CN100427597C - Prokaryotic expression engineering bacteria that can be used to produce human recombinant interleukin-15 and its purification method - Google Patents

Abstract

The invention relates to a prokaryotic system expression and purification technology that can be used for large-scale production of human recombinant interleukin-15 (recombinant human interleukin-15, rhIL-15). More specifically, the technology of the present invention includes an engineering bacterium and a brand-new expression and purification process. The engineering bacterium contains a brand-new recombinant expression vector, in which there is a newly synthesized DNA segment.

Description

Prokaryotic expression engineering bacteria that can be used to produce human recombinant interleukin-15 and its purification method

technical field

The invention relates to two prokaryotic expression engineering bacteria for human recombinant interleukin-15 (recombinant human interleukin-15, rhIL-15) and the recombinant protein purification technology thereof. More specifically, the engineered bacterium of the present invention contains a brand-new recombinant expression vector, in which there is a newly synthesized DNA segment. The invention also relates to the induced expression and purification technology of the recombinant protein.

Background technique

Interleukin (IL)-15 is a cytokine of about 12-14kD discovered by Grabstein et al. in 1994. With the deepening of its research, people have updated the structure and function of IL-15 understanding. Similar to most cytokines, IL-15 can play a role in the body's normal immune response, such as promoting the development of T cells, B cells, and natural killer (NK) cells. IL-15 can also exert broad and pleiotropic effects on various tissues and cells outside the immune system. Due to the diversity of IL-15's role, it can produce cross-regulatory effects in innate and acquired immune responses, so it can be regarded as a bridge between innate and acquired immune systems.

In the functional research of IL-15, its anti-tumor effect has been paid attention to. Experiments have confirmed that low doses of exogenous IL-15 can induce the development, proliferation and activation of NK cells, NK-T cells and CD8 ⁺ memory T cells without obvious toxic effects. When comparing the efficacy of IL-15 and IL-2 on lung adenocarcinoma metastasis models, it was found that although the specific killing effect induced by IL-15 in vivo was only 1/3 of that of IL-2, its NK killing activity was higher than that of IL-2. Induced 3-4 times higher. In addition, only 6 times the dose of IL-15 can cause toxic side effects such as pulmonary vascular leakage. It was also found in the rat colon cancer model that IL-15 can significantly reduce the gastrointestinal toxicity caused by chemotherapy and enhance the maximum tolerated dose of the drug. Therefore, it is speculated that IL-15 may be effective in the treatment of solid tumors, especially the inhibition of IL-15. 2 Potent candidate for the growth of resistant broad-spectrum solid tumors.

Contents of the invention

In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide the new engineering bacteria obtained by the present invention. Specifically, the engineering bacteria described in the present invention are two strains of Escherichia coli (Escherichia coli.). The Escherichia coli has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee on November 25, 2003, and its preservation number (CGMCC) is: 1049 or preservation number (CGMCC) is: 1050.

The invention also provides a method for purifying recombinant human interleukin-15.

In order to accomplish the purpose of the present invention, technical scheme of the present invention is as follows:

The present invention relates to a DNA sequence encoding human interleukin-15, the sequence number of which is 15 or 28.

The present invention also relates to a recombinant vector containing the DNA sequence of sequence number 15 or sequence number 28, preferably, the vector is a plasmid.

The present invention also relates to Escherichia coli containing the DNA sequence vector with sequence number 15 or sequence number 28.

In addition, the present invention relates to a method for separating and purifying recombinant human interleukin-15 by reverse phase chromatography, comprising the following steps:

(A) In the reverse-phase column, use buffer A to equilibrate the chromatography column for at least 2 column volumes, wherein the buffer A is an aqueous solution containing 0.005% to 0.01% trifluoroacetic acid: 0.5 to 2% acetonitrile;

(B) Recombinant human interleukin-15 crude product injection;

(C) Use buffer A and buffer B for gradient elution at a linear flow rate of 2.5 cm/min, and collect a target protein collection according to the absorption peak of the target protein, wherein buffer B contains 0.005% to 0.01% of trifluoroacetic acid: 85-95% aqueous solution of acetonitrile;

(D) lyophilizing the target protein collection to obtain a lyophilizate;

(E) The freeze-dried product was dissolved in physiological saline at 4-10° C., and centrifuged to obtain a purified recombinant human interleukin-15 solution.

