CN105669871A - Fusion protein of thymulin alpha1 - Google Patents

Abstract

The invention discloses a fusion protein of thymosin α1, which is formed by linking thymosin α1 or its active fragment with the constant region of immunoglobulin through connecting peptide or directly. The fusion protein can be expressed in both eukaryotic cells and prokaryotic cells. The present invention uses recombinant DNA technology to prepare a prokaryotic vector containing the target gene, and expresses, isolates and purifies it in Escherichia coli. Compared with the prototype thymosin α1 fusion protein, it has a longer half-life in vivo and a more efficient anti-tumor effect Effect.

Description

A Fusion Protein of Thymosin α1

technical field

The invention belongs to the technical field of bioengineering and pharmacy, and in particular relates to a fusion protein of thymosin α1.

Background technique

Thymosin α1 is a small-molecule active polypeptide with immunomodulatory effects. Goldstein isolated and purified a small-molecule biologically active polypeptide from thymopoietin 5 component (TF5) in the late 1970s, which has a thymosin-like biological Activity, named thymosin α1, it consists of 28 amino acids, Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys- Glu-Lys-Lys-Glu-Val-Val-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH. Thymosin α1 has been widely used clinically, mainly for primary and secondary immunodeficiency diseases, such as chronic hepatitis B, severe hepatitis B, antiviral treatment of AIDS, etc., as well as pre-chemotherapy and radiotherapy for various malignant tumors adjuvant medication. However, the currently clinically used thymosin α1 has disadvantages such as short half-life stimulation or low immune resistance at the tumor site.

Immunoglobulin IgG is one of the most abundant proteins in the blood, with a half-life in the blood of up to 21 days. Immunoglobulins are generally composed of tetrapeptide chains, that is, two identical light chains (L chains, about 25kd) with a smaller molecular weight and two identical heavy chains (H chains, about 50kd) with a larger molecular weight. Sulfur bonds form a tetrapeptide chain molecule. The L chain of IgG has a variable region (LH) and a constant region LH.

The H chain of IgG consists of a variable region (VH), three constant regions (CH1, CH2 and CH3) and functional regions such as the hinge region between CH1 and CH2. VL and VH are the sites that bind to antigens. CH2 has the complement Clq binding point, which can activate the classic activation pathway of complement; CH3 has the function of binding to the Fc receptors of monocytes, macrophages, granulocytes, B cells and NK cells.

The immunoglobulin constant region Fc is mainly composed of CH2 and CH3. When the antigen is combined with the antibody, Fc can bind to the Fc receptor (FcR), activate the complement system, and play an important role in antibody-dependent cell phagocytosis, antibody-dependent cell-mediated cytotoxicity, and stimulation of inflammatory responses. It has been reported in the literature that combining Fc with some clinically important cytokines, such as interleukin-2, coagulation factor VIII, or with soluble receptors, such as the extracellular domain of tumor necrosis factor receptor, into a fusion protein, can prolong the prototypic factor or receptor Circulation half-life and or increase the original biological activity (PeppelK, CrawfordD, BeutlerB. Atumornecrosisfactor (TNF) receptor-IgGheavychainchimericproteinasbivalentantagonistofTNFactivity.JExpMed.1991Dec1; 174 (6): 1483-9.).

Contents of the invention

The purpose of the present invention is to improve a fusion protein of thymosin α1, which is formed by linking thymosin α1 or its active fragment with the constant region of immunoglobulin through connecting peptide or directly. The fusion protein can be expressed in both eukaryotic cells and prokaryotic cells. The present invention uses recombinant DNA technology to prepare a prokaryotic vector containing the target gene, and expresses, isolates and purifies it in Escherichia coli. Compared with the prototype thymosin α1 fusion protein, it has a longer half-life in vivo and a more efficient anti-tumor effect Effect.

Concrete technical scheme of the present invention is as follows:

A fusion protein of thymosin α1 is formed by linking thymosin α1 or its active fragment with the constant region of immunoglobulin through connecting peptide or directly.

Preferably, said immunoglobulin constant region is the constant region of an immunoglobulin heavy chain.

Preferably, the immunoglobulin is human IgG4, further preferably comprising an immunoglobulin hinge region, constant region CH2 and constant region CH3.

Preferably, the amino acid sequence of the thymosin α1 or its active fragment is shown in SEQ ID NO: 1 or 2, or, the C-terminal amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 or 2 is replaced by an amino acid residue other than the original amino acid residue Any of the nineteen natural amino acids, or the absence thereof. Twenty natural amino acids are glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine , Asparagine, Glutamine, Threonine, Aspartic Acid, Glutamic Acid, Lysine, Arginine and Histidine.

Preferably, the N-terminus of the thymosin α1 or its active fragment is acetylated or non-acetylated.

Preferably, the amino acid sequence of the immunoglobulin constant region is shown in SEQ ID NO:3.

The fusion protein of thymosin α1 according to the present invention preferably has an amino acid sequence as shown in SEQ ID NO:4.

