CN103088051B - Method for preparing thymic peptide alpha 1 - Google Patents
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- CN103088051B CN103088051B CN201310058144.8A CN201310058144A CN103088051B CN 103088051 B CN103088051 B CN 103088051B CN 201310058144 A CN201310058144 A CN 201310058144A CN 103088051 B CN103088051 B CN 103088051B
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Abstract
The invention discloses a method for preparing thymic peptide alpha 1. The method provided by the invention sequentially comprises the following steps of: (1) culturing a recombinant expression vector containing a thymic peptide alpha 1 gene and guiding host bacteria to recombinant bacteria, and collecting bacteria and performing ultrasonication; (2) heating to 75 DEG C and standing for 30 minutes and collecting supernate; (3) performing ultrafiltration with a filter membrane with the cut-off molecular weight of 10000 Dalton, collecting the filtrate, namely the thymic peptide alpha 1 liquor; treating at 75 DEG C for 30 minutes to degenerate and centrifugally remove most mycoproteins; and then using the filter membrane with the cut-off molecular weight of 10000 Dalton to further remove impurities so as to obtain the high purity and high activity final product which is equivalent to thymic peptide alpha 1 (zadaxin) chemically synthesized in biological activity. Compared with a conventional synthetic method or chromatographic purification method, the method for preparing the thymic peptide alpha 1 provided by the invention is simple to operate, and the cost is greatly lowered, so that the method is very suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method of preparing Thymosin alpha 1.
Background technology
The people such as nineteen sixty-five Goldestein have been separated to bioactive polypeptide quasi-molecule, called after Zadaxin (Thymosin) from animal thymus.Goldestein in 1972 etc. are further purified the component of obtained main molecules amount at 1-15KD, are referred to as Zadaxin component 5 (Thymosin, TF5), for zooscopy and clinical observation, find that this component has compensation thymus function lowly to act on.
Thymosin alpha 1 (Thymosin α 1, T α 1) be one of the strongest active polypeptide in Zadaxin component 5 (TF5) polypeptide mixture, there is the function that promotes the differentiation of T-lymphocyte, maturation and strengthen cellular immunization, its peptide sintetics is applied at present clinically abroad, good effect has been studied and obtained in the treatment of tumour, hepatitis B, the third liver and immune deficiency etc.The Acid polypeptide that T α 1 is made up of 28 amino-acid residues, iso-electric point 4.2, relative molecular weight 3108.
Main by tissue extraction method and chemical synthesis acquisition T α 1 at present.Tissue extraction method is subject to the restriction of material source, and in product, active constituent content is low conventionally, thereby affects result for the treatment of.T α 1 purity that chemical synthesis obtains is high, but cost is high, expensive, has limited the clinical application of T α 1.Have researchist to attempt preparing T α 1 by genetic engineering technique, but the recombinant protein obtaining need to carry out protease treatment, and purification process adopt the method such as affinity chromatography or ion-exchange, so production cost is still high.
Summary of the invention
The object of this invention is to provide a kind of method of preparing Thymosin alpha 1.
The method of preparing Thymosin alpha 1 provided by the invention, in turn includes the following steps:
(1) after cultivation recombinant bacterium, collect thalline and carry out ultrasonication; Described recombinant bacterium is that the recombinant expression vector that contains Thymosin alpha 1 gene is imported to the recombinant bacterium that Host Strains obtains;
(2) be warming up to 75 DEG C and standing 30 minutes, collect supernatant liquor;
(3) be that 10000 daltonian filter membranes carry out ultrafiltration with molecular weight cut-off, collect filtrate, be Thymosin alpha 1 solution;
Described Thymosin alpha 1 is for being as follows following (a) or (b): the protein (a) being made up of the aminoacid sequence shown in sequence in sequence table 1; (b) by the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and there is the protein being derived by sequence 1 of Zadaxin function.
Described Thymosin alpha 1 gene can be following 1) or 2) or 3) or 4) or 5) or 6) DNA molecular:
1) coding region is if sequence in sequence table 2 is from the DNA molecular as shown in the 7th to 90 Nucleotide of 5 ' end;
2) in sequence table sequence 2 from the DNA molecular shown in the 7th to 91 Nucleotide of 5 ' end;
3) in sequence table sequence 2 from the DNA molecular shown in the 4th to 90 Nucleotide of 5 ' end;
4) in sequence table sequence 2 from the DNA molecular shown in the 4th to 93 Nucleotide of 5 ' end;
5) under stringent condition with 1) to 4) and in the DNA sequence dna hybridization of arbitrary restriction and coding there is the DNA molecular of the albumen of Zadaxin function;
6) with 1) to 4) in the DNA sequence dna of arbitrary restriction there is 90% above homology and coding and have the DNA molecular of the albumen of Zadaxin function.