The present invention also relates to another method for chromatographically separating and refolding recombinant human interleukin-15, comprising the following steps:

(1) at least 2 column volumes of the equilibrated gel filtration chromatography column with buffer A; wherein said buffer A contains 20～100mM Tris, 1～5mM EDTA, 50～100mM β-mercaptoethanol, 30～80mM Glycine, GSSG-GSH 0.4～0.6: 4～6mM, 0～1M urea, pH8.0;

(J) Use buffer B to gradually increase the concentration of urea to form a downward gradient of 8M to 0-1M urea in the 0.6 column at the upper end of the chromatography column, with a linear flow rate of 0.5-1cm/min. At this time, the lower end of the chromatography column The 0.4 column bed is 0-1M urea buffer, the upper 0.6 column bed is 8M-0-1M urea buffer, wherein the buffer B contains 20-100mM Tris, 1-5mM EDTA, 30-80mMβ -Mercaptoethanol, 30~80mM Glycine, GSSG-GSH 0.4~0.6: 4~6mM, 8M urea, pH8.0;

(K) Loading crude recombinant human interleukin-15, the volume does not exceed 5% of the column bed, and the concentration does not exceed 10 mg/ml;

(L) 0.5 column volume was eluted with buffer B, and then replaced with buffer A for elution at a linear flow rate of 0.05-0.1 cm/min, and the target protein was collected;

(M) In the DEAE anion exchange column, equilibrate the chromatography column with buffer C for at least 2 column volumes; wherein said buffer C contains 20～100mM Tris, 1～5mM EDTA, 20～80mM β-mercaptoethanol, pH6～ 8.0;

(N) After loading the target protein obtained in step D, wash the chromatography column with buffer C for at least 2 column volumes;

(O) Gradient elution of buffer D, and collection of recombinant human interleukin-15 was collected according to the absorption peak, wherein said buffer D contained 20～100mM Tris, 1～5mM EDTA, 20～80mM β-mercaptoethanol, 1～2M NaCl, pH6.0～8.0,;

(P) Desalting the collected material in step G through a desalting column to obtain purified recombinant human interleukin-15.

It can be seen that, according to one aspect of the present invention, the present invention provides two kinds of novel engineering bacteria, which contain the DNA sequence (sequence 15) or derivatives thereof (sequence 28) encoding human interleukin-15, and also It contains a recombinant vector that is effectively connected with the recombinant DNA molecule and can be stably and highly expressed in Escherichia coli.

Another aspect of the present invention also provides two technological methods for inducing the engineering bacteria to express human interleukin-15, and two technological methods for purifying human interleukin-15.

with sequence description

Sequences 1-14 represent 4 pairs of primers used for splicing full-length human interleukin-15 mature gene DNA sequence I by PCR method, and also listed 3 pairs of short primers that may be used in splicing. The 3' ends of primers L1s, L2s, L3s, and L4s are complementary to the 3' ends of primers L1a, L2a, L3a, and L4a, and the 5' ends of primers L2s, L3s, and L4s are paired with the 5' ends of primers L1a, L2a, and L3a, respectively end complementary pairing. The small primers are used in the second and third rounds of PCR amplification and serve as auxiliary primers. In the first round of PCR reactions, L1s paired with L1a, L2s with L2a, L3s with L3a, and L4s with L4a. In the second round of PCR reaction, the first round of PCR products L1 is paired with L2, and L3 is paired with L4. In the third round of PCR reaction, the PCR product L1-2 of the second round is paired with L3-4, that is, the mature gene sequence of full-length human interleukin-15 is synthesized.

Sequence 15 represents the fully synthetic human interleukin-15 DNA sequence I obtained by splicing primers of sequences 1-14 by the PCR method.

Sequences 16-27 represent derivatives of primers of sequences 1-14 (partial bases are different from FIG. 2 ), and the primers can be used to obtain human interleukin-15 mature gene DNA sequence II shown in sequence 28 by bridging PCR.

Sequence 28 represents the fully synthetic human interleukin-15 DNA sequence II obtained by splicing primers of sequences 16-27 by the PCR method.

Sequence 29 represents the amino acid sequence of human interleukin-15 obtained from fully synthesized human interleukin-15 DNA sequence I or DNA sequence II.

Sequence 30 represents the DNA sequence determination result of the part of the recombinant expression vector pBV ₂₂₀ -hIL-15 inserted into the target gene, which shows that the mature gene sequence of fully synthesized human interleukin-15 is correct and the position of the insertion vector is correct. The fully synthesized human interleukin-15 DNA sequence I is inside the box, and its two ends are restriction sites and the vector pBV ₂₂₀ sequence.

Description of drawings

Fig. 1 shows the principle of fully synthesizing human interleukin-15 mature gene by PCR method. These 4 pairs of primers can be paired complementary in two parts, and use each other as a template to synthesize DNA fragments under the action of Taq enzyme. Similarly, the synthesized DNA fragments can also be partially complementary to each other to synthesize longer DNA fragments. Therefore, short fragments, sub-full-length and full-length human interleukin-15 mature gene DNA can be synthesized by stepwise bridge splicing. According to the amino acid sequence of hIL-15 and the codon preference of E. coli, design and synthesize 4 pairs of oligonucleotide fragments, and the overlap between adjacent two fragments is about 18bp

Fig. 2 shows the construction process of the expression vector pBV ₂₂₀ -hIL-15 for prokaryotic expression of human interleukin-15.

