A recombinant fusion protein for targeting tumor therapy and a preparation method thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a recombinant fusion protein for targeted treatment of tumors and a preparation method thereof.The present application combines the protein transduction domain TAT with GLIPR1 to form a fusion protein to improve the targeting and penetration ability of GLIPR1 and optimize the defect that GLIPR1 protein mainly enters the cytoplasm by endocytosis.The recombinant fusion protein TAT-GLIPR1 prepared by the present application has significantly enhanced cell penetration ability compared with GLIPR1 protein, and improves the efficiency of GLIPR1 into tumor cells.The drug use concentration can be significantly reduced, and the drug side effects can be reduced;compared with traditional gene therapy, the problems of possible toxicity and immunogenicity can be avoided, and the recombinant fusion protein can be used as a drug for targeted treatment of tumors, and provides important technical support for the application of GLIPR1 targeted treatment of tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a recombinant fusion protein for targeted treatment of tumors and a preparation method thereof. BACKGROUND

[0002] Glioma pathogenesis-related protein 1 (GLIPR1), also known as RTVP1, was first discovered in human glioblastoma by Murphy, E.V. et al. in 1995. It has a full length of 266 amino acid residues, a signal peptide and a transmembrane segment, and is homologous to the PR family (pathogenesis-related protein family) and the CRISP family (cysteine-rich secretory protein family). At the end of the 20th century, GLIPR1 was found to act as a tumor suppressor gene in prostate cancer by inducing apoptosis and degradation of oncogenic proteins, and was regulated by p53. After high expression of GLIPR1 in lung cancer A549 cells, the growth of A549 cells was significantly inhibited, and GLIPR1 could also inhibit the growth of mouse lung xenograft tumors. In addition, the expression level of GLIPR1 was found to be low in osteosarcoma and bladder cancer, indicating that GLIPR1 plays a tumor suppressor role in various solid tumors. Adenovirus vector-mediated GLIPR1 (AdGlipr1) and GLIPR1 gene-modified tumor cell vaccine showed significant anti-tumor activity in a mouse model of prostate cancer. The GLIPR1 protein after removing the transmembrane segment (GLIPR1-ΔTM) can be specifically absorbed by mouse prostate cancer cells, increase the production of reactive oxygen species and induce apoptosis, and inhibit the growth of prostate cancer cell xenograft tumors. The absorption rate of GLIPR1-ΔTM in normal prostate cells and other tissue cells is low, and there is no obvious killing effect. Therefore, the GLIPR1 protein has strong targeting ability for cancer cells and can be used as a potential anti-tumor drug development. However, so far there is no GLIPR1 receptor on the cell surface, and how to improve the cell penetration ability of GLIPR1 protein is a problem that needs to be solved urgently.

[0003] Currently, there are many methods for introducing proteins into cells, including physical methods (such as microinjection, electric shock, etc.), chemical or biological punching (punching protein, ATP treatment), particle absorption or fusion (liposome method, cell fusion method). In order to make these methods widely used, the following problems must be considered: whether a large number of cells can be treated with reagents for a long period of time; whether external action is needed; how much protein enters the cells; whether the dose absorbed by each cell is the same; how much technical repeatability; whether the cells are damaged or changed during treatment; whether the protein can reach the target in the cell. The above methods may be very suitable in a certain specific situation, but have great limitations.

