1. The thrombopoietin peptide mimetic fusion protein (FC-TMP) coding gene and the application thereof are characterized in that the FC-TMP protein is applied to promoting the generation of platelets and remarkably improving the level of the platelets, and the amino acid sequence of the FC-TMP protein is the sequence 1 in the sequence table.

2. The gene and use of the fusion protein of thrombopoietin mimetic peptide (FC-TMP) of claim 1 wherein the nucleotide molecules encoding the FC-TMP protein are used in the product of promoting the production of platelets and significantly increasing the level of platelets; the nucleotide molecule is a nucleotide molecule with a coding region shown as a sequence 2 in a sequence table.

3. The thrombopoietin peptidomimetic fusion protein (FC-TMP) coding gene and use of claim 1, wherein the use of a recombinant vector, recombinant bacterium, transgenic cell line or expression cassette comprising a nucleotide molecule encoding the FC-TMP protein is in a product that promotes platelet production and significantly increases platelet levels.

4. The thrombopoietin peptidomimetic fusion protein (FC-TMP) coding gene and use of claim 1 wherein a thrombopoietin product is provided.

Background

The thrombopoietin peptide mimetic fusion protein (FC-TMP) coding gene and the application thereof are a fusion protein of FC-TMP and the coding gene and the application thereof, which are widely used in the field of biotechnology pharmacy; the existing thrombopoietin peptide mimetic fusion protein (FC-TMP) coding gene and application find that Idiopathic Thrombocytopenic Purpura (ITP), also called primary immune thrombocytopenia, is an acquired autoimmune hemorrhagic disease, and the existing treatment means usually comprise: glucocorticoid, gamma globulin for intravenous infusion, splenectomy and the like, and medicaments for treating thrombocytopenic purpura mainly comprise the following types: prednisone, dexamethasone, azathioprine, cyclosporine, danazol, cyclophosphamide, vincristine and the like, in the existing medicines for treating ITP patients, corticoid medicines can cause adverse reactions or side effects such as behavior change, secondary serious infection, weight gain, osteoporosis, positive urine glucose and the like; the immune preparation inhibits cellular and humoral immune response, has more adverse reactions, is not suitable for long-term use, comprehensively analyzes the existing treatment condition, has the standard treatment scheme and medicines for newly diagnosed and acute ITP patients at present, has no good treatment medicine at present for chronic intractable ITP (ITP) patients through glucocorticoid, immunoglobulin or splenectomy, and has few medicines for patients to select.

Disclosure of Invention

In order to solve the technical problems, the invention provides a thrombopoietin peptidomimetic fusion protein (FC-TMP) coding gene and application thereof, wherein the gene can promote the generation of platelets, remarkably improve the level of platelets and reduce the bleeding risk, and in addition, the gene contains a human IgG1Fc fragment, so that the half-life period of the gene in vivo can be prolonged.

The invention relates to a thrombopoietin peptide-mimetic fusion protein (FC-TMP) coding gene and application, and provides a nucleotide sequence for coding the fusion protein FC-TMP, wherein a nucleotide molecule for coding the fusion protein is a sequence 2.

The nucleotide molecule is any one of the following nucleotide molecules 1) to 3):

1) the coding region is a nucleotide molecule shown as a sequence 1 in a sequence table;

2) a nucleotide molecule which is hybridized with the nucleotide sequence defined in 1) under strict conditions and has the same function.

3) A nucleotide molecule having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the nucleotide sequence defined in 1) or 2) and having the same function.

The above stringent conditions are hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The nucleotide sequence of 1-668bp from the 5 'end in the sequence 2 is IgG1Fc gene, and the nucleotide sequence of 669-789bp from the 5' end in the sequence 2 is 2 TMP genes (containing a linker) connected in series.

The nucleotide molecules shown in the sequence 2 can code the protein shown by the amino acid residues in the sequence 1 in the sequence table.

In the embodiment of the strain, the expression vector is pET22b, and the recombinant vector is a vector obtained by inserting the nucleotide molecule shown in the sequence 2 in the sequence table between the NheI enzyme cutting sites and the Xho I enzyme cutting sites of the pET22b vector and is named as pET22 b/FC-TMP.