Above-mentioned stringent condition can be at 6 × SSC, in the solution of 0.5%SDS, under 65oC, hybridizes, and then uses 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively to wash film once.
Described " recombinant expression vector that contains Thymosin alpha 1 gene " can be described Thymosin alpha 1 gene is inserted to pET15a(+) recombinant plasmid that obtains of the multiple clone site (between NdeI and HindIII restriction enzyme site) of carrier.
Described Host Strains specifically can be e. coli bl21 (DE3).
In described step (1), the method for described " cultivation recombinant bacterium " is: will in described recombinant bacterium liquid medium within, be cultured to bacterium liquid OD
600nm=1, add inductor inducing culture 12 hours; Described inductor is isopropyl-β-D-thiogalactoside(IPTG).The starting point concentration of described inductor specifically can be 0.15mM.Described liquid nutrient medium specifically can be LB substratum.Adding before described inductor, the culture condition of recombinant bacterium specifically can be: 25 DEG C, 200 revs/min (rotation radius is 13mm) shaking culture.The culture condition of described inducing culture specifically can be: 25 DEG C, 200 revs/min (rotation radius is 13mm) shaking culture.
In described step (1), the parameter of described ultrasonication specifically can be: adopt Φ 10 probes to process 30 minutes, ultrasonic 7 seconds 5 seconds, intervals.
In described step (2), the implementation method of described " collection supernatant liquor " specifically can be centrifugal.Described centrifugal parameter specifically can be: centrifugal 10 minutes of 4 DEG C, 10000g.
The Thymosin alpha 1 that arbitrary described method prepares above also belongs to protection scope of the present invention.
In the present invention, Thymosin alpha 1 gene is imported to pET15a(+) carrier, then in e. coli bl21 (DE3), carry out abduction delivering, then ultrasonication collect whole bacterial protein, 75 DEG C of whole bacterial proteins are processed after 30 minutes to centrifugal collection supernatant liquor again with 10000 daltonian membrane filtrations and collect filtrate, be the solution that contains Thymosin alpha 1.Processing for 75 DEG C 30 minutes can be by most tropina sex change centrifugal removal, with obtaining high purity, highly active finished product after the further impurity elimination of 10000 daltonian filter membrane, suitable with Thymosin alpha 1 (Zadaxin) biologic activity of chemosynthesis again.Compared with traditional chemical synthesis or chromatography purification method, adopt method provided by the invention to prepare Thymosin alpha 1, simple to operate, cost significantly reduces, and is applicable to very much industrial production.
Brief description of the drawings
Fig. 1 is the body weight increase ratio of each group piglet every day.
Fig. 2 is the expression level of each group of piglet IL-4.
Fig. 3 is the expression level of each group of young porcine IFN γ.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
Thymosin alpha 1 is as shown in the sequence 1 of sequence table.The open reading frame of Thymosin alpha 1 gene is if the sequence 2 of sequence table is from as shown in 5 ' end 7-93 position Nucleotide.
Zadaxin (a kind of Thymosin alpha 1 disinfection dry powder preparation refining, chemosynthesis): purchased from Saisheng Pharmaceutical Co.,Ltd. of the U.S., catalog number: H20100767.
PET15a(+) carrier: purchased from Novagen company of the U.S., catalog number: 69740-3.
E. coli bl21 (DE3): purchased from China Veterinery Drug Inspection Office.
Sheep red blood cell (SRBC) (product specification: 100ml/ bottle): purchased from Bai Ji bio tech ltd, Zhengzhou.
PBS damping fluid (pH8.0): solvent is water, contains 137mmol/L NaCl, 2.7mmol/L KCl, 4.3mmol/L Na
2hPO
4with 1.4mmol/L KH
2pO
4.
Embodiment 1, prepare Thymosin alpha 1
One, construction recombination plasmid
1, the double chain DNA molecule shown in the sequence 2 of composition sequence table.
2, the double chain DNA molecule obtaining by restriction enzyme NdeI and HindIII double digestion step 1, reclaims enzyme and cuts product.
3, with restriction enzyme NdeI and HindIII double digestion pET21a(+) carrier, reclaims the carrier framework of about 5443bp.
