TW201531485A - A fusion protein, a method of making the same, and a method to deliver the antigenic peptide into the endoplasmic reticulum by the fusion protein - Google Patents

Abstract

A method to deliver the antigenic peptide into the endoplasmic reticulum by the fusion protein includes: (a) preparing a fusion protein with a Tat peptide, a (His)6peptide, a ubiquitin, and an antigenic peptide provided in turn from the N-terminal end to the C-terminal end; (b) the fusion protein crossing the cell membrane to the cytosol through the Tat peptide; (c) cleaving the antigenic peptide from the fusion protein by cytosolic ubiquitin C-terminal hydrolases; and (d) transporting the antigenic peptide into the ER by the TAP1/2 transporter on the ER membrane.

Description

Fusion protein, method for producing same, and method for transferring antigenic peptide to endoplasmic reticulum using fusion protein

The present invention relates to a method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein, which can be applied to clinical studies for treating ankylosing spondylitis.

The pathogenesis of ankylosing spondylitis (Akylosing Spondylitis) is associated with the expression of human leukocytic antigen-B27 (HLA-B27), and HLA-B27 belongs to the first major tissue. MHC (major histocompatibility complex, simply referred to as MHC class I) molecules, which is formed from a length-based heavy chain (heavy chain, abbreviated as HC), and a b ₂ microglobulin (b ₂ -microglobin, simply referred to as b ₂ m Composition, and when synthesized in the endoplasmic reticulum, HLA-B27 will bind to an antigenic peptide, after which the binding complex of HLA-B27 and antigenic peptide will be trans-Golgi. It is transported to the cell membrane and the antigenic peptide is presented on the cell surface.

According to a study by Kollnberger S et al. ( Arthritis Rheum , 46:2972-82, 2002) and Dangoria NS et al. ( J Biol Chem , 277:23459-68, 2002), the heavy chain of HLA-B27 is included. Before the binding of b ₂ microglobulin and antigenic peptide in the plastid network, there is a tendency to slowly fold, which leads to the accumulation of misfolded HLA-B27 heavy chains in the endoplasmic reticulum, triggering denatured protein reaction and forming a HLA-B27 heavy chain dimer (B27-HC) ₂ , and HLA-B27 heavy chain dimer (B27-HC) ₂ does not bind to b ₂ microglobulin, when HLA-B27 heavy chain dimer When displayed on the cell membrane, natural killer-cells (NK cells) and T-helper 17 cells are activated, which triggers an inflammatory response and becomes a major potential for ankylosing spondylitis. Pathological factors ( J Immunol , 173: 1699-710, 2004; J Immunol , 186:2672-80, 2011).

In addition, another literature indicates that HLA-B27-bound antigenic peptides help the HLA-B27 heavy chain to fold correctly in the endoplasmic reticulum ( J Immunol, 163:6665-70, 1999; J Exp Med, 180:2163- 71, 1994), therefore, the HLA-B27 heavy chain has become an important target for the treatment of ankylosing spondylitis, such as the efficient delivery of HLA-B27-bound antigenic peptides into the endoplasmic reticulum to promote the HLA-B27 heavy chain. Folding is the potential mechanism for the treatment of ankylosing spondylitis. However, in the current research field, an appropriate technical method has not been developed to deliver antigenic peptides of the correct size and sequence into the endoplasmic reticulum.

In order to solve the above problems, the present invention provides a method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein, which can deliver an antigenic peptide of the correct size and sequence into the endoplasmic reticulum to promote the HLA-B27 heavy chain. Correctly folded inside the endoplasmic reticulum.