Another object of the present invention is to provide the application of the thymosin α1 described in the present invention in the preparation of drugs for treating primary and secondary immunodeficiency and antitumor.

Preferably, the immunodeficiency disease includes chronic hepatitis B, severe hepatitis B, and AIDS, and the tumor is melanoma.

The fusion protein of thymosin α1 according to the present invention can be prepared by the following method:

(1) Acquisition of genes

The method of gene synthesis is used to obtain thymosin α1 or its active fragment, by linking the peptide or directly with the nucleotide sequence corresponding to the immunoglobulin constant region and its complementary sequence.

(2) Linking the synthesized gene with a prokaryotic expression vector, transforming Escherichia coli, and inducing expression with lactose.

(3) Separating and purifying the expression product.

Advantages of the present invention:

Pharmacodynamic tests prove that the fusion protein of the present invention has stronger anti-tumor effect and longer half-life in vivo than the prototype thymosin α1.

Description of drawings

Fig. 1 is the SDS-PAGE analysis and purification of the thymosin α1 fusion protein of the present invention (wherein M: molecular weight marker, 1: the recombinant thymosin α1 fusion protein induced to express for 1 hour, 2: the recombinant thymosin α1 fusion protein induced to express for 2 hours, 3 : recombinant thymosin α1 fusion protein induced for 4 hours, 4: recombinant thymosin α1 fusion protein induced for 4 hours.

Fig. 2 shows the effect of the thymosin α1 fusion protein of the present invention on inhibiting melanoma in vivo.

detailed description

Embodiment 1 Construction of thymosin α1 fusion protein expression vector

Use the method of gene synthesis to obtain the complementary sequence of the nucleotide sequence corresponding to SEQIDNO: 1 and SEQIDNO: 3. NcoI and HindIII enzyme cutting sites are designed at both ends of the SEQIDNO: 5 sequence, and the 5' of NcoI and HindIII enzyme cutting sites Three guanine protection bases were designed at each end, the complementary sequence was denatured at 94°C for 5 minutes, annealed at 54°C for 10 minutes, then digested with NcoI and HindIII, cloned into the prokaryotic expression vector pET32a, transformed into Escherichia coli, and polymerase chain method Screen for positive transformants. The polymerase chain method primers are:

P1: 5'-CCATGGCTAGCGATGCGGCCGTGGAT-3' SEQ ID NO: 6

P2: 5'-AAGCTTTTATTTACCCGGAGACAGGG-3' SEQ ID NO: 7

The polymerase chain method amplification system is:

10 μl each of primer P1 (25 μmol/L) and primer P2 (25 μmol/L), 5 μl of 10×PCR reaction buffer (Mg2+free), 3 μl of MgCl2 (25 mmol/L), 0.5 μl of dNTPsMix (25 mmol/L), TaqDNA polymerization Enzyme 0.5μl, make up to 50μl with ddH2O. The reaction conditions of the polymerase chain method were denaturation at 94°C for 1 min, annealing at 54°C for 2 min, and extension at 72°C for 2 min, for a total of 30 cycles.

Finally, the nucleotide sequence was determined and analyzed, and the result of the sequence determination was consistent with SEQ ID NO: 5, indicating that the sequence was successfully loaded into the expression vector.

Induction and separation and purification of the recombinant protein of embodiment 2

Inoculate the recombinant bacteria from the single bacterium colony on the plate line to 5ml of LB medium, shake overnight, inoculate into 500ml of ampicillin-resistant liquid LB medium with 1% (V/V) inoculation amount, shake at 37 degrees After incubation for 2.5 hours, when the OD600 is about 0.6, add 1% (V/V) 0.5mol/L lactose and shake for 1, 2, 3, 4 hours to induce the expression of the target protein, and detect it by electrophoresis, see Figure 1.4 Centrifuge at 13,000 rpm for 10 minutes to collect the bacteria, add 20 times the volume of the bacteria in a buffer solution (20mmol/L sodium phosphate at pH 7.4, 0.5mol/L NaCl) to suspend the bacteria, and then perform ultrasonic disruption in an ice bath Bacteria, then 4 degrees of 13000 rpm, centrifuged for 10 minutes, sucked the supernatant and loaded the sample on the metal chelate chromatography column, and first used 10 times of column volume buffer solution (sodium phosphate of 20mmol/LpH7.4, 0.5mol /LNaCl) wash, then elute with 5 times column volume of elution buffer (sodium phosphate of 20mmol/LpH7.4, 0.5mol/LNaCl, 0.1mol/LNaCl, 0.1mol/Lmidazole), and collect the eluate, then load the sample on SephadexG-25 gel column, eluted with 0.01mmol/LNH4HCO3 (pH8.6), collected the penetrating liquid, detected with a spectrophotometer at 280nm to calculate the protein concentration, and finally freeze-dried and stored in a 4-degree refrigerator. As a result, the target protein of electrophoretic purity level was obtained, and the molecular weight was consistent with the theoretical one.