[0004] Protein transduction domain (PTD) is a domain that can efficiently cross the biological membrane in the process of protein transport in recent years, which can realize the transport of protein without relying on receptor and transporter, and almost considers all the above problems. Therefore, PTD has very wide research prospects in both theoretical research and practical application of gene therapy. There are three common PTDs: TAT, ANTP and VP22. The common point of PTDs is that they are all positively charged polypeptide fragments, rich in basic amino acids (arginine and lysine), can bring biomolecules into cells to play a role, have no selectivity to cells, have wide tissue compatibility, stability, low immunogenicity, can be artificially synthesized, and the program is simple. Among them, TAT comes from HIV-1 virus, the sequence is YGRKKRRQRRR, which is the most widely used and the most efficient one. In vivo and in vitro experiments have confirmed that the strategy of using protein transduction domain to conduct the drug needed for treatment into cells and animal bodies can greatly reduce the concentration of the drug used, reduce side effects, and has important practical application value compared with traditional gene therapy, but there is no related research report on the fusion protein of protein transduction domain and GLIPR1. SUMMARY

[0005] Therefore, in order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a recombinant fusion protein for targeted treatment of tumors and a preparation method thereof. The protein transduction domain TAT and the GLIPR1 protein are fused to form a recombinant fusion protein. It has the advantages of wide tissue compatibility, stability, low immunogenicity, and strong membrane penetration ability and targeting.

[0006] The scheme of the present application is realized by the following technical means:

[0007] The present application provides a recombinant fusion protein for targeted treatment of tumors, wherein the recombinant fusion protein is TAT-GLIPR1, which is a fusion protein formed by modifying GLIPR1 with a protein transduction domain, and the amino acid sequence is shown in Seq_1.

[0008] The recombinant fusion protein TAT-GLIPR1 in the present application has a coding gene nucleotide sequence as shown in Seq_2.

[0009] Further, the recombinant fusion protein TAT-GLIPR1 in the present application contains a protein transduction domain sequence as shown in Seq_3, and a TAT coding gene as shown in Seq_4.

[0010] On the other hand, the present application also provides a preparation method of the recombinant fusion protein TAT-GLIPR1, which comprises the following steps:

[0011] (1) taking the cDNA clone plasmid pLX304-GLIPR1 containing GLIPR1 gene as a template, designing specific primer sequences, and obtaining a gene fragment of a recombinant fusion protein TAT-GLIPR1 by PCR amplification coding;

[0012] (2) connecting the gene fragment after double enzyme digestion with an expression vector pET-15b to form a recombinant expression vector pET-15b- tat-glipr1 ;

[0013] (3) performing heat shock transformation on a host cell by using the recombinant expression vector pET-15b- tat-glipr1 obtained in step (2), constructing and screening a positive host bacterium with high expression;

[0014] (4) identifying the positive host bacterium, culturing and inducing expression of the recombinant protein in the host cell, collecting expression products, crushing, centrifuging, denaturing, renaturing, purifying to obtain the recombinant fusion protein TAT-GLIPR1.

[0015] Further, the upstream primer sequence of the specific primer sequence in step (1) is shown as Seq_5, and the downstream primer sequence is shown as Seq_6, and Nde I and BamH I enzyme digestion site sequences are added to the 5' ends of the upstream and downstream primers, and the upstream primer sequence contains a TAT sequence.

[0016] The double enzyme digestion in step (2) is double enzyme digestion by using Nde I and BamH I.

[0017] The host cell in step (3) is E. Coli BL21 (DE3) Codon Plus.

[0018] The application further provides application of the above-mentioned recombinant fusion protein TAT-GLIPR1 in preparation of a targeted drug for treating cancer.

[0019] Further, the cancer is prostate cancer or lung cancer, and the recombinant fusion protein has stronger cell penetration ability compared with GLIPR1 without a conductive domain.

[0020] Compared with the prior art, the application has the beneficial effects that:

[0021] The present application combines the protein transduction domain TAT with GLIPR1 to form a fusion protein to improve the targeting and penetration ability of GLIPR1, and optimize the defect that GLIPR1 protein mainly enters the cytoplasm by endocytosis. The recombinant fusion protein TAT-GLIPR1 prepared by the present application has significantly enhanced cell penetration ability compared with GLIPR1 protein, and improves the efficiency of GLIPR1 into tumor cells. The drug use concentration can be significantly reduced, and the drug side effects can be reduced; compared with traditional gene therapy, the problems of possible toxicity, immunogenicity and the like can be avoided, and the recombinant fusion protein TAT-GLIPR1 can be used as a drug for targeted treatment of tumors, and provides important technical support for the application of GLIPR1 targeted treatment of tumors. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the construction and identification electrophoretogram of the recombinant expression vector pET-15b- tat-glipr1

