CN104313028A

CN104313028A - Small peptide gene for resisting rheumatoid arthritis and production method of small peptide gene

Info

Publication number: CN104313028A
Application number: CN201410521516.0A
Authority: CN
Inventors: 李相鲁; 张严冬; 孟建军; 梁庄严
Original assignee: BEIJING SHENGKE DONGFANG TECHNOLOGY Co Ltd
Current assignee: BEIJING SHENGKE DONGFANG TECHNOLOGY Co Ltd
Priority date: 2014-09-30
Filing date: 2014-09-30
Publication date: 2015-01-28

Abstract

The invention discloses a small peptide gene for resisting rheumatoid arthritis, and the nucleotide sequence is indicated as SEQIDNO.1. The derivative M-DNAJP1 of the DNAJP1 is produced by utilizing a gene recombination method, the gene of the dnaJP1 is artificially established, and M-dnajp1-M-dnajp1-M-dnajp1-M-dnajp1-M-dnajp1-M-dnajp1 is formed in a series connection manner by virtue of a method for connecting in an end-to-end manner and adding methionine, cloned into plasmid pet31b(+), then converted to enter colibacillus to be expressed to obtain the DNAJP1 modified polypeptide, and the repeated sequence of the M-DNAJP1 is indicated as SEQIDNO.2. The small peptide is prepared in a fermenting manner, the free polypeptide is obtained by utilizing a cyanogen bromide dissociation method, the dissociated cyanogen bromide is processed at a high temperature and high pressure to generate ammonia water and sodium formate, the environmental pollution is reduced, the cost is low in the entire process, the yield is high, simplicity in purification is realized, and the industrialization is facilitated.

Description

The arthritic little peptide gene of resisting rheumatoid disease and production method thereof

Technical field

The present invention relates to the restructuring fermentative production of the arthritic little peptide of a kind of resisting rheumatoid disease, and purification process.

Background technology

Rheumatoid arthritis be a kind of chronic recurrent exerbation change into main painful diseases with general joint inflammation, English name Rheumatoid arthritis, writes a Chinese character in simplified form RA, is a kind of common disease, frequently-occurring disease.Disease time can continue a few days, a few weeks or months, and with reactivity in various degree, severe patient patient can't take care of oneself, often involve throughout one's life, form chronic pain, greatly endanger the life and health of the mankind, people expect research and develop and produce the medicine of more high-efficiency high-qualities in clinical application.

The RA medicine of present application has antiphlogiston, as the medicine of acetylsalicylic acid, reflunomide and the symptom that palliates a disease by suppressing or eliminate organism immune response, though these medicines can improve symptom, but the basic pathological changes of rheumatism can not be removed, but also there is the harm weakening the resistance against diseases of body.

DNAJP1 is a new drug polypeptide developed by Computer-aided Design Technology, and it originates from heat shock protein(HSP) dnaJ., and the basis of this albumen is obtained by synthesis method.Heat shock protein white corpuscle is when standing such as inflammation, autoimmune disorder as produced heat shock protein(HSP) when rheumatoid arthritis.The mechanism of action of dnaJP1 is to reduce inflammation by the function of regulatory T-cell, strengthen regulation and control immunity system, the inflammatory reaction caused by rheumatoid arthritis is suppressed, the routine phase II clinical trials of the U.S. 160 shows, this polypeptide oral can reduce inflammatory reaction, achieves good effect.

The arthritic little peptide dnaJP1 of resisting rheumatoid disease, compared with traditional treatment rheumatoid arthritis thing, has unrivaled advantage.This medicine can not produce the immunity system of patient and suppress, and by oral application dnaJP1, because the mucomembranous immune system in intestines can be tamed again to the immunity system of patient, thus effectively improves immunity system.Pass through simulation reaction; patients immune system is helped again to identify Self substances and exogenous antigen; the focus that protection was damaged originally is deeply recovered; symptom is eased; therapeutic action is played by making the immune system tolerates dnaJ of patient; have that security is high, effect is directly absorbed by the body rapidly, easily, specificity is high, the time length is long, can be oral etc. advantage, there are very tempting bright prospects.