Example 3 Anti-melanoma Experiment in Animals

Murine melanoma B16F10 cells were digested with 0.05% trypsin and centrifuged at 1000rpm for 5 minutes, resuspended in PBS, adjusted to 5,000,000 cells per ml, and inoculated subcutaneously on the side of C57BL/6 (6-8 weeks) mice for about 500,000 cells. When the average volume of the tumor reached 100 mm ³ , the mice were randomly divided into 4 groups, 8 mice/group, except the negative control PBS group and the positive control paclitaxel group, one group was given thymosin α1 (dose 0.25 mg/kg/d), one group Thymosin α1 fusion protein (0.25 mg/kg/d, equimolar relationship with the dose of thymosin α1) was administered to the group, and the administration method was subcutaneous injection on the contralateral side of the tumor every day (except paclitaxel group, which was injected every 3 days) Once, each time is 10mg/kg). The final therapeutic effect is expressed by tumor weight inhibition rate: (1-tumor tumor weight in treatment group/tumor tumor weight in negative control group)×100%

The results are shown in Figure 2, when the treatment reached the 11th day, the tumor inhibition rate of thymosin α1 fusion protein was 30.4%, while the tumor inhibition rate of thymosin α1 was 53.4% (p<0.05). The experimental results show that the thymosin α1 fusion protein designed in the present invention can indeed significantly inhibit the tumor growth in mice, and the anti-tumor growth effect is better than that of the prototype thymosin α1.

Embodiment 4 animal pharmacokinetics determination experiment in vivo

SD rats, clean grade, half male and half female, fasting, drinking water freely, administered thymosin α1 fusion protein in a single tail vein at a dose of 2.632 mg/kg, respectively at 0.5, 1, 3, 5, 10, 24 , 36h, 48h, 60h, 72h rat orbital blood, 12000r/min centrifuge 2min, get the supernatant. ELISA method detection. First, the monoclonal antibody against thymosin α1 was diluted to 1 ug/mL with phosphate buffer, added to a 96-well ELISA plate with a volume of 100 ul per well, and coated overnight at 4 degrees. The next day, the coating solution was discarded and washed 3 times with washing solution. Add blocking solution, 300uL per well, incubate at 37°C for 4h, discard blocking solution, and wash 3 times with washing solution. Add diluted serum samples, 100uL per well (blank wells, negative control wells and positive control wells at the same time), incubate at 37 degrees for 1h, and then wash 3 times. Add 100uL of HRP-labeled goat anti-rat IgG (diluted with blocking solution 1:8000) to each reaction well, incubate at 37°C for 1 hour, and wash 3 times. Then add 0.1 ml of TMB substrate solution to each well, and keep at 37°C for 20 minutes. Add 0.05ml of stop solution 2M sulfuric acid, measure A450 on a microplate reader (reference wavelength 630nm) and measure the absorbance of each well. The results are shown in Table 1. After analyzing the data, it was found that the plasma drug peak time of the thymosin α1 fusion protein of the present invention is about 36 hours, which is 22 times longer than the plasma drug peak time (1.67 hours) of the thymosin α1 fusion protein reported in the literature.

Table 1 Fusion protein concentration (ng/L) of the present invention

Claims (10)

1. the fusion protein of a Thymosin alpha 1, it is characterised in that by Thymosin alpha 1 or its active fragment, by connection peptides or be directly formed by connecting with constant region for immunoglobulin.

2. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterised in that described constant region for immunoglobulin is the constant region of heavy chain immunoglobulin.

3. the fusion protein of Thymosin alpha 1 as claimed in claim 2, is characterized in that described immunoglobulin is human IgG 4.

4. the fusion protein of Thymosin alpha 1 as claimed in claim 3, it is characterised in that described in be comprise immunoglobulin hinge region, constant region CH2 and constant region CH3.

5. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterized in that shown in the aminoacid sequence of described Thymosin alpha 1 or its active fragment such as SEQIDNO:1 or 2, or, the C terminal amino acid residue of aminoacid sequence shown in SEQIDNO:1 replaces with any of 19 kinds of natural amino acids outside original acid residue or disappearance.

6. the fusion protein of Thymosin alpha 1 as claimed in claim 5, it is characterised in that the N end of described Thymosin alpha 1 or its active fragment is acetylizad or non-acetylizad.

7. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterised in that described constant region for immunoglobulin aminoacid sequence is such as shown in SEQIDNO:3.

8. the fusion protein of Thymosin alpha 1 as claimed in claim 1, it is characterised in that the fusion protein aminoacid sequence of Thymosin alpha 1 is such as shown in SEQIDNO:4.

9. the fusion protein of the Thymosin alpha 1 as described in any one of claim 1-8 application in preparation treatment constitutional and secondary immunodeficiency disease and antitumor drug.

10. the fusion protein of Thymosin alpha 1 as claimed in claim 9 application in preparing antitumor drug, it is characterized in that described immunodeficiency symptoms includes chronic hepatitis B, chronic hepatitis A, severe hepatitis, serious symptom hepatitis A and acquired immune deficiency syndrome (AIDS), described tumor includes melanoma, pulmonary carcinoma, breast carcinoma, gastric cancer and colon cancer.