CN115850512A - Recombinant fusion protein for targeted therapy of tumors and preparation method thereof - Google Patents

Abstract

The invention relates to the field of biomedicine, in particular to a recombinant fusion protein for targeted therapy of tumors and a preparation method thereof. According to the invention, a protein transduction domain TAT and GLIPR1 are combined to form a fusion protein, so that the targeting property and the penetrating capability of GLIPR1 are improved, and the defect that GLIPR1 protein enters cytoplasm mainly by virtue of endocytosis is optimized. Compared with GLIPR1 protein, the recombinant fusion protein TAT-GLIPR1 prepared by the invention has the advantages that the cell penetration capacity is obviously enhanced, and the efficiency of the GLIPR1 entering tumor cells is improved. The use concentration of the medicine can be obviously reduced, and the side effect of the medicine is reduced; compared with the traditional gene therapy, the gene therapy avoids the problems of possible toxicity, immunogenicity and the like, can be used as a medicament for targeted therapy of tumors, and provides important technical support for the application of GLIPR1 in targeted therapy of tumors.

Description

Recombinant fusion protein for targeted therapy of tumors and preparation method thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a recombinant fusion protein for targeted therapy of tumors and a preparation method thereof.

Technical Field

Glioma pathogenesis-related protein 1 (GLIPR 1), also known as RTVP1, was first discovered in human glioblastoma by Murphy, e.v. et al 1995, 266 amino acid residues in full length, with a signal peptide and a transmembrane segment, homologous to the PR family (pathogenesis-related protein family) and to the CRISP family (cysteine-rich secretory protein family). At the end of the 20 th century, GLIPR1 was found to exert a cancer-suppressing effect as a cancer-suppressing gene in prostate cancer by inducing apoptosis of cells and degradation of oncogene proteins, and was regulated by p 53. After the lung cancer A549 cells highly express GLIPR1, the growth of the A549 cells is obviously inhibited, and GLIPR1 can also inhibit the growth of mouse lung xenograft tumors. In addition, the expression level of GLIPR1 is low in osteosarcoma and bladder cancer, which indicates that GLIPR1 plays a cancer inhibition role in various solid tumors. The tumor cell vaccine modified by GLIPR1 (AdGlipr 1) and GLIPR1 genes mediated by adenovirus vectors shows significant anti-tumor activity in a prostate cancer mouse model. The GLIPR1 protein (GLIPR 1-delta TM) after removing the transmembrane segment can be specifically absorbed by the prostate cancer cells of mice, the generation of active oxygen is increased, the apoptosis is induced, and the growth of the prostate cancer cell xenograft tumor is inhibited. GLIPR 1-. DELTA.TM has a low absorption rate in normal prostate cells and other tissue cells and no significant killing effect. Therefore, the GLIPR1 protein has strong targeting property on cancer cells and can be developed as an anti-tumor drug with potential. However, no GLIPR1 receptor exists on the cell surface, and how to improve the cell penetrating capacity of the GLIPR1 protein is an urgent problem to be solved.

At present, there are many methods for introducing proteins into cells, including physical methods (e.g., microinjection, electroporation, etc.), chemical or biological perforation (perforin, ATP treatment), particle absorption or fusion (liposome method, cell fusion method). For these methods to be widely used, the following issues must be considered: whether a large number of cells can receive the agent for a considerable period of time; whether an external action is required; how much protein enters the cell; whether the dose absorbed by each cell is the same; how repeatable the technology is; whether the cell is damaged, or altered during the treatment; whether the protein can reach the target in the cell. The above method may be very suitable in a particular situation, but has a large limitation.