The recombinant strain is a transgenic recombinant strain obtained by introducing the recombinant vector into a host cell; in the examples of the present invention, the host cell is Escherichia coli BL21(DE3) plys S.

The invention relates to a thrombopoietin peptide-mimetic fusion protein (FC-TMP) coding gene and application, and a second purpose is to provide a product for increasing blood platelets.

The active ingredients of the product provided by the invention are any one of the following 1) -3): 1) the above-mentioned protein; 2) the above-mentioned nucleotide molecule; 3) the recombinant vector, the recombinant strain, the transgenic cell line or the expression cassette are used.

Compared with the prior art, the invention has the beneficial effects that: experiments prove that the novel fusion protein FC-TMP is obtained by optimizing codons, and the nucleotide molecules of the fusion protein FC-TMP are introduced into cells through vectors to express and purify the fusion protein FC-TMP suitable for commercial production; FC-TMP is a Thrombopoietin (TPO) receptor agonist, and through specific binding with TPO receptor (c-Mpl), activates cell pathway, stimulates proliferation, differentiation and maturation of megakaryocytes, promotes generation of platelets, remarkably improves the level of platelets, and reduces bleeding risk. In addition, the human IgG1Fc fragment contained can prolong the half-life in vivo.

Drawings

FIG. 1 schematic diagram of FC-TMP expression vector construction;

FIG. 2 expression analysis of FC-TMP genetically engineered bacteria;

FIG. 3 immunoblotting of expression products of FC-TMP genetically engineered bacteria;

FIG. 4 analysis of the digestion pattern of the FC-TMP plasmid;

FIG. 5 shows the digestion results of the plasmids of the FC-TMP engineering bacteria of each generation;

FIG. 6 shows the expression of engineering bacteria F-TMP of each generation;

FIG. 7 Effect of FC-TMP on splenectomized mouse PLT at different times before (0d) and after administration.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1 construction of fusion protein FC-TMP Gene engineering bacteria

1. Optimizing and designing the nucleotide sequence of FC-TMP:

the FC-TMP amino acid sequence in the sequence table 1 is optimized by codon, and Nde I (CATATG) and Xho I (CTCGAG) enzyme cutting sites are respectively added at the 5 ' end and the 3 ' end of the nucleotide according to the requirement of the cloning site of an expression system, wherein ATG in the Nde I (CATATG) is an initiation codon, and a termination codon TAA is added before Xho I (CTCGAG) at the 3 ' end. See in particular sequence table 2.

2. Synthesis of FC-TMP Gene:

finishing the optimally designed nucleotide sequence in the sequence table 2 by entrusting the Enjiki (Shanghai) trade company, adding Nde I and Xho I enzyme cutting sites at two ends of the nucleotide sequence in the sequence table 2 to synthesize a single-chain oligo, carrying out splicing reaction on the synthesized oligo by utilizing PCR, connecting a spliced PCR product to a T carrier, transferring the carrier into host bacteria, and obtaining positive clone by colony PCR. And (4) sequencing and verifying the positive clone, if mutation exists, carrying out mutation repair, and then verifying again until the sequence is correct.

3. Construction and transformation of recombinant vector containing FC-TMP gene:

the construction of the FC-TMP expression vector is schematically shown in FIG. 1.

The synthesized nucleotide sequence of sequence table 2 was double-digested with Nde I and Xho I, the target gene was recovered by agarose electrophoresis, ligated to plasmid pET22b double-digested with Nde I and Xho I in large fragments, transformed into BL21(DE3) plys S to obtain positive clones, and confirmed by nucleotide sequencing of the positive clones. We named pET22b/FC-TMP the expression plasmid of thrombopoietin peptidomimetic cDNA obtained. The genetically engineered bacterium is pET22b/FC-TMP/BL 21.

