 37 DEG C of cultures in culture medium containing LB
16h is inoculated in 1% inoculum concentration containing 3L TB culture solution (kanamycin sulfate containing 50ug/ml, egg as kind of a liquid, seed liquor
White peptone 12g/L, glycerol 5g/L, K2HPO43H2O 15.2g/l, yeast extract 24g/L, KH2PO4 2.33g/l), 5L fermentor
In 37 DEG C of cultures, revolving speed is set as 200rpm, and tank presses 0.03Mpa, and initial ventilatory capacity is 3L/min, passes through and adjusts ventilatory capacity and maintain DO
It is 25%, starts to be cooled to 25 DEG C when bacteria concentration OD600 reaches 2, after final concentration of 0.2mM IPTG Fiber differentiation 6h is added
Fermentation is terminated, thallus is collected after centrifugation, the final wet thallus that obtains is 20.7g/L.
Embodiment 3
Recombinate the purifying of SAA albumen
Clasmatosis: taking the thallus 10g being collected by centrifugation after fermentation that 1 × PBS, the pH7.4 of 15 times of volumes (150ml) is added,
After 20mM Imidazole, 1%Triton X-100,1mM DTT, 0.2mM PMSF is mixed, slow stream is added to high pressure homogenizer
In, pressure control afterwards by homogenate in 12500rpm, 4 DEG C of centrifugation 15min, abandons precipitating and retains supernatant twice in 700Mpa, circulation；
NI-IDA balance: 5mL NI-IDA filler is taken, with 10 times of column volume equilibration buffers 1 × PBS, pH7.4,20mM
Imidazole balances filler；
It is incubated for: filler being mixed with pillar, is incubated for 60min under the conditions of 4 DEG C；
Purifying: incubation terminates, and separates filler and unbonded foreign protein using gravity pipe and peristaltic pump, is then utilized respectively
Buffer1:1 × PBS, pH7.4,20mM Imidazole removal of impurities, buffer2:1 × PBS, pH7.4,50mM Imidazole are washed
De-, buffer3:1 × PBS, pH7.4,500mM Imidazole elution are collected each elution fraction and are detected using SDS-PAGE,
The SAA albumen of finally obtained fusion sumo label.
Embodiment 4
Digestion and recycling without label SAA albumen
The digestion of fusion protein: collecting the fusion protein that purifying obtains, and dialyses by 1:20 (volume ratio) to the PBS of 5L,
Then SUMO protease is added, in 20 DEG C of conditions in 4h in pH7.4 according to 1:400 (mass ratio) into the albumen after dialysis
Lower digestion 2h, digestion terminate to take supernatant to recycle mixed liquor 12500rpm centrifugation 15min；
NI-IDA balance: taking 2mL NI-IDA filler, is filled out with 10 times of column volume equilibration buffer 1 × PBS, pH7.4 balances
Material；
It is incubated for: filler being mixed with pillar, is incubated for 40min under the conditions of 4 DEG C；
Purifying: incubation terminates, and is separated filler and unbonded foreign protein using gravity pipe and peristaltic pump, and collection flows through liquid,
It is detected using SDS-PAGE, finally obtained no label SAA albumen, purity of protein reaches 95% or more.
Correlated performance test is carried out to gained SAA recombinant protein:
Fig. 1 is PCR product agarose gel electrophoresis figure, and Lane 1,2 is PCR product, and Lane M is DNA Marker.
Fig. 2 is SAA albumen in BL21 (DE3) expression, Lane M:SDS-PAGE Protein Marker, Lane1:
It compares (IPTG is not added), Lane2:SAA albumen induces the expression of 6h at 25 DEG C.
Fig. 3 is SAA protein purification figure, and Lane M:SDS-PAGE Protein marker, Lane 1: full bacterium breaks bacterium centrifugation
Supernatant afterwards, Lane 2: efflux after supernatant is incubated for Ni-IDA, Lane 3-13:SAA albumen wash-out component.
Fig. 4 is SAA proteolytic cleavage recycling figure, Lane M:SDS-PAGE Protein marker, Lane 1:SAA fusion
Albumen, supernatant after Lane 2: digestion 2h centrifugation, Lane 3: efflux after supernatant mixing is incubated for Ni-IDA, Lane 4: elution
Component.