The Protein Transduction Domain (PTD) is a domain that has been found in recent years to be highly efficient across biological membranes during protein transport, and it can achieve transport of proteins without relying on receptors and transport proteins, and almost all of the above problems are taken into consideration. Thus, PTDs have a very broad prospect of study, both in theoretical studies and in practical applications of gene therapy. There are three common PTDs: TAT, ANTP and VP22. The PTDs have the common characteristics that the PTDs are polypeptide fragments with positive charges, are rich in basic amino acids (arginine and lysine), can bring biomolecules into cells to play a role, have no selectivity on the cells, have wide histocompatibility, stability and low immunogenicity, can be artificially synthesized, and have simple procedures. Among them, TAT is derived from HIV-1 virus, has the sequence YGRKKRRQRRR, and is the most widely used and most efficient one. In vitro and in vivo experiments prove that the strategy of transferring the medicament required by treatment into cells and animals by using the protein transfer domain can greatly reduce the use concentration of the medicament and reduce side effects, and compared with the traditional gene therapy, the protein transfer domain has important practical application value, but no relevant research report on the fusion protein of the protein transfer domain and GLIPR1 is found.

Disclosure of Invention

In view of the above, in order to overcome the defects of the prior art, the present invention aims to provide a recombinant fusion protein for targeted therapy of tumors and a preparation method thereof. The invention fuses protein transduction domain TAT and GLIPR1 protein to form recombinant fusion protein. It has the advantages of wide histocompatibility, high stability, low immunogenicity, high membrane penetrating capacity, high targeting property, etc.

The scheme of the invention is realized by the following technical means:

the invention provides a recombinant fusion protein for targeted therapy of tumors, wherein the recombinant fusion protein is TAT-GLIPR1, is a fusion protein formed by modifying GLIPR1 through a protein conduction domain, and has an amino acid sequence shown as Seq _ 1.

The recombinant fusion protein TAT-GLIPR1 has a coding gene nucleotide sequence shown as Seq _ 2.

Furthermore, the recombinant fusion protein TAT-GLIPR1 contains a protein transduction domain sequence shown as Seq _3 and a TAT coding gene shown as Seq _ 4.

In another aspect, the present invention also provides a method for preparing a recombinant fusion protein TAT-GLIPR1, said method comprising the steps of:

(1) Taking cDNA clone plasmid pLX304-GLIPR1 containing GLIPR1 gene as a template, designing a specific primer sequence, and obtaining a gene fragment of recombinant fusion protein TAT-GLIPR1 through PCR amplification coding;

(2) The gene fragment is connected with an expression vector pET-15b after double enzyme digestion to form a recombinant expression vector pET-15b-tat-glipr1；

(3) The recombinant expression vector pET-15b obtained in the step (2)tat-glipr1Transforming host cells by heat shock, and constructing and screening high-expression positive host bacteria;

(4) And identifying the positive host bacteria, carrying out amplification culture in host cells, carrying out induced expression on the recombinant protein, collecting an expression product, crushing, centrifuging, denaturing, renaturing and purifying to obtain the recombinant fusion protein TAT-GLIPR1.

Furthermore, the upstream primer sequence of the specific primer sequence in the step (1) is shown as Seq _5, the downstream primer sequence is shown as Seq _6, nde I and BamH I enzyme cutting site sequences are respectively added to the 5' ends of the upstream primer and the downstream primer, and the upstream primer sequence contains TAT sequence.

The double enzyme digestion in the step (2) is carried out by Nde I and BamH I.

The host cell described in step (3) is E. Coli BL21 (DE 3) Codon Plus.

The invention also provides application of the recombinant fusion protein TAT-GLIPR1 in preparation of a targeted drug for treating cancer.

Further, the cancer is prostate cancer or lung cancer. The recombinant fusion protein has stronger cell penetrating capacity compared with GLIPR1 without a conduction domain.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, a protein transduction domain TAT and GLIPR1 are combined to form a fusion protein, so that the targeting property and the penetrating capability of GLIPR1 are improved, and the defect that GLIPR1 protein enters cytoplasm mainly by virtue of endocytosis is optimized. Compared with GLIPR1 protein, the recombinant fusion protein TAT-GLIPR1 prepared by the invention has the advantages that the cell penetration capacity is obviously enhanced, and the efficiency of GLIPR1 entering tumor cells is improved. The use concentration of the medicine can be obviously reduced, and the side effect of the medicine is reduced; compared with the traditional gene therapy, the gene therapy avoids the problems of possible toxicity, immunogenicity and the like, can be used as a medicament for targeted therapy of tumors, and provides important technical support for the application of GLIPR1 in targeted therapy of tumors.