Fig. 3 shows the construction process of the expression vector pET ₄₂ -hIL-15 prokaryotically expressing human interleukin-15.

Figure 4 shows the 2% agarose electrophoresis results of the DNA fragments obtained at different stages of splicing the new DNA sequence 1-14 of the full-length human interleukin-15 mature gene or the new DNA sequence 15 by PCR method, and 1 and 2 are the first round of PCR Splicing results, 3 and 4 are the results of the second round of PCR splicing, 5 and 6 are the results of the third round of PCR splicing, and M is the DNA molecular weight standard (DL2000) of Dalian Bao Biological Company.

Figure 5 shows that the constructed recombinant expression vector pBV ₂₂₀ -hIL-15 was positively identified by double enzyme digestion, indicating that the human interleukin-15 mature gene was inserted correctly. 1 is the pBV ₂₂₀ -hIL-15 vector, 3 and 4 are pBV ₂₂₀ empty vectors without inserting the target gene, and M is the DNA molecular weight standard (DL2000) of Dalian Bao Biological Company.

Fig. 6 shows the sequencing map of the part of the recombinant expression vector pBV ₂₂₀ -hIL-15 inserted into the target gene.

Fig. 7 shows the results of SDS-PAGE gel electrophoresis of bacterial proteins before and after induced expression of engineered bacteria BL ₂₁ Star ^TM (DE3) PlysS/pBV ₂₂₀ -hIL-15. M is a low-molecular-weight protein standard, 1 and 3 are the total protein of the bacterial body after the induction of the engineered bacteria, and 2 is the total protein of the bacterial body before the induction of the engineered bacteria. The arrow points to the expressed target protein.

Figure 8 shows the percentage content of the target protein in the total bacterial protein. 16 is the absorption peak of the target protein, and the index indicated by the arrow is the expression level of the target protein, which is 15.2%.

Figure 9 shows the Western Blot method of the human interleukin-15 monoclonal antibody to detect the existence form of the target protein in the bacterium. 1 and 3 are inclusion body precipitates, and 2 is supernatant. The arrow points to the expressed target protein.

Figure 10 shows the determination of the amino acid sequence of the preliminary purified recombinant protein. The six amino acids at the N-terminal of the preliminary purified protein are M-N-W-V-N-V in sequence, and the results show that it is human interleukin-15.

Figure 11 shows the removal of impurities from inclusion bodies after different washing processes, 1 and 2 are supernatants after washing with washing solution A; 3 are supernatants after washing with 2% TritonX-100; 4 and 5 are supernatants after washing with 2M urea clear; 6, 7 are inclusion body precipitation after washing; M is low molecular weight protein standard; 8 is bacterial protein after induction; 9 is bacterial protein before induction.

Figure 12 shows the elution gradient and absorption curve of reverse phase chromatography separation of crude recombinant human interleukin-15, and the absorption peak indicated by the arrow is the absorption peak of recombinant human interleukin-15.

Figure 13 shows the SDS-PAGE gel electrophoresis results of each collection peak separated by reverse phase chromatography of recombinant human interleukin-15 crude product, 5, 6, and 7 are the front, middle, and rear parts of the target protein absorption peak respectively, 1, 2 , 3 and 4 are absorption peaks other than the target protein absorption peak, M is a low molecular weight protein standard, 8 and 9 are crude recombinant human interleukin-15. The arrow points to recombinant human interleukin-15.

Fig. 14 shows the SDS-PAGE gel electrophoresis results of the purified protein obtained by lyophilizing and dissolving the target protein collected after reverse phase chromatography separation of crude recombinant human interleukin-15. 1 is the supernatant after the freeze-dried product is dissolved in water, 2 is the supernatant after the freeze-dried product is dissolved in normal saline, 4 is the precipitate after the freeze-dried product is dissolved in water, 5 is the freeze-dried product, 9 and 11 are recombinant human leukocytes Interleukin-15 is crude, and 10 is a low molecular weight protein standard.

Figure 15 shows the purity of purified recombinant human interleukin-15 measured by optical density method, 1 is the absorption peak of impurities, 2 is the absorption peak of recombinant human interleukin-15, and the others are noise. in:

Figure 16 shows the SDS-PAGE results of the crude recombinant human interleukin-15 separated by Amersham Sephacryl S100 gel filtration chromatography and collected after renaturation. M is a low molecular weight protein standard, and 1 to 10 are the front, middle and back parts of the target protein absorption peak, respectively. The position indicated by the arrow is recombinant human interleukin-15.

specific implementation plan

The engineering bacteria, induced expression method and purification process of the present invention can be used to produce recombinant human interleukin-15.