Fig. 5 be SAA albumen quality inspection figure, Lane M:SDS-PAGE Protein marker, Lane 1:BSA, Lane 2:
SAA protein。
The concentration and activity identification of SAA recombinant protein
It measures SAA protein concentration and uses BCA determination of protein concentration kit.Its standard curve is as shown in Fig. 6.Dilution five
Its OD=0.521 is measured again, and final converted score is 1.37mg/mL, thus obtains the yield without label SAA albumen after final digestion
For 50-60mg/L, yield height.
The recombination SAA albumen and natural SAA protein active that the present invention obtains are compared using indirect elisa method, identical anti-
In the case of physical examination is surveyed: this experimental antigen plate peridium concentration is 1ug/ml (i.e. every hole is coated with 10ng antigen), and primary antibody is that immune SAA is small
Mouse whole blood serum, is diluted using 1*PBS, and three times serial dilution is detected after 1:1000 dilution, and secondary antibody is sheep anti mouse two
It is anti-, it is diluted using 1*PBS, dilution ratio 1:250000, negative control 1*PBS.Two Duplicate Samples of every group of setting, respectively
For coating recombination SAA albumen -1, coating recombination SAA albumen -2, the natural SAA albumen -1 of coating, natural albumen -2 SAA of coating, knot
Fruit is as shown in table 1 below, under same antibody detection case, the recombination SAA albumen potency that the present invention obtains with natural SAA albumen
Potency is close, illustrates that recombination SAA protein active is strong.
1 activity comparison of table
The invention is not limited to specific embodiments above-mentioned.The present invention, which expands to, any in the present specification to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
Claims (10)
1. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen, which is characterized in that including following step
It is rapid:
1) codon optimization is carried out to SAA genetic fragment, SAA genetic fragment must be optimized;
2) the optimization SAA genetic fragment building of the expression plasmid containing SAA and the expression bacterial strain containing SAA: is connected to expression
On plasmid, the expression plasmid containing SAA is obtained；Then the expression plasmid containing SAA is transferred in expression bacterial strain, obtains the expression bacterium containing SAA
Strain is genetic recombination SAA；
3) fermentation of genetic recombination SAA: by the expression bacterial strain containing SAA cultivated in 37 DEG C of fermentation medium to OD600=2~
3, IPTG Fiber differentiation is then added, obtains fermentation liquid；
4) collection and cracking of cell: the fermentation liquid being carried out thallus is collected after centrifugation, and is then 1:10 by mass volume ratio
~15 are added lysis buffer, and cell cracking, centrifugation are collected supernatant at 4 DEG C followed by high pressure homogenizer；
5) purifying of SAA: being incubated for 60~80 min for the supernatant and affinity chromatography filler, and it is pure then to carry out affinity chromatography
Change, obtain the SAA of fusion SUMO label, the SAA of SUMO label is then merged using the digestion of SUMO protease, again affinity chromatography
Purification and recovery obtains genetic recombination high activity serum amyloid protein SAA antigen.
2. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, the codon optimization software that codon optimization described in the step 1) is developed using the safe biology of moral
MaxCodonTM Optimization Program V13。
3. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, expression plasmid described in the step 2 is pSUMO.
4. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, expression bacterial strain described in the step 2 is E. coli BL21(DE3).
5. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, fermentation medium described in the step 3) is TB culture medium, the condition of the culture are as follows: revolving speed 200~300
Rpm, tank pressure are 0.03~0.05Mpa, and initial ventilatory capacity is 3L/min, maintain DO 25%~40% by adjusting ventilatory capacity；Institute
State the final concentration of 0.1~0.5mmol/L of IPTG.
6. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
Be characterized in that, lysis buffer described in the step 4) include 1 × PBS, 0.5%~2% Triton X-100,1~
2mmol/L DTT, 0.2~0.6 mmol/L PMSF and 20~30 mmol/L Imidazole.
7. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, the homogenization pressure of high pressure homogenizer described in the step 4) is 700~900Mpa.
8. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, filler used in affinity chromatography described in the step 5) is NI-IDA, equilibration buffer used in the affinity chromatography
For 1 × PBS and 20~50 mmol/L Imidazole.
9. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, the temperature of digestion described in the step 5) is 4~30 DEG C, and the time of digestion is 1~3h；The fusion SUMO mark
The mass ratio of SAA and the SUMO protease of label is 1:400~800.
10. a kind of preparation process of genetic recombination high activity serum amyloid protein SAA antigen according to claim 1,
It is characterized in that, recycling filler used described in the step 5) is NI-IDA, 30~60 min of incubation time of the recycling,
Recycling equilibration buffer used is 1 × PBS.
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