Drawings

FIG. 1 is the recombinant expression vector pET-15b-tat-glipr1Constructing and identifying an electrophoretogram;

FIG. 2 is an SDS-PAGE electrophoretic identification of recombinant fusion protein TAT-GLIPR 1;

FIG. 3 is an SDS-PAGE electrophoretic identification of the purified recombinant fusion protein TAT-GLIPR 1;

FIG. 4 is a graph of a comparative analysis of the uptake rates of GLIPR1 protein and TAT-GLIPR1 protein by different cells;

FIG. 5 is a graph showing the activity of GLIPR1 protein and recombinant fusion protein TAT-GLIPR1 on different cells. Wherein (A) is a growth curve comparison graph; (B) is a graph comparing the number of cells after 6 days of treatment.

Detailed Description

The invention provides a recombinant fusion protein for targeted therapy of tumors and a preparation method thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention. The methods, devices and materials used in the examples which follow, if not specifically indicated, are all conventional and commercially available methods, devices and materials used in the art.

Reagent: pLX304-GLIPR1 was purchased from DNASU, USA.

Example 1: construction of recombinant fusion protein TAT-GLIPR1 recombinant vector

Designing a primer by taking cDNA clone plasmid pLX304-GLIPR1 containing a GLIPR1 gene (HsCD 00441029) as a template, wherein Nde I and BamH I enzyme cutting site sequences are respectively added to the 5' end of the designed primer sequence, the upstream primer sequence contains a TAT sequence, and the upstream primer sequence is shown as Seq _ 5; the sequence of the downstream primer is shown in Seq _ 6. The nucleotide sequence encoding the recombinant fusion protein TAT-GLIPR1 was amplified by PCR using the high fidelity DNA polymerase Pfu. The PCR reaction program is: pre-denaturation at 94 ℃ for 45s; (94 ℃ denaturation 45sec,50 ℃ annealing 45sec,72 ℃ extension 1 min) cycle 3 times; (94 ℃ denaturation 45sec,56 ℃ annealing 45sec,72 ℃ extension 1 min) cycle 30 times; extending for 10min at 72 ℃; storing at 4 ℃. Recovering PCR product and expression vector pET-15b from glue, double enzyme digestion with Nde I and BamH I, purifying PCR enzyme digestion product by phenol-ethanol method, connecting with pET-15b vector enzyme digestion product recovered from glue overnight at room temperature by T4DNA ligase, thermally converting the connection product intoE.The next day, positive clones were picked from the competent cells of coli BL21-CodonPlus (DE 3) to obtain the recombinant expression vector pET-15b-tat- glipr1The sequence is shown in SEQ 7.

And carrying out PCR and double enzyme digestion verification on the obtained recombinant expression vector. FIG. 1 shows a recombinant expression vector pET-15b-tat- glipr1Constructing and identifying an electrophoretogram. Wherein M represents a molecular weight marker; in FIG. 1, the left side is of a recombinant expression vectorThe PCR product identification picture is shown on the right side, the recombinant expression vector pET-15b-tat-glipr1The restriction enzyme map of (1). As can be seen from FIG. 1, the size of the amplified PCR product was approximately 861 bp, and the recombinant expression plasmid pET-15b-tat-glipr1The construction was successful. Meanwhile, the DNA sequencing analysis confirms that pET-15b-tat-glipr1The sequence is correct. The amino acid sequence is shown as Seq _1, and the nucleotide sequence is shown as Seq _ 2.

Note that pET-15b in the present invention-tat-glipr1The recombinant plasmid is not limited to the amino acid sequence shown in the Seq _1 in the sequence table, but also can be the amino acid sequence of a protein which is derived from the sequence shown in the Seq _1 and has the same protein activity by substituting, deleting or adding one or more amino acid residues in the amino acid sequence shown in the Seq _ 1.

