WO2010075770A1

WO2010075770A1 - A recombinant fusion protein, the preparation method and use thereof

Info

Publication number: WO2010075770A1
Application number: PCT/CN2009/076177
Authority: WO
Inventors: 文勇; 陈悦; 殷晓进; 原雪; 陈亚利
Original assignee: 江苏先声药物研究有限公司
Priority date: 2008-12-30
Filing date: 2009-12-29
Publication date: 2010-07-08
Also published as: CN101768219B; CN101768219A

Abstract

Provided is a fusion protein comprising of a 27-amino-acid peptide derived from the NH2-terminal domain of endostatin and Kringle5 domain derived from plasminogen. The amino acid sequence of said fusion protein is the sequence as shown in SEQ ID No.2. Said fusion protein can inhibit the proliferation of endothelial cell, and further inhibit the formation of tumor neovessels.

Description

Instruction manual

Recombinant fusion protein and preparation method and application thereof

Technical field

The invention belongs to the field of biomedicine and relates to a recombinant fusion protein for inhibiting proliferation of endothelial cells, a preparation method thereof and application in pharmacy. Background technique

To date, cancer remains one of the greatest threats to human health, so anti-tumor drugs have always been the focus of new drug development for pharmaceutical manufacturers around the world. Conventional tumor treatment methods target tumor cells, but during treatment, resistance is often caused by genetic mutations in tumor cells, or the entire treatment fails due to toxic side effects of chemotherapy drugs. Scientists have been pursuing effective ways to treat cancer. In the 1970s, the anti-angiogenic therapy proposed by Professor Verkman of Harvard University found a new way for the treatment of cancer. This view has been confirmed by a large number of research results. Targeting blood vessels as a target has become one of the hotspots in cancer research, and a variety of angiogenesis inhibitors have entered the clinical trial phase. Among them, angiostatin and endostatin have attracted more and more attention due to their specificity and non-resistance. In September 2005, rH-Endostatin (Endostar) was approved by the State Food and Drug Administration for the treatment of non-small cell lung cancer. The researchers found that about 30 synthetic short peptides from the N-terminus of endostatin have endomycin-like anti-tumor and anti-metastatic activity. Kringle5, also derived from plasminogen, which has 57.5% homology with Kringlel in angiostatin, has a better inhibitory effect on proliferation of capillary endothelial cells than angiostatin. Endostatin and angiostatin are not only attracting attention for their own advantages in tumor treatment, but also gradually noticed that their combined use has more synergistic anti-angiogenic effects and anti-tumor growth effects in the course of continuous experiments. The existing experimental results show that the combination of endostatin and angiostatin has a more obvious inhibitory effect on tumor growth. At the same time, the anti-tumor effect of the combination treatment group is 2 times stronger than the expected effect, which further indicates the combination of endostatin and angiostatin for cancer growth. Inhibition has synergistic effects. However, the cost of producing endostatin and angiostatin separately is too high, and if the endostatin and angiostatin directly form a fusion protein with a relatively large molecular weight (molecular weight of about 65 KD), too many disulfide bonds (13 pairs of disulfide bonds) will seriously affect the purpose. The yield and yield of protein are not conducive to subsequent industrialization.

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It is an object of the present invention to provide a recombinant fusion protein which inhibits proliferation of endothelial cells.

Another object of the invention is to provide a process for the preparation of said recombinant fusion protein. Still another object of the invention is to provide the use of the recombinant fusion protein in pharmaceuticals.

The object of the invention is achieved by the following technical measures:

A recombinant fusion protein, the amino acid sequence of which is SEQ ID No. 2.

A nucleotide sequence encoding the recombinant fusion protein, preferably having a nucleotide sequence of SEQ ID No. 1.

A genetically engineered strain capable of expressing the recombinant fusion protein.

The preparation method of the recombinant fusion protein comprises the following steps:

a PCR reaction is carried out by inserting a whole-gene synthetic fragment (a nucleotide sequence encoding the recombinant fusion protein) into a template, and using SEQ ID No. 3 and SEQ ID No. 4 as upstream and downstream primers. The PCR product is collected; b. The PCR product is inserted into the expression vector to construct a recombinant expression vector, and the recombinant vector is transformed into Escherichia coli; c transformant is fermented and cultured, the target protein is induced to be expressed, and the target protein is collected and purified.

The preparation method, wherein the expression vector is pET25b.

The preparation method, wherein the inducer is lactose, IPTG.