The purification process of the arthritic little peptide dnaJP1 of existing resisting rheumatoid disease is chemical synthesis.U.S. patent Nos US5773570A, this technology utilizes the phagemid of polymerization chain reaction (PCR) working and restructuring to comprise in dnaK and dnaJ the fragment increased containing dnaJ gene, then this fragment is subcloned into the Xba1 site of PCG808fX, express as maltose-binding fusion albumen, then with containing the rabbit anti-serum for this fusion rotein, the rdnaJ carrying out expressing with pUC19 plasmid is carried out purifying, finally, use immobilized albumin A, IgG-MBL-DnaJ is carried out purifying, then wash-out is carried out out with the glycine buffer of pH2.5, SDS electrophoresis result shows to only have a band, fusion rotein meets expection, molecular weight is at 41KD.This patent have employed fusion rotein and desired polypeptides merges the method for producing, the molecular weight of fusion tag itself is 40KD, and the molecular weight of desired polypeptides only has 1.76KD, differ more than 20 times, even if that is produce a large amount of fusion roteins, output very low (accounting for 4% of total protein content) in fact containing target protein, causes unnecessary waste, is unfavorable for scale operation.That he is purified in addition is fusion rotein (41KD), instead of real dnaJP1 polypeptide (1.76KD).

Summary of the invention

The technical problem to be solved in the present invention is: the small peptide that the arthritic little peptide dnaJP1 of resisting rheumatoid disease is made up of several amino acid, and the expression of small peptide directly in Host Strains will be realized and separate very difficult, and take amalgamation and expression mode also because adopting larger fusion tag and lower anti-inflammation gene copy to cause expression amount very low.The present invention builds the gene with suitable high copy number, increase yield is carried out by its gene being carried out 6 methods repeating to connect, and adopt less fusion tag to carry out this gene of amalgamation and expression to be increased in relative proportion in expression product, to the high expression realizing the little peptide of resisting rheumatoid disease sacroiliitis, relative proportion is about 50%, because the fusion rotein generated is inclusion body, so be very easy to purifying, inclusion body protein after purifying, adopt bromize fluoride crack, specificity is strong, cost is low, the present invention adopts the method for decomposition under high pressure to remove cyanogen bromide, cost is low, pollute little, simple to operate, be easy to industrialization.

Technical scheme of the present invention is: the arthritic little peptide gene of a kind of resisting rheumatoid disease, its nucleotide sequence is as shown in SEQ ID NO:1.

The arthritic little peptide gene of described resisting rheumatoid disease, the polypeptide coded by nucleotide sequence is as shown in SEQ ID NO:2.

The polypeptide series production method of the arthritic little peptide gene of described expression resisting rheumatoid disease, utilize colibacillary pET31b (+) expression system, SEQ ID NO:1 gene order is subcloned into after the gene order of plasmid pET31b (+) fusion tag KSI, restriction enzyme is AwnI, then being transformed in e. coli bl21 (DE3) plysS of being obtained by subclone is expressed, and carry out fermentative production, the thalline obtained obtains inclusion body by cryogenic high pressure cracking, inclusion body after washing utilizes cyanogen bromide (CNBr) or L-Methionine γ-lyase to carry out being cracked into single polypeptide, buck quencher reaction is added after cracking, adopt high-temperature high-pressure hydrolysis, the impurity such as removing cyanogen bromide, by nanofiltration desalination, obtain sterling.

The polypeptide series production method of the arthritic little peptide gene of described expression resisting rheumatoid disease, the condition of described high-temperature high-pressure hydrolysis is, 103.4kPa, 1.05kg/cm2, under the condition of temperature 121.3 ° of C, process 15 ~ 20 minutes.

The invention has the beneficial effects as follows: the present invention utilizes the method for gene recombination to produce the derivative M-DNAJP1 of DNAJP1, the gene of artificial constructed dnaJP1, and carry out series connection formation as under type by the middle method of adding methionine(Met) that joins end to end: M-dnajp1-M-dnajp1-M-dnajp1-M-dnajp1-M-dnajp1-M-dnajp1, be cloned in plasmid pet31b (+), be then transformed in intestinal bacteria and carry out expressing the polypeptide after obtaining DNAJP1 modification: the tumor-necrosis factor glycoproteins of M-DNAJP1 is as shown in SEQ ID NO:2.By fermentation for this little peptide, then utilize the method for bromize fluoride crack to obtain free polypeptide, the cyanogen bromide after cracking passes through high temperature high pressure process, generate ammoniacal liquor and sodium formiate, reduce the pollution to environment, whole process cost is low, output is high, and purifying is simple, is beneficial to industrialization.

The present invention utilizes His label fragment amalgamation and expression of series 6 little peptides of object less on pet-31 (b+), add the ratio (~ 50%) in the recombinant protein expressed shared by desired polypeptides, form fusion rotein inclusion body, to reduce the cytotoxicity of expressing protein, considerably increase expression efficiency and the separating difficulty reducing little peptide.