Example 2: expression of recombinant fusion protein TAT-GLIPR1

Respectively picking out the recombinant plasmid containing pET-15b-tat-glipr1And of the empty plasmid pET-15bE.A single colony of coli BL21-Codonplus (DE 3) was grown in 2 mL LB liquid medium (ampicillin 100. Mu.g/mL) and shaken overnight at 37 ℃. Transferring the strain into 20 mL of LB culture solution according to the proportion of 1: 100 the next day, shaking at 37 ℃ until Optical density 600 (OD 600) is 0.6, adding IPTG until the final concentration is 1 mmol/L, shaking at 30 ℃ for 4 h, collecting the strain after ice bath for 10min, washing, then resuspending the strain with 500 ul PBS buffer solution, taking out 100 mu L of the strain for whole-strain protein electrophoresis, carrying out ultrasonic disruption on the residual strain at 4 ℃, centrifuging (12 000 r/min,15 min,4 ℃), taking out the whole strain, and carrying out SDS-PAGE electrophoresis on supernatant and precipitate. FIG. 2 is the SDS-PAGE electrophoresis identification picture of the recombinant fusion protein TAT-GLIPR1. Wherein M represents a molecular weight marker; numbers 1 to 6 are respectively empty plasmid transformation bacterium whole bacterial proteins; supernatant of the empty plasmid transformed bacteria; empty plasmid transformed bacterial inclusion bodies; recombinant expression bacteria whole bacterial protein; recombinant expression bacteria supernatant; recombinant expression of bacterial inclusion body. As can be seen from FIG. 2, the bands numbered 4 and 6 gave a clear band of interest at 30kd, indicating that the recombinant plasmid pET-15b-tat-glipr1In thatE.Successfully expressed in coli BL21-CodonPlus (DE 3), which is predominantly present in the form of inclusion bodies.

Example 3: purification of recombinant fusion protein TAT-GLIPR1

Since TAT-GLIPR1 protein is mainly present in inclusion bodies, 5 mL of equilibration buffer (100 mM NaH) was used ₂ PO ₄ 10 mM Tris-Cl, 8M urea, pH 8.0), mixing and ice-bathing for 30 min. Bacteria were lysed ultrasonically during ice-bath (1 mmol/L PMSF added protease inhibitor). Centrifuging at 12 000 r/min at 4 deg.C for 20 min, and collecting supernatant. The supernatant was transferred to a centrifuge tube and 1 mL of NTA-Ni was added ²⁺ Column, rotate gently without interruption, room temperature 2 h. The mixture was transferred to a5 mL syringe, the bottom of which was previously sealed with filter paper. The syringe needle is pulled out to ensure that the liquid can flow out smoothly. Elution buffer (100 mM NaH) was added ₂ PO ₄ 10 mM Tris-Cl, 8M urea, pH 6.3) to remove the hetero-protein, OD280 as the washing solution flows out<0.01 to NTA-Ni ²⁺ And (3) adding 1 mL of eluent into the column, eluting for 4 to 5 times, collecting the eluent, and measuring the protein concentration. SDS-PAGE checks the purity of the purified protein. FIG. 3 is an SDS-PAGE identification of the purified recombinant fusion protein TAT-GLIPR1. Wherein M represents a molecular weight marker; the number 1 is TAT-GLIPR1 protein after gel cutting and purification; 5 to 6 are TAT-GLIPR1 proteins after Ni column affinity purification. As shown in FIG. 3, a clear target band was observed at 30kb, and it can be seen that the purified TAT-GLIPR1 protein was highly pure.