In the present invention, the terms used are defined as follows:

The term "prokaryotic expression" refers to Escherichia coli BL ₂₁ Star ^TM (DE3) plysS transfected with the recombinant expression vector pBV ₂₂₀ -hIL-15 plasmid under high temperature induction or transfected with the recombinant expression vector pET ₄₂ -hIL-15 plasmid Escherichia coli BL ₂₁ Star ^TM (DE3) plysS expresses human interleukin-15 under the induction of 1.0mmol/l IPTG.

The term "engineering bacterium" refers to the Escherichia coli BL ₂₁ Star ^TM (DE3) plysS transfected with the recombinant expression vector pBV ₂₂₀ -hIL-15 plasmid and the Escherichia coli BL ₂₁ Star transfected with the recombinant expression vector pET ₄₂ -hIL-15 plasmid ^TM (DE3)plysS.

The term "recombinant human interleukin-15 (rhIL-15)" refers to the human interleukin-15 produced in Escherichia coli through genetic engineering, which may have one more amino acid than the interleukin-15 produced in the human body (ie The first amino acid at the N-terminal - methionine), in addition, the amino acid sequence is the same as that of interleukin-15 in the human body.

The terms "new DNA sequence I/II of full-length human interleukin-15 mature gene" and "total synthetic human interleukin-15 DNA sequence I/II" both refer to the full-length DNA encoding human interleukin-15 mature peptide The derivative of the sequence, the expressed polypeptide has the same amino acid sequence as human interleukin-15 except that the first amino acid at the N-terminal is methionine.

The term "expression and purification process" refers to the method and process of inducing engineering bacteria to express and purify recombinant human interleukin-15.

The content of the present invention can be understood more easily by referring to the following examples, which are only for further illustration and are not meant to limit the scope of the present invention.

Example 1

Total synthesis of full-length human interleukin-15 mature gene

According to the principle of fully synthesizing human interleukin-15 mature gene by the PCR method shown in FIG. 1 . The two parts of the four pairs of primers are complementary to each other, and each uses the other as a template to synthesize DNA fragments under the action of Taq enzyme. Similarly, the synthesized DNA fragments can also be partially complementary to each other to synthesize longer DNA fragments. Therefore, short fragments, sub-full-length and full-length human interleukin-15 mature gene DNA sequences can be synthesized by stepwise bridge splicing. The 3' ends of primers L1s, L2s, L3s, and L4s are complementary to the 3' ends of primers L1a, L2a, L3a, and L4a, and the 5' ends of primers L2s, L3s, and L4s are paired with the 5' ends of primers L1a, L2a, and L3a, respectively end complementary pairing. The small primers are used in the second and third rounds of PCR amplification and serve as auxiliary primers. In the first round of PCR reactions, L1s paired with L1a, L2s with L2a, L3s with L3a, and L4s with L4a. In the second round of PCR reaction, the first round of PCR products L1 is paired with L2, and L3 is paired with L4. In the third round of PCR reaction, the PCR product L1-2 of the second round is paired with L3-4, that is, the mature gene sequence of full-length human interleukin-15 is synthesized.

Example 2

Construction of recombinant expression vector

The technical route for the construction of the recombinant expression vector is shown in Figure 2 and Figure 3. First, the full-length human interleukin-15 mature gene was spliced by polymerase chain reaction (PCR) bridging, and enzyme cutting sites were added at its 3' and 5' ends, and primers such as sequence 12 (containing Pst I enzyme cutting site) and sequence 9 (containing EcoR I restriction site). When pBV ₂₂₀ is used to construct the recombinant expression vector, PstI and EcoRI restriction sites, namely CTGCAG and GAATTC, are added to the 3' and 5' ends of rhIL-15, respectively. When using pET _42a to construct a recombinant expression vector, add XhoI and NdeI restriction sites, namely CTCGAG and CATATG, to the 3' and 5' ends of rhIL-15, respectively. The full-length human interleukin-15 mature gene with restriction sites was loaded into pGEM-T plasmid and sequenced, and the pGEM-T plasmid with full-length human interleukin-15 mature gene was encoded with PstI and EcoRI Double digestion, or double digestion with XhoI and NdeI. The DNA fragment containing the full-length human interleukin-15 mature gene was recovered. At the same time, the pBV ₂₂₀ plasmid was double-digested with PstI and EcoRI, or the pET _42a plasmid was double-digested with XhoI and NdeI to recover a large fragment. The digested pBV ₂₂₀ vector fragment or pET _42a vector fragment was ligated with the DNA fragment containing the full-length human interleukin-15 mature gene to form pBV ₂₂₀ -hIL-15 plasmid and pET ₄₂ -hIL-15 plasmid. Then, transformation of the ligated product, mini-extraction and purification of plasmid DNA was carried out.