To achieve the foregoing object, the present invention provides a method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein, comprising the steps of: (a) producing a fusion protein, wherein the sequence of the fusion protein is from N to C Including a Tat peptide, a (His) ₆ peptide, a ubiquitin and an antigenic peptide; (b) delivering the fusion protein from the cell to the cytoplasm, wherein, fusion The protein is introduced into the cytoplasm of the cell from the outside of the cell by the Tat peptide; (c) the antigenic peptide is cleaved, wherein a ubiquitin C-terminal hydrolase in the cytoplasm can be used. An antigenic peptide is cleaved from the fusion protein; and (d) an antigenic peptide is introduced into the endoplasmic reticulum, wherein the excised antigenic peptide is accessible via a TAP1 on the membrane of the endoplasmic reticulum in the cytoplasm /2 delivers the protein and feeds it into the endoplasmic reticulum.

According to the above, one of the effects of the present invention is that the present invention can effectively deliver the antigenic peptide into the endoplasmic reticulum, and the antigenic peptide obtained can help the HLA-B27 heavy chain to fold correctly and reduce HLA- The amount of B27 heavy chain dimer, which in turn slows down the inflammatory response, therefore, the present invention can be applied to the clinical study of treating ankylosing spondylitis.

By the above, the present invention achieves the second effect, and the present invention can also be applied to the research of cancer treatment. If the antigenic peptide derived from a bacterium is sent to the endoplasmic reticulum of human cells, the antigenic peptide can be expressed on the surface of the cell by HLA-B27 of the human cell, thereby deactivating CD8 ⁺ T cells, thereby causing cell cytotoxicity. The reaction can be applied to the field of cancer treatment research by the present invention. If a specific antigenic peptide is introduced into a cancer cell by the present method, the MHC class I molecule of the cancer cell is presented with the antigenic peptide which is fed to the cell membrane. At this time, the cancer cells are poisoned by activated CD8 ⁺ T cells to achieve the effect of treating cancer.

The foregoing and other technical aspects, features, and advantages of the present invention will be apparent from the description of the accompanying drawings.

Referring to Figures 1 to 3, the present invention provides a method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein, comprising the steps of:

(a) producing a fusion protein 100, wherein the sequence of the fusion protein 100 sequentially contains a Tat peptide 10, a (His) ₆ peptide 20, a ubiquitin 30 and an antigen from the N-terminus to the C-terminus. The sex peptide 40, as shown in FIG. 2, or may sequentially contain (His) ₆ peptide 20, Tat peptide 10, ubiquitin 30 and antigenic peptide 40, as shown in FIG. 3, wherein the antigen The sequence of the sex peptide 40 may be RRFKEGGRGGKY or RRYLENGKETL. When the sequence is RRFKEGGRGGKY, the antigenic peptide 40 is derived from the DNA primase of Chlamydia trachomatis . At this time, the fusion protein 100 is called THUC fusion protein 100, and when the sequence is RRYLENGKETL, the antigenic peptide 40 is derived from a human HLA-B27 heavy chain, and at this time, the fusion protein 100 formed is referred to as THUB fusion protein 100.

The method for producing the fusion protein 100 comprises the following steps: (1) designing a cDNA of the fusion protein 100 and encoding the cDNA via PCR; (2). cloning the cDNA into a vector DNA and transforming the vector DNA Transformation into an E. coli cell strain, allowing the cDNA to be expressed in an E. coli cell line and displaying a large amount of the fusion protein 100; (3) extracting the protein in the E. coli cell strain; and (4). Step (3) extracting the protein for purification step, since the fusion protein 100 contains (His) ₆ peptide 20, the fusion protein 100 is easily purified by the Ni ²⁺ -Sepharose chromatography column, therefore, the first step (3) The extracted protein is subjected to affinity chromatography on a Ni ²⁺ -Sepharose chromatography column, and then subjected to cation exchange chromatography through a fast protein liquid chromatography column (FPLC) to purify and extract the fusion protein 100.

(b) The fusion protein 100 is delivered from the outside of the cell to the cytoplasm. After extracting human peripheral blood mononuclear cells (PBMCs) from patients with ankylosing spondylitis, the patient's PBMCs culture solution is added to fusion protein 100 (5 mm/ml fusion protein 100 per 5 10 ⁶ cells) for PBMCs cell culture. . According to the prior art, the amino acid sequence of Tat peptide 10 is GRKKRRQRRR, which is a densely charged positive peptide, so that Tat peptide 10 can carry the protein fused with it through the cell membrane into the cytoplasm (Adv Drug Deliv). Rev. 57:559-577, 2005), therefore, fusion protein 100 can be taken from the extracellular domain of PBMCs into the cytoplasm of PBMCs by Tat peptide 10.