The preparation method, wherein the purified protein is subjected to affinity chromatography.

The use of the recombinant fusion protein for the preparation of a medicament for inhibiting proliferation of endothelial cells,

In the application, the drug for inhibiting proliferation of endothelial cells is a tumor neovascular inhibitor.

Specifically, the technical route of the present invention is as follows:

1. Design and acquisition of SIMEK gene

The SIMEK gene includes the first 27 amino acids of Endostatin and two active fragments of Kringle5, which are ligated with three overlapping linker Gly-Gly-Ser-Gly-Ser, so as not to affect the active regions of the fusion protein. The entire sequence of the gene of interest was synthesized by Shanghai Yingjun Biotechnology Co., Ltd. and inserted into the vector pMD 18-T. According to the SIMEK sequence, select the prokaryotic preferred codon, design two oligonucleotide strands by computer, respectively as upstream and downstream primers, and add the restriction endonuclease Nco l enzyme at the 5' end of the upstream primer. The cleavage site, the 5' end of the downstream primer plus the restriction endonuclease 1⁄21 I restriction site, and the PCR-chain reaction (PCR) method to obtain SIMEK using pMD 18-T (inserted target gene) as a template The nucleotide sequence of the gene.

2. Construction of BL21-pET25b-SIMEK recombinant genetic engineering bacteria

The SIMEK gene was digested with Nco I and Xhol I and inserted into the vector pET25b which was also digested with Nco I and Xhol I to construct the recombinant plasmid pET25b-SIMEK. Transformed into E. coli BL21 (DE3), colonized

Positive clones were obtained by PCR, double enzyme digestion and sequencing, and the recombinant genetically engineered strains were preserved. The expression vector pET25b used in the present invention fuses a gene sequence encoding 6 histidines at the 3' end of the multiple cloning site, and after translation, the C-terminus of the fusion protein contains 6-His-Tag, which is convenient for expression. Identification and separation purification. 3. Engineering bacteria fermentation and acquisition of fusion protein SIMEK

The liquid LB was used as the fermentation medium, and the fermentation conditions were as follows: freshly activated recombinant bacteria, inoculated in LB medium (ampicillin 50 g/mL), cultured at 37 ° C, 215 r/min for 3 h, and added with a final concentration of 0.5% lactose. At 20 °C, 150 r/min for 12 h, the cells were collected. The cells were sonicated, nickel column affinity chromatography and samples treated with endotoxin-free affinity filler to obtain a pure product with an endotoxin content of less than 2.5 EU/mg. The purity of the expressed product SIMEK was measured by 15% SDS-PAGE.

A second aspect of the present invention is the use of the recombinant protein, which, as described above, inhibits proliferation of endothelium cells and further inhibits tumor angiogenesis.

The beneficial effects of the invention:

The present invention creatively links the active fragment of the N-terminus of endostatin with Kringle5, and constructs the recombinant protein SIMEK (having a molecular weight of only about 16 KD, wherein only three pairs of disulfide bonds are present) by genetic engineering to exert both in inhibiting endothelial cell proliferation and Anti-tumor effect. DRAWINGS

Figure 1 is a schematic diagram showing the construction of the recombinant plasmid pET25b-SIMEK.

Figure 2 shows the PCR amplification of the SIMEK gene. (The lane M is DNA Marker: 1200 bp, 900 bp, 700 bp, 500 bp, 300 bp, 100 bp; Lane 1 is a PCR amplification sample; Lane 2 is a PCR amplification sample)

Figure 3 shows the colony PCR identification map. (lane M is DNA Marker; lanes 1-9 are single colony clones) Figure 4 shows the electrophoresis pattern of SIMEK expression in BL21 (DE3). (Way 1 is a non-inducible whole bacterium; Lane 2 is a protein Marker: 94.0 KDa, 66.2 KDa, 45.0 KDa, 35.0 KDa, 26.0 KDa, 20.0 KDa, 14.4 KDa; Lane 3 is induced at 37 °C; Lane 4 is Induction of whole bacteria at 20 °C)

Figure 5 shows the SIMEK cell localization electrophoresis map (lane 1 is whole bacteria; lane 2 is ultrasonic precipitation; lane 3 is ultrasonic supernatant)

Figure 6 shows the effect of different lactose concentrations on the expression of SIMEK

Figure 7 shows the purification of SIMEK protein (lane 1-2 is purified SIMEK; lane 3 is protein Marker). Figure 8 shows the results of different concentrations of SIMEK inhibiting HUVEC cell proliferation.