4. Expression analysis of FC-TMP genetically engineered bacteria:

inoculating a single colony of an engineering bacterium pET22b/TMP/BL21 into an ALB culture solution, culturing at 37 ℃ overnight to be used as a seed, inoculating into an ALB culture medium according to a ratio of 1: 10, carrying out shake culture at 37 ℃ until OD600 is 0.4-0.7, adding 1mM IPTG (isopropyl thiogalactoside) into the ALB culture medium, carrying out induced expression for 4 hours, centrifuging at 4 ℃ and 10000rpm for 5min, and harvesting the thallus. After the sample was lysed and analyzed by SDS-PAGE (see FIG. 2), a distinct thrombopoietin peptidomimetic protein band was visible at a molecular weight of about 29.5kD, consistent with theory. Calculated by optical density scanning, the expressed thrombopoietin peptide mimics account for more than 30% of the total protein of the thallus, which indicates that the thrombopoietin peptide mimics are efficiently expressed.

5. Western Blot analysis of FC-TMP:

carrying out SDS-PAGE electrophoresis on the expression product of pET22b/TMP/BL21, a negative control and a positive control (the negative control is a stock solution of the recombinant human granulocyte stimulating factor, and the positive control is a foreign similar product Nplate medicine), transferring the electrophoresis product to a nitrocellulose membrane, putting the nitrocellulose membrane on a flat plate, adding a confining liquid, and gently shaking the nitrocellulose membrane for 1 hour at 37 ℃. After blocking, the membrane was washed 3 times with PBS and the nitrocellulose membrane was soaked in the primary antibody solution overnight at room temperature. The nitrocellulose membrane was washed 3 times with PBS for 8 minutes each. The membrane was soaked in secondary antibody solution and gently shaken at room temperature for 40 min. The nitrocellulose membrane was taken out, washed with PBS 3 times for 8 minutes each, put into the developing solution, gently shaken at room temperature, observed for color reaction, when the band reached the desired depth, the color development was stopped immediately with water, and then the membrane was transferred to PBS for storage.

The primary antibody is a mouse antiplatelet erythropoietin peptidomimetic polyclonal antibody, the secondary antibody is a horse radish peroxidase-labeled goat anti-mouse immunoglobulin antibody, a substrate DAB is used for developing color, a Western-blot test is carried out, and the immune hybridization analysis is shown in figure 3.

6. Identifying the characteristics of the FC-TMP high-efficiency expression engineering bacteria:

6.1 detection of bacterial morphology, biochemical reactions and antibiotic resistance:

selecting FC-TMP engineering bacteria colony, inoculating in LB culture solution containing ampicillin, performing shake culture at 37 deg.C for 20h, transferring into LB culture solution containing ampicillin at a ratio of 1: 10, and performing amplification culture for 3 h. One part of the plasmid is used for extracting plasmid and performing enzyme digestion map identification; the other part was inoculated on two kinds of LB medium with or without ampicillin and each specific medium, and antibiotic resistance and colony, bacterial type and biochemical reaction were observed.

A lyophilized strain BL21(DE3) plys S was inoculated in LB medium without ampicillin, cultured with shaking at 37 ℃ for 20 hours, then expanded at a ratio of 1: 10, and then inoculated in a medium containing ampicillin or not and various specific media, and antibiotic resistance, colony formation and biochemical reaction were observed. The experiment refers to the culture medium for the X IV bacterial biochemical reaction in the appendix of the three parts (2015 edition) of the Chinese pharmacopoeia. The results of the above experiments are summarized in tables 1, 2 and 3.

TABLE 1 bacterial morphology

TABLE 2 resistance of bacteria to ampicillin

TABLE 3 Biochemical reaction of the strains

Note:indicating the gas production and acid production; + indicates positive; -represents negativity

The above examination revealed that the FV-TMP-engineered bacterium is Escherichia coli having ampicillin resistance.

6.2 plasmid digestion map analysis:

quickly extracting plasmid DNA by an alkaline lysis method, extracting by phenol/chloroform, precipitating by ethanol, drying in vacuum and dissolving in TE buffer solution containing RNase, acting for 30min at 37 ℃ and performing enzyme digestion identification. After digestion analysis with Nde I, Nde I/Xho I, Pst I/Bgl II, Mlu I/Xho I, respectively, and electrophoresis in 1% agarose, and image observation in a gel imaging UV analyzer (see FIG. 4), it can be seen that pET22b/TMP is cut into one band with size 6170bp with Nde I, and two bands with size of 807bp and 5363bp, 22061 bp and 2209bp, 1633bp and 4537bp, respectively, are cut out with Mlu I/Xho I, respectively, which indicates that pET22b/TMP is successfully constructed.