Example 3

Obtaining, Screening and Identification of Engineering Bacteria BL ₂₁ Star ^TM (DE3)plysS/pBV ₂₂₀ -hIL-15 and BL ₂₁ Star ^TM (DE3)plysS/pET ₄₂ -hIL-15

Step 1. Screen engineering bacteria with stable and high expression

(1) Transform the pBV ₂₂₀ -hIL-15 plasmid and the pET ₄₂ -hIL-15 plasmid obtained in Example 2 above into the host bacterium BL ₂₁ Star ^TM (DE3)plysS, which is the engineering bacterium of the present invention;

(1) Pick a monoclonal colony BL ₂₁ Star ^TM (DE3)plysS/pBV ₂₂₀ -hIL-15 from a solid LB agar medium plate, shake the bacteria overnight in 4ml LB liquid medium at 28°C and 250 rpm;

(3) Inoculate in 4ml LB medium at a ratio of 1:100, shake the bacteria at 28°C and 250 rpm until the OD ₆₀₀ value is 0.4-0.6, then rapidly raise the temperature to 42°C to induce expression, and continue to shake the bacteria for 4-5 hours , to recycle the cells.

(4) As for the engineered bacteria BL ₂₁ Star ^TM (DE3)plysS/pET ₄₂ -hIL-15, the same is true, except that the culture condition is changed to conventional 37°C, and the induction condition is changed to OD ₆₀₀ value of 0.4-0.6 when it is added to the culture medium IPTG was added at a final concentration of 1 mmol/l.

(5) Collect the bacteria by centrifugation, wash once with buffer A (Buffer A: 50mM Tris, 5Mm EDTA, 150Mm NaCl, pH8.0), then resuspend in 1× loading buffer, cook at 95C for 5 minutes, and then Load the sample for SDS-PAGE gel electrophoresis, Coomassie blue staining, and compare the changes in bacterial protein before and after induction, and select the strain with the highest expression of the target protein and save it as the seed bacteria.

Step 2. Western-Blot identification of target protein

The bacterial protein before and after induction was subjected to SDS-PAGE gel electrophoresis, and then transferred to a nitrocellulose membrane, and the target protein was identified as recombinant human interleukin-15 by Western Blot with anti-human interleukin-15 monoclonal antibody.

Example 4

Mass preparation and preliminary purification of target protein

(1) The preserved seed fungus BL ₂₁ Star ^TM (DE3)plysS/pBV ₂₂₀ -hIL-15 was inoculated in 100ml LB medium at a ratio of 1:1000, at 28°C, 250 rpm, and shaken for 8 hours;

(2) The bacteria solution is then inoculated in a large bottle of LB medium at a ratio of 1:100, at 28°C, 200 rpm, shake the bacteria until the OD ₆₀₀ value is 0.4-0.6 (2.5-3 hours), and quickly heat up to 42°C Induce the expression, continue to shake the bacteria for 4-5 hours, and recover the bacteria.

(3) The induction method of engineering bacteria BL ₂₁ Star ^TM (DE3) plysS/pET ₄₂ -hIL-15 is the same, except that the culture condition is changed to conventional 37°C, and the induction condition is changed to OD ₆₀₀ value of 0.4-0.6 when adding to the culture medium IPTG was added at a final concentration of 1 mmol/l.

(4) Collect the bacteria by centrifugation; wash 2 times with Buffer A (50mM Tris, 5mM EDTA, 150mM NaCl, pH8.0); ultrasonically disrupt the bacteria in an ice bath (360 watts, work for 10 seconds, stop for 30 seconds, 80 cycles ); centrifuged to separate the precipitate; the precipitate was successively washed with Buffer B containing 2% TritonX-100 and Buffer C containing 2M urea. After washing, the precipitate was dissolved in 6M guanidine hydrochloride to obtain crude recombinant human interleukin-15.

Example 5

Separation and Purification of Recombinant Human Interleukin-15 by Reverse Phase Chromatography

Reverse-phase column RPC Resource15 was used to purify recombinant human interleukin-15.

(1) Buffer A (trifluoroacetic acid: acetonitrile: water=0.065%: 2%: 97.35%) equilibrates chromatographic column with 5 column volumes (column volume, CV);

(2) Recombinant human interleukin-15 crude product injection;

(3) Use Buffer A and Buffer B (trifluoroacetic acid: acetonitrile: water = 0.1%: 90%: 9.9%) to elute with a linear flow rate of 2.5cm/min, and the elution gradient is 0% B 2CV40% B 5CV 40-50% B 8CV 50-65% B 1CV 65% B5CV 100% B 5CV, collected according to the absorption peak of the target protein.