Figure 9 shows the results of different concentrations of SIMEK to inhibit the proliferation of BCE cells.

The invention is further illustrated by the following examples. Material:

The host strain E.Coli BL21 (DE3) is a commonly used tool strain for genetic engineering and is generally preserved in laboratories associated with genetic engineering research. Plasmid pET25b was purchased from Novagen.

Enzymes and reagents: Molecular cloning tools enzymes and reagents are products of Takara; plasmid extraction kits, PCR recovery kits and PCR purification kits are products of Qiagen.

PCR templates and primers: synthesized by Invitrogen.

LB medium (g/ml): peptone 1.0%, yeast powder 0.5%, sodium chloride 1.0%, and the rest is water. Peptone, yeast powder is a product of Oxoid.

Method:

Molecular biological methods of operation: Plasmid extraction, polymerase chain reaction, restriction endonuclease digestion, DNA fragment recovery, ligation and E. coli transformation experiments are routine methods of operation in the field of genetic engineering research, see the Guide to Molecular Cloning 》.

Recombinant protein expression was determined by SDS-PAGE: See the Protein Technology Manual. Example 1 : Construction of recombinant plasmid pET25b-SIMEK

The SIMEK gene sequence is artificially synthesized according to the present invention, and according to the sequence, two oligonucleotide fragments are designed with the aid of a computer using E. coli preferred codon. The upstream primer sequence was: 5 '-GCCCATGGCATAGCCACCGCGACTTC-3 ' (the cleavage site of Nco I at the scribe) (SEQ ID No. 3). The downstream primer sequence was: 5,-GCCTCGAGGGCCGCACACTGAGGGAC -3, (the cleavage site of 3⁄4ol I at the scribe) (SEQ ID No. 4). A schematic diagram of the construction of the recombinant plasmid is shown in Figure 1.

The SIMEK gene was obtained by PCR: 94 ° C, 1 min, 60 ° C, 50 s, 72 ° C, 1 min, a total of 30 cycles; ITC, 10 min. The amplification results are shown in Figure 2. There is a significant amplification band around 400 bp. The PCR product obtained by amplification was purified by a kit, digested with restriction endonucleases Nco I and hol I, and ligated with the pET25b plasmid digested with the same restriction enzyme. The ligated product was transformed into E. coli TOP 10 host strain, and positive clones were screened by colony PCR amplification and double digestion. The PCR amplification map of the colony is shown in Fig. 3. It can be seen from the figure that the monoclonal samples 6, 8, and 9 have amplified bands at 400 bp, and the preliminary identification is a positive clone. The SIMEK gene sequence inserted into the plasmid was determined to be correct. Example 2: Expression of SIMEK fusion protein in Escherichia coli BL21 (DE3)

After the E. _CO / BL21 (DE3 ) expressing bacteria were transformed with the correct plasmid pET25b-SIMEK, single colonies were inoculated. In LB liquid medium (containing 50 g / mL ampicillin) overnight at 37 ° C, 1% of the inoculum was transferred to fresh LB liquid medium (containing 50 g / mL ampicillin), cultured at 37 ° C At 3 h, the final concentration of 1.0 mM IPTG was added to induce E. coli expression of the target protein SIMEK5. The cells were recovered by centrifugation and detected by SDS-PAGE. The expression of the recombinant fusion protein SIMEK was about 30% of the total bacterial protein. The results are shown in Figure 4. Cellular localization of the expression of the target protein SIMEK was performed according to the pET operating manual. The results are shown in Figure 5. As can be seen from the figure, the target protein portion is expressed in a soluble form and partially in a bulk form. The formation of inclusion bodies in E. coli is mainly due to the strong promoter, high protein expression efficiency, and inability to fold correctly. Therefore, lowering the induction temperature can appropriately increase the expression of soluble forms. Example 3: Optimization of expression of SIMEK

Previous experiments have shown that lactose can be a good substitute for IPTG to induce the expression of the target protein. Moreover, lactose is not only an inducer, but also a good carbon source, which is beneficial to increase the biomass of engineered bacteria. The present inventors examined the effect of different lactose concentrations on the amount of protein expressed. The specific results are shown in Fig. 6. It can be seen from the figure that both lactose and IPTG can induce the expression of the target protein. Lactose as an inducer can significantly increase the biomass of the bacteria and the expression of the target protein. Therefore, 0.5% lactose was used as an inducer in the experiment. Example 4: Preparation of SIMEK fusion protein