[0023] Figure 2 is the SDS-PAGE electrophoretogram for identification of the recombinant fusion protein TAT-GLIPR1;

[0024] Figure 3 is the SDS-PAGE electrophoretogram for identification of the recombinant fusion protein TAT-GLIPR1 after purification;

[0025] Figure 4 is a comparative analysis diagram of the absorption rates of GLIPR1 protein and TAT-GLIPR1 protein by different cells;

[0026] Figure 5 is a diagram of activity detection of GLIPR1 protein and recombinant fusion protein TAT-GLIPR1 on different cells. Wherein, (A) is a growth curve comparison diagram; (B) is a comparison diagram of cell number after 6 days of treatment. DETAILED DESCRIPTION

[0027] The present application provides a recombinant fusion protein for targeted treatment of tumors and a preparation method thereof. Those skilled in the art can refer to the content herein and appropriately improve the process parameters. It should be particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology. The methods, devices and materials in the following implementation examples are all conventional methods, devices and materials in the art if not specifically stated, and can be purchased from the market.

[0028] Reagent: pLX304-GLIPR1 purchased from DNASU company in the United States. ​

[0029] Example 1: Construction of recombinant fusion protein TAT-GLIPR1 recombinant vector

[0030] The cDNA cloning plasmid pLX304-GLIPR1 containing the GLIPR1 gene (HsCD00441029) was used as a template to design primers. The 5' end of the designed primer sequence was added with Nde I and BamH I restriction site sequences. The upstream primer sequence contained a TAT sequence, and the upstream primer sequence is shown in Seq_5. The downstream primer sequence is shown in Seq_6. High-fidelity DNA polymerase Pfu was used to amplify the nucleotide sequence encoding the recombinant fusion protein TAT-GLIPR1 by PCR. The PCR reaction program was as follows: 94°C pre-denaturation for 45 s; (94°C denaturation for 45 s, 50°C annealing for 45 s, 72°C extension for 1 min) for 3 cycles; (94°C denaturation for 45 s, 56°C annealing for 45 s, 72°C extension for 1 min) for 30 cycles; 72°C extension for 10 min; 4°C storage. The PCR product and the expression vector pET-15b were recovered and digested with Nde I and BamH I. The PCR enzyme digestion product was purified by the phenol-ethanol method, and then ligated with the pET-15b vector enzyme digestion product recovered by gel recovery using T4 DNA ligase at room temperature overnight. The ligation product was heat-shocked and transformed into E. coli BL21-CodonPlus (DE3) competent cells. The next day, positive clones were selected to obtain the recombinant expression vector pET-15b- tat- glipr1 , the sequence of which is shown in SEQ 7.

[0031] PCR and double enzyme digestion were performed on the obtained recombinant expression vector. Figure 1 is the recombinant expression vector pET-15b -tat- glipr1 The construction and identification electrophoretogram of the recombinant expression vector pET-15b Figure 1 , wherein the left side is the PCR product identification diagram of the recombinant expression vector, and the right side is the enzyme digestion identification diagram of the recombinant expression vector pET-15b tat-glipr1 . As can be seen from Figure 1 , the amplified PCR product is about 861 bp in size, and the recombinant expression plasmid pET-15b tat-glipr1 is successfully constructed. At the same time, DNA sequencing analysis confirms that the sequence of pET-15b tat-glipr1 is correct. The amino acid sequence is shown in Seq_1, and the nucleotide sequence is shown in Seq_2.