(c) Excision of the antigenic peptide 40. By the ubiquitin C-terminal hydrolase in the cytoplasm of PBMCs, it can be accurately and automatically cleaved at the C-terminus of ubiquitin protein 30. Therefore, the ubiquitin C-terminal hydrolase can be obtained from the fusion protein. In 100, the antigenic peptide 40 of the present invention is excised.

(d) The antigenic peptide 40 is delivered to the endoplasmic reticulum. The antigenic peptide 40 which is cleaved in the above step (c) can be transported into the endoplasmic reticulum via the endoplasmic reticulum in the cytoplasm of the PBMCs, and a TAP1/2 on the membrane transports the protein.

In addition, the present invention also delivers the fusion protein 100 into the C1R-B2704 cell population, wherein the C1R-B2704 cell line is a human lymphoid cell line, and the cell surface exhibits a misfolded HLA-B27 weight. The method and principle of feeding the antigenic peptide 40 into the endoplasmic reticulum after the fusion protein 100 of the present invention is introduced into the C1R-B2704 cells is the same as the above-mentioned PBMCs.

The above description is the method and principle of the present invention, and the effects that can be achieved by the present invention are as follows:

4A to 4D are data diagrams of the THUC fusion protein 100 or the THUB fusion protein 100 of the present invention, which are fed into cells, wherein FIGS. 4A and 4B are fed into C1R-B2704 cells, and FIGS. 4C and 4D are shown. PBMCs were fed, and the Tf receptors in Figures 4A and 4B were used as an internal control. THU, HUB, and HUC were used as experimental controls. THU was a fusion protein 100 but not The antigenic peptide 40 is contained, while the HUB and HUC are another fusion protein 100 but do not contain Tat peptide 10. When the THUC fusion protein 100 of the present invention is administered into a cell, the amount of the HLA-B27 heavy chain dimer (B27-HC) ₂ can be significantly reduced, as shown in Figures 4A, 4C and 4D, and the THUB fusion protein 100 is sent. The same effect can be obtained after entering the cells, as shown in Fig. 4B.

5A to 5C, after the THUC fusion protein 100 or THUB fusion protein 100 of the present invention is introduced into C1R-B2704 cells, the antigenic peptide 40 is measured by flow cytometry on the surface of C1R-B2704 cells. Data sheet, in which THU, HUB, and HUC are used as experimental control groups, and the Y-axis data is the fluorescence intensity (Mean channel fluorescence, abbreviated as MCF) data of W6/32 monoclonal antibody, and the W6/32 single used in the experiment. The antibody of the strain can bind to the HLA-B27 heavy chain (HC) linked to b ₂ microglobulin (b ₂ m) on the surface of C1R-B2704 cells, so the fluorescence intensity of W6/32 monoclonal antibody can be reacted. The correct amount of HLA-B27 heavy chain folding, and the amount of antigenic peptide 40 bound to HLA-B27 present on the surface of C1R-B2704 cells.

When the THUC fusion protein 100 of the present invention is administered into the C1R-B2704 cells, the amount of the antigenic peptide 40 present on the surface of the C1R-B2704 cells is significantly increased, as shown in FIG. 5A, and the THUB fusion protein 100 is sent. After entering C1R-B2704 cells, the same effect can be obtained. As shown in Figure 5B, if TAP1 shRNA is used to knockdown the TAP1 transport protein on the endoplasmic reticulum of C1R-B2704 cells, either THUC fusion protein 100 or THUB fusion protein 100 was delivered to C1R-B2704 cells, and the amount of antigenic peptide 40 present on the surface of C1R-B2704 cells did not increase, as shown in Fig. 5C.