Single colonies were picked from freshly-transferred dishes and placed in LB liquid medium, cultured at 37 °C to log phase as primary seeds, and 1% inoculated into 500 mL shakes containing 70 mL of LB medium. After incubating for 3 h at 37 °C and 215 rpm in the flask, 0.5% lactose was added at 20 °C for 12 h, and the cells were collected by centrifugation at 8500 rpm for 5 min. The cells were disrupted by ultrasonic wave, ie 10 mL of phase A buffer (25 mM imidazole, 1 mol/L sodium chloride, 0.01 mol/L PBS, pH 7.2-7.4) per 1 g of wet cells, sonicated for 30 min, 10000 The supernatant was collected by centrifugation at rpm, 4 °C for 45 min. Column chromatography with Ni-NTA was carried out with a gradient of 45 mmol/L imidazole to 270 mmol/L imidazole. The specific elution conditions were as follows: 45 mmol/L, 70 mmol/L, 170 mmol/L, 270 mmol The volume of 5 bed was eluted by /L imidazole concentration. The distribution was collected and detected by SDS-PAGE. The purity of the sample was detected by SDS-PAGE after SIMEK was eluted at 170 mmol/L imidazole. As shown in Figure 7, the sample purified by Ni-NTA can be electrophoretically pure. Example 5: SIMEK inhibits proliferation of human umbilical vein endothelial cells (HUVEC) and calf aortic endothelial cells (BCE)

HUVEC (purchased from Cascade) cultured to the fourth generation with medium (M200+5.0% FBS+2.0% LSGS) The cells were resuspended, seeded in a 96-well plate at a cell volume of 100 μl, 3000 cells/well, and cultured overnight to allow the cells to adhere. Discard the culture medium, add new complete medium (M200+5.0% FBS+2.0% LSGS) to induce cell proliferation, and add the drug and the control. The positive control is 5-FU. The negative control replaces the drug with the same dose of PBS. , cultured for 72h. After incubation for a corresponding period of time, 10 l of CCK8 reagent was added, and after 4 h of incubation, the absorbance at 450 nm was measured. M200 (Media 200) is a commonly used medium for HUVEC, CCK8 (cell counting kit), similar to MTT, for cell counting.

BCE cells were purchased from the American clones company, and the cell proliferation inhibition assay was the same as the HUVEC cell proliferation assay. The results are shown in Fig. 8, and the results show that the fusion protein SIMEK can significantly inhibit the proliferation of HUVEC cells. When the dose is 250 g/mL, the inhibition rate can reach 58.86%. Similarly, SIMEK also inhibited the proliferation of BCE cells, but there was no significant dose-dependent relationship. The same dose of protein did not significantly inhibit normal mouse fibroblast NIH3T3, indicating that the protein acts mainly on endothelial cells.

Claims

Claim

A recombinant fusion protein, the amino acid sequence of the recombinant fusion protein being SEQ ID No. 2.

2. A nucleotide sequence encoding the recombinant fusion protein of claim 1.

3. The nucleotide sequence of the recombinant fusion protein according to claim 2 is SEQ ID No. 1.

A genetically engineered strain characterized in that the strain is capable of expressing the recombinant fusion protein of claim 1.

5. The method for preparing a recombinant fusion protein according to claim 1, comprising the steps of:

a PCR product is obtained by inserting pMD 18-T which encodes the nucleotide sequence encoding the recombinant fusion protein into a template, and using SEQ ID No. 3 and SEQ ID No. 4 as upstream and downstream primers to collect PCR products;

b. Inserting the PCR product into an expression vector to construct a recombinant expression vector, identifying and transforming the recombinant vector into Escherichia coli; c transforming the transformant to induce expression of the target protein, and collecting and purifying the target protein.

6. The preparation method according to claim 5, wherein the expression vector is pET25b.

7. The preparation method according to claim 5, wherein the inducer is lactose, IPTG.

8. The preparation method according to claim 5, characterized in that the purified protein is subjected to affinity chromatography.

9. The use of the recombinant fusion protein of claim 1 for the preparation of a medicament for inhibiting proliferation of endothelial cells,

10. Use according to claim 9, characterized in that the drug which inhibits the proliferation of endothelial cells is a tumor neovascular inhibitor.

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