[0032] It should be noted that the pET-15b -tat-glipr1The recombinant plasmid is not limited to the amino acid sequence shown in Seq_1, but can also be an amino acid sequence of a protein derived from Seq_1 with one or more amino acid residues substituted, deleted or added and having the same protein activity.

[0033] Example 2: Expression of recombinant fusion protein TAT-GLIPR1

[0034] The recombinant plasmid pET-15b -tat-glipr1 and empty plasmid pET-15b E. coli BL21-CodonPlus (DE3) single colony in 2 mL LB liquid medium (ampicillin 100 µg / mL), 37 ℃ shaking overnight. The next day, 1:100 scale into 20 mL of LB culture, 37 ℃ shaking to Optical density 600 (OD600) is 0.6, adding IPTG to a final concentration of 1 mmol / L, 30 ℃ shaking for 4 h, ice bath for 10 min, collect bacteria, washed with 500ul PBS buffer resuspended bacteria, take out 100 µL for whole bacterial protein electrophoresis, the remaining bacteria 4 ℃ ultrasonic broken, centrifugation (12000 r / min, 15 min, 4 ℃), take whole bacteria, supernatant and precipitate for SDS-PAGE electrophoresis. Figure 2 is the SDS-PAGE electrophoresis identification diagram of recombinant fusion protein TAT-GLIPR1. Wherein M represents molecular weight marker; No. 1~6 are empty plasmid transformed bacteria whole bacterial protein; empty plasmid transformed bacteria supernatant; empty plasmid transformed bacteria inclusion body; recombinant expression bacteria whole bacterial protein; recombinant expression bacteria supernatant; recombinant expression bacteria inclusion body. From Figure 2 It can be seen that No. 4 and No. 6 get an obvious target band at 30 kd, which shows that the recombinant plasmid pET-15b -tat-glipr1 is successfully expressed in E. coli BL21-CodonPlus (DE3), which mainly exists in the form of inclusion body.

[0035] Example 3: Purification of recombinant fusion protein TAT-GLIPR1

[0036] Since the TAT-GLIPR1 protein mainly exists in inclusion bodies, the inclusion bodies were resuspended with 5 mL of equilibration buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, pH 8.0), mixed well and then ice-bathed for 30 min. During the ice-bathing, the bacteria were ultrasonically lysed (1 mmol / L PMSF was added). The mixture was centrifuged at 12 000 r / min for 20 min at 4°C, and the supernatant was collected. The supernatant was transferred into a centrifuge tube, 1 mL of NTA-Ni 2+ column was added, and the mixture was gently rotated for 2 h at room temperature. The mixture was transferred into a 5 mL syringe, and the bottom was covered with filter paper in advance. The needle of the syringe was removed to allow the liquid to flow out smoothly. Elution buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, pH 6.3) was added to remove impurities, and when the OD280 of the eluate was less than 0.01, 1 mL of elution buffer was added to the NTA-Ni 2+ column for 4-5 times, and the eluate was collected and the protein concentration was measured. SDS-PAGE was used to detect the purity of the purified protein. Figure 3 is an SDS-PAGE electrophoresis identification diagram of the purified recombinant fusion protein TAT-GLIPR1. M represents the molecular weight marker; No. 1 is the TAT-GLIPR1 protein after gel cutting purification; and 5-6 are the TAT-GLIPR1 proteins after Ni column affinity purification. As shown in Figure 3 indicated, there is a relatively obvious target band at 30 kb, and it can be seen that the purity of the purified TAT-GLIPR1 protein is high.