Accompanying drawing explanation

Fig. 1 is 5 liters of pilot plant test results during engineering bacterium fermentation is expressed;

Fig. 2 is growth curve in pilot scale 50 liters of fermenting processs;

Fig. 3 is fermentation SDS-PAGE;

Fig. 4 is the sample electrophoresis figure of thalline after cryogenic high pressure fragmentation;

Fig. 5 is inclusion body washing after product electrophorogram;

Fig. 6 is little peptide bromize fluoride crack electrophorogram;

Fig. 7 is little peptide MALDI-TOF/TOF mass spectrum On-line measurement figure.

Embodiment

The structure of engineering bacteria:

Content comprises according to codon-bias implementation sequence, synthesizes complete genome sequence, utilizes Alwn I digested plasmid, import aim sequence, construct be expression vector with pet-31 (b+), e. coli bl21 (DE3) pLysS is the MDNAJP1 amalgamation and expression system of Host Strains.The aminoacid sequence of 6 MDNAJP1 repeated, according to e. coli codon preferences.Determine that gene order is as shown in SEQ ID NO:1.

Gene order is synthesized by this laboratory oneself.Cut by plasmid AlwnI restriction endonuclease, goal gene fragment is carried out subclone, is connected in plasmid and goes, be then transformed in intestinal bacteria, Host Strains and expression vector are all purchased from Novagen company.After engineering bacteria has built, cut qualification, the qualification of object fragment DNA sequencing through expression plasmid enzyme, it is correct that result shows that engineering bacteria builds.

Engineering bacterium fermentation is expressed

After constructing engineering strain, gene recombined escherichia coli BL21 (DE3) pLysS streak inoculation, in containing on the LB solid plate of penbritin (80ug/ml), is first carried out lab scale expression: shake-flask culture, has then been carried out 5 liters of fermentor cultivation.To grope seed culture medium, inoculum density.

5 liters of pilot plant test results as shown in Figure 1.From left to right point sample order be: secondary kind, 0hr, 3 hr, 5 hr(induce 0hr), MARK, 8 hr(induce 3hr), 11 hr(induce 6hr), 17 hr(induce 12hr), 20 h(induce 15hr) finally determine that processing step is as follows:

One-level, secondary seed medium are LB, and fermention medium is TB(revision), before inoculation, Amp concentration is 100 μ g/mL

This project carbon source is glycerine, because it does carbon source produce less impurity protein.

Pilot scale 50 liters of fermenting processs:

One fermenting process

(1) by gene recombined escherichia coli BL21 (DE3) pLysS streak inoculation in containing on the LB solid plate of penbritin (80ug/ml), put 37 DEG C of thermostat containers and be inverted and cultivate 12h;

(2) e. coli bl21 (DE3) pLysS on picking flat board

Be inoculated into 20ml containing in the LB substratum of penbritin (80ug/ml), at 37 DEG C, 200rpm.min-1 shaking culture 8h is to logarithmic phase, above-mentioned nutrient solution 20ml is inoculated in 200ml containing in the LB substratum of penbritin (80ug/ml), at 37 DEG C, 220rpm.min-1 shaking culture 4h, obtains fermentation seed liquid;

(3) after ullage of fermenting disappears, correct pH electrode, dissolved oxygen electrode, pour the TB substratum dissolved into, carry out sterilizing (121 DEG C, 30min) by fermentor tank disinfecting action, when feed temperature is down to 37 DEG C, add ready penbritin (80ug/ml) and glucose (3%), tap into seed liquor in 10% ratio, control temperature 37 DEG C, dissolved oxygen more than 30%, regulate pH7.0 with ammoniacal liquor, stir speed (S.S.) and air flow regulate and are determined by oxyty.Add inductor IPTG when OD600 value is 15 reach 0.2mM when strain density in fermented liquid reaches, continue to cultivate about 10-16h, final OD about 42.

Table 1 ferments record

Fermentation results

1. consume feed supplement 2000mL, (feed rate 1/ minute at the beginning, afterwards furnishing 2/ minute, mean flow rate, 3 ml/min) obtain thalline weight in wet base 1000g;

2. growth curve as shown in Figure 2, and fermentation SDS-PAGE as shown in Figure 3;

3, the purifying process of target protein, research contents comprises the purifying of the preparation of fusion rotein, cyanogen bromide cleavage of fusion proteins, little peptide cleavage mixture.Purifying process of the present invention simplifies flow process, saves time, reagent consumptive material, is easy to amplify.Concrete steps are as follows: (1) fermentation ends, collected by centrifugation thalline, after distilled water wash 2 ~ 3 times, add the buffer A re-suspended cell of fermentating liquid volume 1/10th.Utilize cryogenic high pressure cytoclasis cell to not thickness under condition of ice bath, centrifugally abandon supernatant, precipitation inclusion body washings washs four times and obtains inclusion body protein.