4, the carrier framework of the enzyme of step 2 being cut to product and step 3 is connected, and obtains recombinant plasmid pET21-T α 1.According to sequencing result, recombinant plasmid pET21-T α 1 is carried out to structrual description as follows: at pET21a(+) insert in sequence table sequence 2 between the NdeI of carrier and HindIII restriction enzyme site from the DNA molecular shown in the 7th to 91 Nucleotide of 5 ' end.
Two, prepare Thymosin alpha 1
1, recombinant plasmid pET15-T α 1 is imported to e. coli bl21 (DE3), obtain recombinant bacterium.
2, recombinant bacterium step 1 being obtained is seeded to LB substratum, and 25 DEG C, 200 revs/min (rotation radius is 13mm) shaking culture are to bacterium liquid OD
600nm=1; Add isopropyl-β-D-thiogalactoside(IPTG) (making its concentration in system is 0.15mM), 25 DEG C, 200 revs/min (rotation radius is 13mm) shaking culture 12 hours; Centrifugal 10 minutes of 5000g, collects thalline.
Three, the expression of purifying Thymosin alpha 1
1, the thalline obtaining with PBS damping fluid washing step two resuspended thalline, and ultrasonication 30 minutes (producer of Ultrasonic Cell Disruptor is: Nanjing Shun Ma plant and instrument company limited, and model is GL-400SD; Ultrasonication adopts Φ 10 to pop one's head in, ultrasonic 7 seconds 5 seconds, intervals).
2, liquid-phase system step 1 being obtained is warming up to rapidly 75 DEG C, leaves standstill 30 minutes, and centrifugal 10 minutes of then 4 DEG C, 10000g are collected supernatant liquor.
3, supernatant molecular weight cut-off step 2 being obtained is that 10000 daltonian filter membranes carry out ultrafiltration, collects filtrate, is the Thymosin alpha 1 solution that purifying obtains.The protein concentration of Thymosin alpha 1 solution is 5 mg/ml.
4, Thymosin alpha 1 solution step 3 being obtained carries out SDS-PAGE electrophoresis.Result shows, a protein band appears in Thymosin alpha 1 solution in 3kD vicinity.
Four, control treatment first
1, with 1 of step 3.
2, liquid-phase system step 1 being obtained is warming up to rapidly 70 DEG C, leaves standstill 30 minutes, and centrifugal 10 minutes of then 4 DEG C, 10000g are collected supernatant liquor.
3, supernatant molecular weight cut-off step 2 being obtained is that 10000 daltonian filter membranes carry out ultrafiltration, collects filtrate, is contrast solution first.The protein concentration of contrast solution first is 8 mg/ml.
Five, control treatment second
1, with 1 of step 3.
2, liquid-phase system step 1 being obtained is warming up to rapidly 80 DEG C, leaves standstill 30 minutes, and centrifugal 10 minutes of then 4 DEG C, 10000g are collected supernatant liquor.
3, supernatant molecular weight cut-off step 2 being obtained is that 10000 daltonian filter membranes carry out ultrafiltration, collects filtrate, is contrast solution second.The protein concentration of contrast solution second is 2 mg/ml.
Embodiment 2, Thymosin alpha 1 Determination of biological activity (cell experiment)
The biologic activity (Thymosin alpha 1 can activate the de-E thymocyte in fresh pig thymus gland, makes it and sheep cell form rosette) of Thymosin alpha 1 prepared by employing Rose connection mensuration embodiment 1.
1, gather fresh pig thymus gland, prepare lymphocyte suspension with PBS damping fluid.
2,45 DEG C of temperature of the lymphocyte suspension that step 1 obtained are bathed 1h(every jolting in 5 minutes once), with PBS damping fluid washed cell and to adjust cell concn be (3-5) × 10
6individual/ml, is placed in testing tube, every pipe 0.2ml.
3, prepare various solution
(1) the Thymosin alpha 1 solution obtaining with PBS damping fluid gradient dilution embodiment 1, obtains the diluent (concentration of Thymosin alpha 1 is in the total protein concentration of solution) of each Thymosin alpha 1 concentration.
(2) dissolve and gradient dilution Zadaxin with PBS damping fluid, obtain the Zadaxin solution of each concentration.
(3) the contrast solution first obtaining with PBS damping fluid gradient dilution embodiment 1, obtains the diluent of each protein concentration.
(4) the contrast solution second obtaining with PBS damping fluid gradient dilution embodiment 1, obtains the diluent of each protein concentration.