[0037] Example 4: In vitro cell activity detection of the recombinant fusion protein TAT-GLIPR1

[0038] Prostate cancer PC3 cells, lung cancer A549 cells and normal lung epithelial cells BEAS-2B were inoculated in 96-well plates at 5000 cells per well, and were cultured with MEM culture medium (10% fetal bovine serum) to the logarithmic growth phase. The above cells were divided into GLIPR1 protein group, TAT-GLIPR1 protein group, negative control group without drug and zero setting group containing only culture medium, and 40 µM of drug or PBS buffer was added to each group. After 48 hours of drug treatment, the OD value at 570 nm was measured by MTT method, and the cell survival rate was calculated. Figure 4 is a comparative analysis diagram of the absorption rates of GLIPR1 protein and TAT-GLIPR1 protein by different cells. As shown in Figure 4As shown, the normal lung epithelial cell BEAS-2B has low absorption rate of GLIPR1 protein and TAT-GLIPR1, and the prostate cancer PC3 cell and lung cancer A549 cell have significantly higher absorption rate of GLIPR1 and TAT-GLIPR1 than BEAS-2B; and compared with GLIPR1, the prostate cancer PC3 cell and lung cancer A549 cell have significantly increased absorption rate of TAT-GLIPR1, indicating that GLIPR1 and TAT-GLIPR1 can target tumor cells, but TAT-GLIPR1 has higher cell penetration rate than GLIPR1.

[0039] Figure 5 Figure 1 is a diagram of activity detection of GLIPR1 protein and recombinant fusion protein TAT-GLIPR1 on different cells; in the figure, (A) is a growth curve comparison diagram; (B) is a comparison diagram of cell number after 6 days of treatment; and Figure 5 As can be seen, GLIPR1 protein and recombinant fusion protein TAT-GLIPR1 have weak side effects on normal lung epithelial cells and have no obvious killing effect, but have great killing effect on prostate cancer PC3 cells and lung cancer A549 cells and can specifically act on tumor cells. Moreover, the killing effect of recombinant fusion protein TAT-GLIPR1 on prostate cancer and lung cancer cells is significantly stronger than that of GLIPR1 protein, and after TAT-GLIPR1 treatment, the cell number is significantly reduced.

[0040] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A recombinant fusion protein targeting tumor therapy, characterized in that, The recombinant fusion protein is TAT-GLIPR1, which is a fusion protein formed by modifying GLIPR-1 with a protein transduction domain, and the amino acid sequence is shown as SEQ ID NO.

1.

2. A preparation method of a recombinant fusion protein TAT-GLIPR1, the method comprising the following steps: (1) using a cDNA cloning plasmid pLX304-GLIPR1 containing a GLIPR1 gene as a template, designing a specific primer sequence, and obtaining a gene fragment encoding the recombinant fusion protein TAT-GLIPR1 by PCR amplification; (2) double enzyme digestion of the gene fragment and ligation with an expression vector pET-15b to form a recombinant expression vector pET-15b-tat-glipr1; (3) heat shock transformation of the recombinant expression vector pET-15b-tat-glipr1 obtained in step (2) into host cells to construct and screen positive host bacteria with high expression; (4) identification of the positive host bacteria, expansion of the host cells, induction of expression of the recombinant protein, collection of the expression product, crushing, centrifugation, denaturation, renaturation, purification to obtain the recombinant fusion protein TAT-GLIPR1; the amino acid sequence of the recombinant fusion protein TAT-GLIPR1 is shown as SEQ ID NO.

1. In step (1), the 5' end of the upstream and downstream primers of the specific primer sequence is added with Nde I and BamH I enzyme digestion site sequences, and the upstream primer sequence contains a TAT sequence.

3. The preparation method according to claim 2, characterized in that, In step (1), the upstream primer sequence of the specific primer sequence is shown as SEQ ID NO. 5, and the downstream primer sequence is shown as SEQ ID NO.

6.

4. The production method according to claim 2, characterized by, In step (2), the double enzyme digestion is performed with Nde I and BamH I.

5. The preparation method according to claim 2, characterized in that, In step (3), the host cell is E. Coli BL21 (DE3) Codon Plus.

6. The method of claim 2, wherein, 7. Use of the recombinant fusion protein of claim 1 in the preparation of a targeted drug for treating cancer; the cancer is prostate cancer or lung cancer. ​

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