Table 2 experimental procedure

Experimental procedure	Experimental period	Duration	Correlation parameter	Weigh
					1, first time high-pressure homogenization	15:00~16:10	70min	Pressure: 680 ~ 720 Bar	——
2, second time high-pressure homogenization	16:50~17:40	50min	Pressure: 680 ~ 750 Bar	——
					3, broken liquid is centrifugal	18:50~19:40	50min	——	2.170 kg
4, first time washs inclusion body	9:50~10:40	50min	20L inclusion body washings	1.906 kg
					5, second time washs inclusion body	12:00~13:40	100min	15L inclusion body washings	1.270 kg
6, third time washs inclusion body	14:40~16：20	100min	15L pure water	1.085 kg
					7, the 4th washing inclusion body	17:00~17~50	50min	15L pure water	1.065 kg

The fermented liquid produced after fermentation, centrifugal, collect thalline, after distilled water wash 2 ~ 3 times, the resuspended thalline of buffer A of thalline fermentating liquid volume 1/10th.Utilize cryogenic high pressure cytoclasis cell to not thickness under condition of ice bath, centrifugally abandon supernatant, precipitation inclusion body washings washs once, centrifugal (5000 revs/min, 15min), by thalline broken wall, washing, each sample that the step such as centrifugal obtains carries out electrophoretic analysis, result as shown in Figure 4, washed for inclusion body washings inclusion body sample is carried out again 3 washings, centrifugal (5000 revs/min, 15min), removing cell walls, the impurity such as nucleic acid, the sample obtained carries out electrophoretic analysis, and result as shown in Figure 5.

(2) after being weighed by inclusion body protein, add fusion rotein lysate (8M urea) by the mass volume ratio of 1:10, be placed in 4 DEG C, refrigerator and spend the night dissolving inclusion body protein, centrifugal precipitation of abandoning retains supernatant, obtains solubilization of inclusion bodies liquid.Take appropriate cyanogen bromide to be dissolved in acetonitrile (200mg/ml) and to be configured to cyanogen bromide working fluid.Get equal-volume cyanogen bromide working fluid and add above solubilization of inclusion bodies liquid, sealing, lucifuge is in shaking table cracking 24h.In ventilating kitchen, add excessive buck after cracking in scission reaction liquid stop cyanogen bromide reaction, then remove cyanogen bromide with high pressure-temperature cracking, to be cooled to room temperature, high speed centrifugation, collects lysate supernatant, 4 DEG C of preservations.

(3) get little peptide lysate sample and carry out Tricine – SDS-PAGE electrophoretic analysis, do high performance liquid chromatography and MALDI-TOF/TOF mass spectrum On-line measurement simultaneously, the spectrogram obtained after Mass Spectrometric Identification, bioinformatics software is utilized to carry out marking and extracting the specific charge data of peptide section, by retrieval albumen database, identifying lysate Small Peptides is M-DNAJP1.Bromize fluoride crack thing electrophoresis carries out electrophoretic analysis, and electrophorogram as shown in Figure 6.

Claims

1. the arthritic little peptide gene of resisting rheumatoid disease, its nucleotide sequence is as shown in SEQ ID NO:1.

2. the arthritic little peptide gene of resisting rheumatoid disease as claimed in claim 1, the polypeptide coded by nucleotide sequence is as shown in SEQ ID NO:2.

3. polypeptide series production method of expressing the arthritic little peptide gene of resisting rheumatoid disease as claimed in claim 2, it is characterized in that: utilize colibacillary pET31b (+) expression system, SEQ ID NO:1 gene order is subcloned into after the gene order of plasmid pET31b (+) fusion tag KSI, restriction enzyme is AwnI, then being transformed in e. coli bl21 (DE3) plysS of being obtained by subclone is expressed, and carry out fermentative production, the thalline obtained obtains inclusion body by cryogenic high pressure cracking, inclusion body after washing utilizes cyanogen bromide (CNBr) or L-Methionine γ-lyase to carry out being cracked into single polypeptide, buck quencher reaction is added after cracking, adopt high-temperature high-pressure hydrolysis, the impurity such as removing cyanogen bromide, by nanofiltration desalination, obtain sterling.

4. polypeptide series production method of expressing the arthritic little peptide gene of resisting rheumatoid disease as claimed in claim 3, is characterized in that: the condition of described high-temperature high-pressure hydrolysis is, 103.4kPa, 1.05kg/cm ², under the condition of temperature 121.3 ° of C, process 15 ~ 20 minutes.