Example 4: in vitro cell activity detection of recombinant fusion protein TAT-GLIPR1

Prostate cancer PC3 cells, lung cancer a549 cells, and normal lung epithelial cells BEAS-2B were inoculated into 96-well plates, 5000 cells per well, and cultured in MEM (10% fetal bovine serum) to logarithmic phase. Dividing the cells into a GLIPR1 protein group, a TAT-GLIPR1 protein group, a negative control group without the drug and a zero adjustment group only containing a culture medium, adding 40 mu M of the drug or PBS buffer solution into each group, measuring the OD value at 570nm by using an MTT method after the drug is treated for 48 hours, and calculating the cell survival rate. FIG. 4 is a graph showing comparative analysis of the uptake rates of GLIPR1 protein and TAT-GLIPR1 protein by different cells. As shown in FIG. 4, the absorption rates of normal lung epithelial cells BEAS-2B to GLIPR1 protein and TAT-GLIPR1 are lower, and the absorption rates of prostate cancer PC3 cells and lung cancer A549 cells to GLIPR1 and TAT-GLIPR1 are obviously higher than that of BEAS-2B; and compared with GLIPR1, the absorptivity of the prostate cancer PC3 cells and the lung cancer A549 cells to TAT-GLIPR1 is obviously increased, which indicates that GLIPR1 and TAT-GLIPR1 can target tumor cells, but TAT-GLIPR1 has higher cell penetration rate compared with GLIPR1.

FIG. 5 is a graph of the activity of the GLIPR1 protein and the recombinant fusion protein TAT-GLIPR1 on different cells; in the figure, (A) is a growth curve comparison graph; (B) is a graph comparing the number of cells after 6 days of treatment; as can be seen from FIG. 5, the GLIPR1 protein and the recombinant fusion protein TAT-GLIPR1 have weak toxic and side effects on normal lung epithelial cells and no obvious killing property, but have larger killing effect on prostate cancer PC3 cells and lung cancer A549 cells and can specifically act on tumor cells. And the killing effect of the recombinant fusion protein TAT-GLIPR1 on prostate cancer and lung cancer cells is obviously stronger than that of GLIPR1 protein, and the cell number is obviously reduced after the treatment of TAT-GLIPR1.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.

Claims (9)

1. The recombinant fusion protein for targeted therapy of tumors is TAT-GLIPR1, is formed by modifying GLIPR-1 through a protein conduction domain, and has an amino acid sequence shown as Seq _ 1.

2. The recombinant fusion protein of claim 1, wherein TAT-GLIPR1 comprises a protein transduction domain sequence as set forth in Seq _3 and a TAT encoding gene as set forth in Seq _ 4.

3. The recombinant fusion protein of claim 1, wherein the recombinant fusion protein TAT-GLIPR1 has a nucleotide sequence of a coding gene as shown in Seq _ 2.

4. A method of making a recombinant fusion protein TAT-GLIPR1, said method comprising the steps of:

(1) Designing a specific primer sequence by taking cDNA clone plasmid pLX304-GLIPR1 containing GLIPR1 gene as a template, and obtaining a gene fragment of recombinant fusion protein TAT-GLIPR1 through PCR amplification coding;

(2) The gene fragment is connected with an expression vector pET-15b after double enzyme digestion to form a recombinant expression vector pET-15b-tat- glipr1；

(3) The recombinant expression vector pET-15b obtained in the step (2)tat-glipr1Transforming host cells by heat shock, and constructing and screening high-expression positive host bacteria;

(4) And identifying the positive host bacteria, carrying out amplification culture in host cells, inducing and expressing the recombinant protein, collecting an expression product, crushing, centrifuging, denaturing, renaturing and purifying to obtain the recombinant fusion protein TAT-GLIPR1.

5. The method according to claim 4, wherein Nde I and BamH I cleavage site sequences are added to the 5' ends of the upstream and downstream primers of the specific primer sequence in step (1), respectively, and the upstream primer sequence contains TAT sequence.

6. The method according to claim 4, wherein the specific primer sequence in step (1) has an upstream primer sequence shown as Seq _5 and a downstream primer sequence shown as Seq _ 6.

7. The process according to claim 4, wherein the double cleavage in step (2) is performed by using Nde I and BamH I.

8. The method according to claim 4, wherein the host cell used in step (3) is E. Coli BL21 (DE 3) Codon Plus.

9. Use of the recombinant fusion protein TAT-GLIPR1 of claim 1 in the manufacture of a targeted medicament for the treatment of cancer.

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