6A to 6C, the THUC fusion protein 100 or the THUB fusion protein 100 of the present invention is introduced into a C1R-B2704 cell, and the C1R-B2704 cells are presented with an antigenic peptide 40 on the cell membrane thereof, and then the antigen is added. After SDS40-activated CD8 ⁺ T cells, flow cytometry was used to measure the number of C1R-B2704 cells showing apoptosis. The activated CD8 ⁺ T cells were prepared to remove PBMCs from patients with ankylosing spondylitis. After PBMCs were stimulated and activated by interlukin-2 (IL-2) and THUC fusion protein 100 or THUB fusion protein 100, activated CD8 ⁺ T cells were isolated, and THU was used as an experimental control group. TAP1 shRNA knockdown the TAP1 delivery protein in C1R-B2704 cells. In addition, the C1R-B2704 cell line exhibiting apoptosis is calibrated with anti-active caspase 3 antibody, and the data line is represented by the Y-axis in Figures 6A to 6C. .

When the THUC fusion protein 100 of the present invention is administered into C1R-B2704 cells, the number of C1R-B2704 cells exhibiting apoptosis is significantly increased as shown in FIG. 6A, and the apoptosis reaction is confirmed to be activated with CD8. ⁺ T cell correlation, as shown in Figure 6B, the activated CD8 ⁺ T cell line causes a cytotoxicity reaction, causing C1R-B2704 cells to exhibit apoptosis, and conversely, if the THUB fusion protein 100 of the present invention is fed After C1R-B2704 cells, the number of C1R-B2704 cells exhibiting apoptosis did not increase, as shown in Fig. 6C.

Therefore, it is confirmed from the data of Figs. 6A to 6C that if the antigenic peptide 40 is derived from bacteria, the antigenic peptide 40 activates CD8 ⁺ T cells, causing a cytotoxic reaction, and the host cell C1R- B2704 cells exhibit apoptosis. Conversely, if the antigenic peptide 40 is derived from the same human as the host cell, the antigenic peptide 40 does not activate CD8 ⁺ T cells, and thus does not cause a cytotoxic reaction.

In summary, the present invention provides a method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein, which can deliver an antigenic peptide 40 of the correct size and sequence into the endoplasmic reticulum of the cell to promote HLA-B27. The heavy chain is correctly folded in the endoplasmic reticulum, and then the correctly folded HLA-B27 heavy chain can bind to b ₂ microglobulin to form HLA-B27, and the HLA-B27 binds to the antigenic peptide 40, and the antigenicity is obtained. Since the peptide 40 is present on the cell surface, the present invention can reduce the number of HLA-B27 heavy chain dimers, thereby slowing down the occurrence of an inflammatory reaction, and therefore, the present invention can be applied to clinical research for treating ankylosing spondylitis.

In addition, if the antigenic peptide 40 derived from bacteria is sent to a human cell, the antigenic peptide 40 can be expressed on the surface of the cell by HLA-B27 of the human cell to deactivate the CD8 ⁺ T cell, thereby causing a cytotoxic reaction. Therefore, the present invention can be applied to the field of cancer treatment research, and if a specific antigenic peptide is introduced into a cancer cell by the present method, the MHC class I molecule of the cancer cell is presented with the antigenic peptide which is fed to the cell membrane, At this point, the cancer cells are poisoned by activated CD8 ⁺ T cells to achieve the effect of treating cancer.

100‧‧‧ fusion protein
10‧‧‧Tat peptide
20‧‧‧(His) ₆ -peptide
30‧‧‧ ubiquitin
40‧‧‧Antigenic peptide

Figure 1 is a flow chart of the present invention. Figure 2 is a structural diagram (I) of the fusion protein of the present invention. Figure 3 is a structural diagram (II) of the fusion protein of the present invention. 4A to 4D are data plots of the fusion protein of the present invention after it is administered into a cell. 5A to 5C are graphs showing data presented on the cell surface by the antigenic peptide of the present invention. Figures 6A to 6C are graphs showing the number of cells exhibiting apoptosis after the fusion protein of the present invention is introduced into a cell.