4, packet transaction
Experimental group-1: the diluent that adds the Thymosin alpha 1 solution that 0.lml step 3 obtains in the each testing tube obtaining in step 2;
Experimental group-2: the Zadaxin solution that adds 0.lml step 3 to obtain in the each testing tube obtaining in step 2;
Experimental group-3: the diluent that adds the contrast solution first that 0.lml step 3 obtains in the each testing tube obtaining in step 2;
Experimental group-4: the diluent that adds the contrast solution second that 0.lml step 3 obtains in the each testing tube obtaining in step 2;
Control group: add 0.lml sterilized water in the each testing tube obtaining in step 2.
5, with PBS damping fluid suspension sheep red blood cell (SRBC), obtain cell concn for (3-5) × 10
7mianyang red cell suspension of individual/ml.
6, Mianyang red cell suspension that adds 0.2ml step 5 to obtain in the each testing tube obtaining in step 4, shakes up, centrifugal 2 minutes of 600g, and 4 DEG C of placements are spent the night.
7, after completing steps 6, discard the supernatant liquor in testing tube, every pipe adds a stationary liquid, shakes up gently and leaves standstill 10 minutes, and then every pipe adds 2 staining fluids, shakes up and leave standstill 15 minutes.
The preparation method of stationary liquid: 1 parts by volume 25% glutaraldehyde solution, 1 parts by volume 3.5% sodium hydrogen carbonate solution and 38 parts by volume Hank ' s liquid are mixed.
The preparation method of staining fluid: 2ml Ji's nurse Sa staining fluid stoste and 6ml are added to Hanks liquid and mix and shake up, 1500 revs/min centrifugal 10 minutes, get supernatant liquor.
The preparation method of Ji's nurse Sa staining fluid stoste: get Giemsa 0.5g, glycerol adding 33ml, 55-60 DEG C is heated to Giemsa dissolving, is chilled to room temperature, adds 33ml methyl alcohol, and room temperature is placed after 24 hours and is used filter paper filtering, gets filtrate, sealing room temperature preservation.
8, after completing steps 7, count under the microscope, all lymphocytic numbers (being no less than 200) on 16 grids in counting field of microscope, statistics E rosette wherein forms number (in conjunction with the thymocyte of more than 3 sheep red blood cell (SRBC)), calculates E rosette and forms percentage.
Carry out repeating for three times experiment, result is got the mean value that repeats experiment for three times.
Biological activity=experimental group E rosette of Thymosin alpha 1 forms percentage-control group E rosette percentage.
Measurement result is in table 1.
The biological activity of table 1 Thymosin alpha 1
Result shows: Thymosin alpha 1 solution prepared by embodiment 1 can significantly improve E rosette rate of formation, and the synthetic Thymosin alpha 1 (Zadaxin) of its action effect and import is suitable; Contrast solution first due to adopt be 70 DEG C leave standstill 30 minutes, farthest do not impel foreign protein sex change, thereby foreign protein content increases in filtrate, target protein purity drop, while measuring the protein content in diluent with total protein concentration, the biological activity of total protein reduces; Contrast solution second due to adopt 80 DEG C leave standstill 30 minutes, also inactivation of part target protein when foreign protein inactivation, thus in filtrate, the content of target protein reduces greatly, the output capacity of target protein greatly reduces.
Embodiment 3, Thymosin alpha 1 Determination of biological activity (experimentation on animals)
Experimental animal: 8 week age sodium selenite, 15.
The kind of piglet is " Du × long × large ", purchased from Daxing, Beijing boar Zhong Qin factory.
Laboratory animal is divided into three groups, 5 every group, is handled as follows respectively:
Experimental group-1: Thymosin alpha 1 solution prepared by intramuscular injection embodiment 1 (adjusting total protein concentration with physiological saline), every pig injection 1ml(is containing 1mg total protein);
Experimental group-2: reach first solution (preparing with physiological saline) intramuscular injection day, every pig injection 1ml(is containing 1mg Thymosin alpha 1);
Experimental group-3: contrast solution first prepared by intramuscular injection embodiment 1 (adjusting total protein concentration with physiological saline), every pig injection 1ml(is containing 1mg total protein);
Experimental group-4: contrast solution second prepared by intramuscular injection embodiment 1 (adjusting total protein concentration with physiological saline), every pig injection 1ml(is containing 1mg total protein);
Control group: intramuscular injection physiological saline, every pig injection 1ml.
Injecting the day before yesterday is the 0th day, and injecting the same day is the 1st day, is followed successively by afterwards the 2nd day, the 3rd day etc.