(4) The collection is freeze-dried;

(5) The freeze-dried product was dissolved in physiological saline at 4-10°C, and centrifuged at 12,000 rpm for 10 minutes to obtain a purified recombinant human interleukin-15 solution.

The dissolved target protein is proved to be recombinant human interleukin-15 with biological activity by cell activity experiments.

Example 6

Separation and Purification of Recombinant Human Interleukin-15 by Reverse Phase Chromatography

Reverse-phase column RPC Resource15 was used to purify recombinant human interleukin-15.

(A) Buffer A (0.005% trifluoroacetic acid: 0.5% acetonitrile) aqueous solution was used to equilibrate the chromatography column for 5 column volumes (column volume, CV);

(B) Recombinant human interleukin-15 crude product injection;

(C) Elute with Buffer A and Buffer B (0.005% trifluoroacetic acid: 85% acetonitrile in water) at a linear flow rate of 2.5cm/min, and the elution gradient is 0% B 2CV40% B 5CV 40-50% B 8CV 50-65% B 1CV 65% B5CV 100% B 5CV, collected according to the absorption peak of the target protein;

(D) the collection is freeze-dried;

(E) The freeze-dried product was dissolved in physiological saline at 4-10°C, and centrifuged at 12,000 rpm for 10 minutes to obtain a purified recombinant human interleukin-15 solution.

The dissolved target protein is proved to be recombinant human interleukin-15 with biological activity by cell activity experiments.

Example 7

The method steps of Example 6 were repeated, except that buffer A was an aqueous solution containing 0.01% trifluoroacetic acid: 2% acetonitrile, and buffer B was an aqueous solution containing 0.01% trifluoroacetic acid: 95% acetonitrile.

Example 8

Separation and refolding of recombinant human interleukin-15 by Amersham Sephacryl S100 (16×100) molecular sieve gel filtration chromatography

(1) Refolding buffer A (Buffer A: 50mM Tris, 1mM EDTA, 50mM β-mercaptoethanol, 50mM Glycine, GSSG-GSH 0.5:5mM, 0.4M urea, pH8.0) Equilibrated gel filtration chromatography column 2 column volume;

(2) Use solution B (50mM Tris, 1mM EDTA, 50mM β-mercaptoethanol, 50mMGlycine, GSSG-GSH 0.5∶5mM, 8M urea, pH8.0) to gradually increase the concentration of urea in the upper 0.6 column of the chromatography column Form a downward descending gradient of 8M to 0.4M urea, and the linear flow rate is 0.5-1cm/min. At this time, the 0.4 times column bed at the lower end of the chromatography column is 0.4M urea buffer, and the 0.6 times column bed at the upper end is 8M-0.4M urea buffer;

(3) Sample loading (volume not exceeding 5% of the column bed, concentration not exceeding 10mg/ml);

(4) 0.5 column volume was eluted with solution B, and then changed to solution A for elution to obtain a target protein collection.

Since the urea gradient moves slower than the recombinant protein, as the elution progresses, the recombinant protein gradually flows through the eluent with a decreasing gradient of urea and is gradually refolded. The linear flow rate during this process is 0.05-0.1cm/min, which is not suitable Too fast, otherwise the protein is easy to precipitate. The recombinant human interleukin-15 was collected according to the absorption peak and separated by ion exchange chromatography.

Example 9

Separation of recombinant protein obtained by molecular sieve separation by DEAE anion exchange column chromatography

(2) Use Buffer C (Buffer A: 50mM Tris, 1mM EDTA, 50mM β-mercaptoethanol, pH8.0) to equilibrate the chromatography column for 2 column volumes;

(3) Wash the chromatographic column with liquid A for 2 column volumes after loading the target protein collection obtained in Example 8;

(4) Buffer D solution (50mM Tris, 1mM EDTA, 50mM β-mercaptoethanol, 1MNaCl, pH8.0) gradient elution, collect recombinant human interleukin-15 according to the absorption peak;

(5) The collected material is desalted through a desalting column to obtain purified recombinant human interleukin-15.

Example 10

Repeat the steps of Example 8, except that the buffer A used contains 20mM Tris, 1mM EDTA, 50mM β-mercaptoethanol, 30mM Glycine, GSSG-GSH 0.4: 4mM, pH8.0. And buffer B is wherein said buffer B contains 20mM Tris, 1mM EDTA, 30mM β-mercaptoethanol, 30mM Glycine, GSSG-GSH 0.4: 4mM, 8M urea, pH8.0.

Example 11

Repeat the steps of Example 8, except that the buffer A used contains 100mM Tris, 5mM EDTA, 100mM β-mercaptoethanol, 80mM Glycine, GSSG-GSH 0.6: 6mM, 1M urea, pH8.0;

And buffer B contains 100mM Tris, 5mM EDTA, 80mM β-mercaptoethanol, 80mM Glycine, GSSG-GSH 0.6:6mM, 8M urea, pH8.0.