100‧‧‧ fusion protein

10‧‧‧Tat peptide

20‧‧‧(His) ₆ -peptide

30‧‧‧ ubiquitin

40‧‧‧Antigenic peptide

Claims (15)

A method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein, comprising the steps of: (a) preparing a fusion protein having a Tat peptide, a (His) ₆ peptide, a pan a protein and an antigenic peptide; (b) delivering the fusion protein from the cell to the cytoplasm, wherein the fusion protein is introduced from one cell to the cytoplasm of the cell by the Tat peptide (c) excising the antigenic peptide, wherein the antigenic peptide is cleaved from the fusion protein by a ubiquitin C-terminal hydrolase in the cytoplasm; and (d) the antigen The sex peptide is sent to the endoplasmic reticulum, wherein the antigenic peptide which is excised can be transported into the endoplasmic reticulum via a TAP1/2 protein on the membrane of one of the cytoplasms. . A method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein according to claim 1, wherein the sequence of the fusion protein sequentially comprises the Tat peptide, the (His) ₆ from the N-terminus to the C-terminus. The peptide, the ubiquitin protein and the antigenic peptide. The method for transmitting an antigenic peptide to an endoplasmic reticulum using a fusion protein according to claim 1, wherein the sequence of the fusion protein sequentially contains the (His) ₆ -peptide from the N-terminus to the C-terminus, the Tat The peptide, the ubiquitin protein and the antigenic peptide. A method for delivering an antigenic peptide to an endoplasmic reticulum using a fusion protein according to claim 1, wherein the amino acid sequence of the antigenic peptide is RRFKEGGRGGKY. The method of claim 1, wherein the amino acid peptide of the antigenic peptide is RRYLENGKETL. A fusion protein having a Tat peptide, a (His) ₆ peptide, a ubiquitin protein and an antigenic peptide, wherein the antigenic peptide is located at the C-terminal position of the fusion protein, and the antigen The sex peptide is linked to the C-terminus of the ubiquitin protein. The fusion protein according to claim 6, wherein the fusion protein comprises the Tat peptide, the (His) ₆ peptide, the ubiquitin protein and the antigenic peptide in sequence from the N-terminus to the C-terminus. . The fusion protein according to claim 6, wherein the amino acid sequence of the antigenic peptide is RRFKEGGRGGKY. The fusion protein according to claim 6, wherein the amino acid sequence of the antigenic peptide is RRYLENGKETL. A method for producing a fusion protein, comprising the steps of: (a) preparing a cDNA of the fusion protein, and encoding the cDNA by PCR; (b) cloning the cDNA into a vector DNA, and transforming the vector DNA Introduced into an E. coli cell strain, the cDNA is expressed in the E. coli cell strain, and the fusion protein is expressed in a large amount; (c) extracting the protein in the E. coli cell strain; and (d) taking the step ( c) The extracted protein is subjected to a purification step to purify the fusion protein. The method for producing a fusion protein according to claim 10, wherein the method for purifying the fusion protein is subjected to affinity chromatography using a Ni ²⁺ -Sepharose chromatography column, and then passed through a fast protein liquid chromatography tube. The column was purified by cation exchange chromatography. The method for producing a fusion protein according to claim 10, wherein the sequence of the fusion protein comprises a Tat peptide, a (His) ₆ peptide, a ubiquitin protein and a sequence from the N-terminus to the C-terminus. Antigenic peptide. The method for producing a fusion protein according to claim 10, wherein the sequence of the fusion protein sequentially comprises a (His) ₆ -peptide, a Tat-peptide, a ubiquitin, and a sequence from the N-terminus to the C-terminus. Antigenic peptide. The method for producing a fusion protein according to claim 12, wherein the amino acid sequence of the antigenic peptide is RRFKEGGRGGKY. The method for producing a fusion protein according to claim 12, wherein the amino acid sequence of the antigenic peptide is RRYLENGKETL.