Weigh piglet body weight every day, taking the mean body weight of the 0th day as 100%, the body weight of calculating each group piglet every day increases ratio, the results are shown in Figure 1.The equal sustainable growth of piglet body weight of experimental group-1 and experimental group-2, rate of growth is suitable, a little more than control group.
Respectively at the 0th day, the 7th day and the 14th day collection peripheral blood, measure the expression level of IFN-γ and IL-4 with cytokine test kit Porcine IFN-γ (PIF00) and IL-4 ELISA Kit (DY654) (RD company product).The results are shown in Figure 2 and Fig. 3, * P<0.05, * * P<0.01, * * * P<0.001.IFN-γ and the IL-4 of experimental group-1, experimental group-2 and experimental group-4 are suitable, are significantly higher than experimental group-3, are more significantly higher than control group.
Claims (3)
1. a method of preparing Thymosin alpha 1, in turn includes the following steps:
(1) after cultivation recombinant bacterium, collect thalline and carry out ultrasonication; Described recombinant bacterium is that the recombinant expression vector that contains Thymosin alpha 1 gene is imported to the recombinant bacterium that Host Strains obtains;
(2) be warming up to 75 DEG C and standing 30 minutes, collect supernatant liquor;
(3) be that 10000 daltonian filter membranes carry out ultrafiltration with molecular weight cut-off, collect filtrate, be Thymosin alpha 1 solution;
Described Thymosin alpha 1 is the protein being made up of the aminoacid sequence shown in sequence in sequence table 1;
The recombinant plasmid that described " recombinant expression vector that contains Thymosin alpha 1 gene " obtains for described Thymosin alpha 1 gene being inserted to the multiple clone site of pET21a (+) carrier;
Described Host Strains is e. coli bl21 (DE3).
2. the method for claim 1, is characterized in that: described Thymosin alpha 1 gene be in sequence table sequence 2 from the DNA molecular shown in the 7th to 91 Nucleotide of 5 ' end.
3. the method for claim 1, is characterized in that: the method for described " cultivation recombinant bacterium " is: will in described recombinant bacterium liquid medium within, be cultured to bacterium liquid OD
600nm=1, add inductor inducing culture 12 hours; Described inductor is isopropyl-β-D-thiogalactoside(IPTG).
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Non-Patent Citations (8)
| Title |
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| 参见对比文件1摘要部分. * |
| 参见对比文件1第3页第5段第2-3行 * |
| 参见对比文件第13页第2段 * |
| 吴盛等.重组人胸腺肽α1 在大肠杆菌中的表达纯化和鉴定.《南京大学学报(自然科学版)》.2004,第40卷(第1期),第49-57页. * |
| 曹颖瑛等.胸腺素α1 的基因合成、表达及活性测定.《中国生物制品学杂志》.2003,第16卷(第1期),第13-15页. |
| 管占利等.重组人胸腺素Tα1工艺的研究.《中国优秀硕士学位论文全文数据库》.2012,参见对比文件1第12页第1段 |
| 胸腺素α1 的基因合成、表达及活性测定;曹颖瑛等;《中国生物制品学杂志》;20030228;第16卷(第1期);第13-15页 * |
| 重组人胸腺素Tα1工艺的研究;管占利等;《中国优秀硕士学位论文全文数据库》;20120131;参见对比文件1第12页第1段；参见对比文件1第3页第5段第2-3行；参见对比文件第13页第2段；参见对比文件1摘要部分 * |
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Inventor after: Liu Wenjun Inventor after: Li Jing Inventor after: Dong Xiukai Inventor after: Yang Limin Inventor after: Lian Weijiang Inventor before: Fu Xianhua Inventor before: Liu Tingjian Inventor before: Cen Junyu |
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Free format text: CORRECT: ADDRESS; FROM: 100220 CHANGPING, BEIJING TO: 116450 DALIAN, LIAONING PROVINCE Free format text: CORRECT: INVENTOR; FROM: FU XIANHUA LIU TINGJIAN CEN JUNYU TO: LIU WENJUN LI JING DONG XIUKAI YANG LIMIN LIAN WEIJIANG |
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Effective date of registration: 20141115 Address after: 116450 standard factory building, No. three, hatchery Park, Dalian garden economy zone, Liaoning, Wanshou Road province A01 Patentee after: DALIAN JINXIU BIOTECHNOLOGY ENGINEERING CO., LTD. Patentee after: Institute of Microbiology, Chinese Academy of Sciences Address before: 100220 Beijing city Changping District small town crown court centre 10-6-601 Patentee before: Beijing Newpep Biological Technology Co., Ltd. |