Example 12

Repeat the steps of Example 9, except that buffer C used contains 20mM Tris, 1mM EDTA, 20mM β-mercaptoethanol, pH6.0; and buffer D contains 20mM Tris, 1mM EDTA, 20mM β-mercaptoethanol, 1M NaCl , pH6.0.

Example 13

Repeat the steps of Example 9, except that buffer C used contains 100mM Tris, 5mM EDTA, 80mM β-mercaptoethanol, pH8.0; buffer D contains 100mM Tris, 5mM EDTA, 80mM β-mercaptoethanol, 2M NaCl, pH8.0.

sequence listing

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gacgcttcta tccacgac 18

<210>12

<211>24

<212>DNA

<213> PstI

<400>12

ctgcagctat taagaagtgt tgat 24

<210>13

<211>21

<212>DNA

<213>NdeI

<400>13

catatgaact gggttaacgt a 21

<210>14

<211>24

<212>DNA

<213>XhoI

<400>14

ctcgagctat taagaagtgt tgat 24

<210>15

<211>351

<212>DNA

<213> EcoRI

<400>15

atgaactggg ttaacgtaat ctctgacttg aaaaaaatcg aagacctgat ccagtctatg 60

cacatcgacg ctactctgta caccgaaagc gacgttcacc cgtcttgcaa agtaaccgct 120

atgaagtgct tcctcctgga gctccaggtt atctctcttg agtccggtga cgcttctatc 180

cacgacaccg tagaaaacct gatcatcctg gctaacaact ctttgtcttc taacgggaac 240

gtaaccgaat ctggttgcaa agaatgcgag gaactggagg aaaaaaacat caaagagttc 300

ttgcagagct tcgtacacat cgttcagatg ttcatcaaca cttcttaata g 351

<210>16

<211>59

<212>DNA

<213> Synthetic

<400>16

atgaactggg ttaacgtaat ctctgacctg aaaaaaatcg aagacctgat ccagtctat 59

<210>17

<211>59

<212>DNA

<213> Synthetic

<400>17

tgaacgtcag attcggtgta cagagtagcg tcgatgtgca tagactggat caggtcttc 59

<210>18

<211>59

<212>DNA

<213> Synthetic

<400>18

caccgaatct gacgttcacc cgtcttgcaa agttactgct atgaaatgtt tcctgctgg 59

<210>19

<211>59

<212>DNA

<213> Synthetic

<400>19

ggatagaagc gtcacctgac tccagagaga taacctggag ttccagcagg aaacatttc 59

<210>20

<211>59

<212>DNA

<213> Synthetic

<400>20

tctatccacg acaccgttga aaacctgatc atcctggcta acaactctct gtcatctaa 59

<210>21

<211>59

<212>DNA

<213> Synthetic

<400>21

tcctcacatt ctttgcaacc tgattcagta acgttaccgt tagatgacag agagttgtt 59

<210>22

<211>59

<212>DNA

<213> Synthetic

<400>22

ggttgcaaag aatgtgagga actggaagaa aaaaacatca aagagttcct ccagtcttt 59

<210>23

<211>59

<212>DNA

<213> Synthetic

<400>23

ctattaagag gtgttgatga acatctgaac gatgtgaacg aaagactgga ggaactctt 59

<210>24

<211>24

<212>DNA

<213> EcoRI

<400>24

gaattcatga actgggttaa cgta 24

<210>25

<211>21

<212>DNA

<213> Synthetic

<400>25

tgtcgtggat agaagcgtca c 21

<210>26

<211>21

<212>DNA

<213> Synthetic

<400>26

gacgcttctatccacgacac c 21

<210>27

<211>26

<212>DNA

<213>Pstl

<400>27

ctgcagctat taagaggtgttgatga 26

<210>28

<211>351

<212>DNA

<213> EcoRI

<400>28

atgaactggg ttaacgtaat ctctgacctg aaaaaaatcg aagacctgat ccagtctatg 60

cacatcgacg ctactctgta caccgaatct gacgttcacc cgtcttgcaa agttactgct 120

atgaaatgtt tcctgctgga actccaggtt atctctctgg agtcaggtga cgcttctatc 180

cacgacaccg ttgaaaacct gatcatcctg gctaacaact ctctgtcatc taacggtaac 240

gttactgaat caggttgcaa agaatgtgag gaactggaag aaaaaaacat caaagagttc 300

ctccagtctt tcgttcacat cgttcagatg ttcatcaaca cctcttaata g 351

<210>29

<211>115

<212>PRT

<213> EcoRI

<400>29

Met Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

1 5 10 15

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

20 25 30

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

35 40 45

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

50 55 60

Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn

65 70 75 80

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

85 90 95

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

100 105 110

Asn Thr Ser

115

<210>30

<211>580

<212>DNA

<213> EcoRI

<400>30

gtgtgtgatg atacgaaacg aagcattggt taaaaattaa ggaggaattc atgaactggg 60

ttaacgtaat ctctgacttg aaaaaaatcg aagacctgat ccagtctatg cacatcgacg 120

ctactctgta caccgaaagc gacgttcacc cgtcttgcaa agtaaccgct atgaagtgct 180

tcctcctgga gctccaggtt atctctcttg agtccggtga cgcttctatc cacgacaccg 240

tagaaaacct gatcatcctg gctaacaact ctttgtcttc taacgggaac gtaaccgaat 300