7. Passage genetic stability data of engineering bacteria

The original strain is inoculated on an LB slant non-selection pressure culture medium after amplification culture, which is F1 generation, and then another slant is inoculated on F1 generation, which is F2 generation, and the like until F100 generation. The following examination was performed by selecting each strain of F1, F10, F20, F40, F80, and F100 at intervals and performing the expanded culture (see Table 4).

A. Streaked LB plates.

B. The smear was gram stained.

C. Ampicillin resistance test.

D. The extracted plasmid was digested with Nde I/Xho I, and electrophoresed to check whether it is a large (5363bp) and small (807bp) band, as shown in FIG. 5.

E. The expression level of FC-TMP in shake culture is shown in FIG. 5.

TABLE 4 passage stability observations

8. And (3) detecting the genetic stability of engineering bacteria plasmid:

the plasmid loss condition in the process of the passage of the engineering bacteria is detected by utilizing the characteristic that the recombinant plasmid contains ampicillin resistance genes and continuously culturing in an LB culture medium without ampicillin. The specific operation is as follows:

a single colony was inoculated from the original strain plate in LB liquid medium and cultured overnight at 37 ℃ for F2 generations, and so on to F100 generations. Cultures of generations F1, F10, F20, F40, F80 and F100 were selected at intervals, diluted to 103 with LB medium, 100. mu.l of each of the cultures was plated on LB plates, incubated overnight at 37 ℃, 100 colonies randomly selected the next day were inoculated on ALB and LB plates, incubated overnight at 37 ℃, the difference between the two was compared, and the percentage of lost plasmid was calculated (see Table 5).

TABLE 5 plasmid loss during passage of thrombopoietin peptidomimetic engineering bacteria

As can be seen from the table, the percentage of plasmid loss after the engineering bacteria are passaged for 100 generations in LB culture medium is 0%, which indicates that the plasmid inheritance of the engineering bacteria is stable.

The thrombopoietin peptide-mimetic fusion protein (FC-TMP) coding gene and the application of the invention, during the work, firstly, the full-length cDNA of FC-TMP with Nde I and Xho I enzyme cutting sites at two ends is synthesized, after Nde I and Xho I enzyme cutting, agarose electrophoresis is carried out for recycling, the full-length cDNA is connected with the large fragment of pET22b plasmid which is subjected to Nde I and Xho I double enzyme cutting, and then the large fragment is transformed into BL21(DE3) plys S, so as to obtain positive clone, and DNA sequencing is carried out on the positive clone strain. Sequencing proves that: the synthesized cDNA is consistent with the optimally designed FC-TMP sequence, the sequence comprises 807bp FC-TMP cDNA, and the total code number is 269 amino acid residues, which proves that the synthesized F-TMP cDNA is correct. The engineering bacteria are cultured in LB liquid culture medium in a shaking table, IPTG induction expression is carried out, the culture solution is centrifuged to obtain thalli, and SDS-PAGE electrophoresis is carried out to obtain a protein expression band with the molecular weight of about 29.5KD, and the expression level is more than 30%. The plasmid restriction map is used to confirm that the constructed expression plasmid meets the design requirements. Westernon blot analysis proves that the product has immunoreaction with mouse anti-FC-TMP polyclonal antibody, which indicates that the expression product is correct. And (4) the engineered bacteria are passaged/genetically stable. Therefore, we can obtain the genetic engineering strain with high expression of FC-TMP and genetic stability.

Example 2 in vivo pharmacodynamic assay of fusion protein FC-TMP

2.1 Effect of FC-TMP injection on platelet number (PLT) in splenectomized mice:

the experiment was performed using the mouse splenectomy model [4 ]. Splenectomized mice were randomly divided into FCT-MP high dose (100. mu.g/kg), medium dose (50. mu.g/kg), low dose (10. mu.g/kg), positive control (100. mu.g/kg) and model control (0. mu.g/kg) for injection, with 20 mice per group. The mice were administered once by subcutaneous injection at the back of the neck 48h after splenectomies in a volume of 0.1ml/10 g.