ctggttgcaa agaatgcgag gaactggagg aaaaaaacat caaagagttc ttgcagagct 360

tcgtacacat cgttcagatg ttcatcaaca cttcttaata gctgcagcca agcttggctg 420

ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg 480

tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc 540

gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg 580

Claims (7)

1. A DNA encoding human interleukin-15, the base sequence of which is shown in SEQ ID NO: 15 or SEQ ID NO: 28. 2. A recombinant vector containing DNA as claimed in claim 1. 3. The vector according to claim 2, characterized in that the vector is a plasmid. 4. contain the escherichia coli (Escherichia coli.) of DNA as claimed in claim 1. 5. The escherichia coli containing the DNA as claimed in claim 1, the preservation number is CGMCC NO: 1049 or CGMCC NO: 1050, which was deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee on November 25, 2003. 6. A method for reverse-phase chromatography separation and purification of recombinant human interleukin-15 encoded by DNA as claimed in claim 1, comprising the steps of: a) In the reverse-phase column, use buffer A to equilibrate the chromatography for at least 2 column volumes, wherein the buffer A is an aqueous solution containing 0.005% to 0.01% of trifluoroacetic acid: 0.5 to 2% of acetonitrile, b) Recombinant human interleukin-15 crude product injection; c) Use buffer A and buffer B for gradient elution at a linear flow rate of 2.5 cm/min, and collect a target protein collection according to the absorption peak of the target protein, wherein buffer B contains 0.005% to 0.01% of trifluoroacetic acid: acetonitrile 85-95% aqueous solution; d) freeze-drying the target protein collection to obtain a freeze-dried product; e) Dissolving the freeze-dried product in physiological saline at 4-10° C., and centrifuging to obtain a purified recombinant human interleukin-15 solution. 7. A method for chromatographic separation and renaturation of the recombinant human interleukin-15 encoded by DNA as claimed in claim 1, comprising the steps of: (A) Balance at least 2 column volumes of the gel filtration chromatography column with buffer A; wherein said buffer A contains 20-100mM Tris, 1-5mM EDTA, 50-100mM β-mercaptoethanol, 30-80mM Glycine, GSSG-GSH 0.4～0.6: 4～6mM, 0～1M urea, pH8.0; (B) Use buffer B to gradually increase the concentration of urea to form a downward decreasing gradient of 8M to 0-1M urea in the 0.6 column at the upper end of the chromatographic column, and the linear flow rate is 0.5-1cm/min. At this time, the chromatographic column The 0.4-fold column bed at the lower end is 0-1M urea buffer, and the 0.6-fold column bed at the upper end is 8M to 0-1M urea buffer, wherein the buffer B contains 20-100mM Tris, 1-5mM EDTA, 30- 80mM β-mercaptoethanol, 30~80mM Glycine, GSSG-GSH 0.4~0.6: 4~6mM, 8M urea, pH8.0; (C) The recombinant human interleukin-15 crude product is loaded as a sample, the volume does not exceed 5% of the column bed, and the concentration does not exceed 10mg/ml; (D) 0.5 column volume was eluted with buffer B, and then replaced with buffer A for elution at a linear flow rate of 0.05-0.1 cm/min, and the target protein was collected; (E) In the DEAE anion exchange column, use buffer C to equilibrate the chromatography column for at least 2 column volumes; wherein said buffer C contains 20～100mM Tris, 1～5mM EDTA, 20～80mM β-mercaptoethanol, pH6～ 8.0; (F) After loading the target protein obtained in step D, wash the chromatography column with buffer C for at least 2 column volumes; (G) Gradient elution of buffer D, and collection of recombinant human interleukin-15 was collected according to the absorption peak, wherein said buffer D contained 20-100mM Tris, 1-5mM EDTA, 20-80mM β-mercaptoethanol, 1 ～2M NaCl, pH6.0～8.0,; (H) Desalting the collected material in step G through a desalting column to obtain purified recombinant human interleukin-15.

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