After splenectomy of the mice, single subcutaneous injection is given to FC-TMP for injection, and after administration, a PLT high-dose group is more than a positive control group, a middle-dose group is more than a low-dose group, and the PLT is more than a model control group; PLT began to rise at day 3 after the administration of splenectomized mice, and the high dose reached the highest value (5430X 109/L) at day 5, which was equivalent to the drug effect of the positive control group. The results are shown in FIG. 6.

2.2 Effect of thrombopoietin mimetic peptides for injection on platelet number (PLT) and bone marrow cell classification in Normal rats:

70 rats were divided by platelet count (PLT) into FC-TMP high dose (100. mu.g/kg), medium dose (30. mu.g/kg), low dose (10. mu.g/kg), positive control (100. mu.g/kg) and vehicle control groups for injection, 10 animals per group. Injecting subcutaneously on the back of neck with the administration volume of 0.5ml/200 g; the administration was 3 times per week and 10 times continuously.

The weight of each group of rats shows a remarkable increasing trend after the rats are injected with FC-TMP with different doses subcutaneously; after the rat is dosed for 3 times (3 times per week and 10 times of continuous dosing), the number of the detected platelets shows an ascending trend, and after the rat is dosed for 10 times, the platelets continuously ascend and show obvious dose correlation; platelet counts rose to a maximum at 25d post-dose: a positive control group > a high-dose group > a medium-dose group > a low-dose group > an auxiliary material control group; the number of the blood platelets begins to decrease on the 27 th day after the administration, and the number of the blood platelets of each administration group is basically recovered to be normal after 33 days after the administration; at the end of the test, the rats in each group dissect and take sternum bone marrow for smear and classified counting. The results show that the myeloproliferation of the FC-TMP dose groups is active, which indicates that the thrombopoietin peptide mimics have the effect of promoting the division and proliferation of bone marrow cells. Specific results are shown in tables 6 and 7.

TABLE 7 Effect of thrombopoietin peptidomimetics on the differential enumeration of bone marrow cells in Normal rats

n＝10， ^*p＜0.05，^**p is less than 0.01 and is compared with a model control group;^★p＜0.05，^★★p is less than 0.01 and is compared with a positive control group

In the experiment, the rats are injected with FC-TMP with different dosages subcutaneously, and the influence of the FC-TMP on the number of platelets and the classified counting of bone marrow cells of normal rats is observed. The results show that the weight of rats in each group shows a remarkable increase trend after the rats are injected with different doses of FC-TMP peptide subcutaneously, which indicates that the rats have no remarkable influence on the weight. The number of the blood platelets detected after the rats are dosed 3 times (3 times per week and 10 times of continuous dosing) shows an ascending trend, and the blood platelets continuously ascend after the rats are dosed 10 times of continuous dosing and show obvious dose correlation. Platelet counts rose to a maximum at 25d post-dose: a positive control group > a high-dose group > a medium-dose group > a low-dose group > an auxiliary material control group; the platelet count began to decline on day 27 after administration, and became substantially normal in each group at day 33 after administration. The number of platelets of rats in each dose group of thrombopoietin peptide mimics increases along with the increase of the administration frequency, and the number of platelets gradually decreases after the administration is stopped, which indicates that FC-TMP has certain dose dependence.

At the end of the test, the rats in each group dissect and take sternum bone marrow for smear and classified counting. The results show that the thrombopoietin peptide mimics have active bone marrow hyperplasia in each dose group, which indicates that the thrombopoietin peptide mimics have the function of promoting the proliferation of bone marrow cell division.

Sequence 1

FC-TMP amino acid sequence

Sequence 2

FC-TMP optimized design nucleotide sequence

Note: the red color indicates the Nde I cleavage site,

green color indicates the Xho I cleavage site,

the position of the double-line is used as an initiator,

the single-scribe line location is the terminator,

the bold part is the FC-TMP gene sequence.

The gene encoding thrombopoietin peptidomimetic fusion protein (FC-TMP) and the use thereof according to the present invention are all conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified, and can be used as long as the advantageous effects thereof are achieved.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.