CN115991789B - Fusion protein based on SH2 super parent and application thereof - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to a fusion protein based on SH2 super parent and application thereof, belonging to the field of biotechnology. The fusion protein contains a chronic granulocytic leukemia cell specific transmembrane peptide and also contains an SH2 super-parent, and the chronic granulocytic leukemia cell specific transmembrane peptide is connected with the SH2 super-parent. The invention is based on the advantages of leukemia cell targeting transmembrane peptide and SH2 super-parent, prepares a fusion protein of leukemia cell targeting transmembrane peptide CAYHRLRRC-SH2 super-parent, namely CR-sSH2 fusion protein, and is used as a targeting candidate medicament for treating CML, the fusion protein has the function of specifically recognizing leukemia cells of leukemia cell targeting transmembrane peptide CAYHRLRRC, CAYHRLRRC polypeptide can help to introduce SH2 super-parent into cells, SH2 super-parent entering the cells can replace natural SH2 structural domain to combine with a plurality of tyrosine phosphorylation sites, and then signal paths related to cell proliferation and apoptosis are blocked.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to fusion protein based on SH2 super parent and application thereof, in particular to protein polypeptide with strong affinity to phosphorylated tyrosine residues (phosphotyrosine, pY) of protein, gene recombination, expression purification of prokaryotic vector fusion protein and the like, and particularly relates to CR-sSH fusion gene and application of prokaryotic expression vector thereof.
Background
1. Current state of treatment for chronic granulocytic leukemia
Chronic myelogenous leukemia (Chronic myelocytic leukemia, CML) is a malignant hematological disease that originates in hematopoietic stem cells, accounting for about 20% of adult leukemias. The diagnosis and treatment standard (2019 edition) of adult chronic granulocytic leukemia reports that the global annual incidence rate of CML is about (1.6-2)/10 ten thousand, and the annual incidence rate of China is about (0.36-0.55)/10 ten thousand. With age, the incidence of CML has a gradual rise and is seriously harmful to human health. Patients often experience extremely high blood granulocyte fractions with concomitant clinical symptoms of immature granulocytes, anemia, splenomegaly, etc., which are commonly found by chance during physical examination and confirmed by karyotyping. Existing data statistics indicate that Philadelphia (Ph) chromosomes are present in over 95% of patient hematopoietic cells, and occur as a result of translocation of the oncogene c-abl on human chromosome 9 to the gene bcr on chromosome 22, i.e., the occurrence of balanced alternate ectopic (t 9, 22) (q 34, q 11) forms the fusion gene bcr-abl. The gene can code fusion protein BCR-ABL with continuous tyrosine kinase activity, so that a plurality of relevant channels in cells are activated, abnormal proliferation of cells, blocked apoptosis capability, reduced intercellular adhesion and abnormal DNA repair are caused, and the occurrence and development of CML are promoted.
The main mode of existing treatment for chronic myeloid leukemia is tyrosine kinase inhibitors (Tyrosine kinase inhibitors, TKIs), of which Imatinib (Imatinib) is the most widely used one approved by the FDA in the united states for the treatment of CML in 2001, and one year later becomes the first line drug for the treatment of CML, leading to a survival rate of 85% -90% in 10 years for CML patients. But with increased patient survival, intolerance and multiple drug resistance. The developed secondary tyrosine kinase inhibitors, such as TKIs (such as nilotinib (Nilotinib) and Dasatinib (Dasatinib) and the like, are used as first-line therapeutic drugs, have stronger inhibition capability on BCR-ABL, and can obtain faster and deeper molecular reactions. Although it has a certain curative effect on most imatinib-resistant patients, its effect still has a certain limitation, such as the curative effect on ABL-T315I mutant patients is not significant. The flumatinib (Flumatinib) is used as a first-second generation TKI in China, is an upgrade version of the imatinib from the molecular structure, has better curative effect, but has similar adverse reaction rate with the imatinib. CML patients with T315I mutation are resistant to all current generation and second generation BCR-ABL inhibitors, so there is a great clinical need for third generation BCR-ABL inhibitors that can effectively treat CML with T315I mutation. The orinbatinib (GZD 824, HQP 1351) is a global second and domestic first oral TKI inhibitor, is incorporated into a priority review and breakthrough treatment variety by Chinese drug administration, is also granted with rapid channel qualification and orphan drug qualification by the U.S. FDA, can target wild BCR-ABL and T315I mutation thereof, and can overcome clinical drug resistance problem of a first-generation BCR-ABL inhibitor. However, TKIs act only on differentiated cancer cells but cannot eliminate leukemia stem cells, so lesions cannot be completely cleared, and their expensive selling price also makes many families affordable. In addition to TKIs, allogeneic hematopoietic stem cell transplantation (allosct) has been the primary method of treatment for CML, but its application conditions are very demanding, and have a relationship with age, donor, disease progression and economic status of the patient, and may also be subjected to severe immune rejection after surgery, gradually becoming a two-line or even three-line treatment option after TKIs treatment failure or intolerance. Therefore, the search for drugs with multiple targets, less susceptibility to drug resistance and lower cost is important for the treatment of CML.
2. Overview of phosphorylated tyrosine and SH2 superparents and their antitumor applications
Tyrosine phosphorylation of proteins plays an important role in many vital activities of cells, and abnormal tyrosine phosphorylation is particularly relevant to cancer. There are many documents reporting that the pathogenesis of CML is closely related to abnormal tyrosine phosphorylation of proteins, and a plurality of signal channels in cells are abnormally activated, so that proliferation of cells is inhibited, and apoptosis is promoted. SH 2domain (Src homolog 2 domain) can specifically bind pY and play an important role in cell signaling. Compared with SH2 structural domain or anti-pY antibody in natural state, the SH2 super parent has better binding capacity to protein or polypeptide containing pY, can bind a large number of different pY proteins, thus blocking various signal paths in cancer cells, further affecting various vital activities such as proliferation, apoptosis and the like of the cells, and effectively killing the cancer cells. Most of the current CML-targeted therapeutic drugs are single targets, and although the early-stage therapeutic effect is better, with wide application, many CML patients gradually develop drug resistance. And because of the multi-target property of SH2 super parent, the drug resistance caused by a series of gene mutation, drug inactivation, DNA damage repair and other reasons is likely to be improved to a certain extent, and the generation of the drug resistance is delayed. In recent years, the subject group is focused on the application research and mechanism exploration of SH2 super-parent for treating cancers, and the fact that SH2 super-parent can strongly bind to various pY proteins in various cells (breast cancer, melanoma, pancreatic cancer, non-small cell lung cancer and the like) has been verified, and with the help of cell penetrating peptide (Arg) 9, SH2 super-parent proteins enter cancer cells to block various signal paths mediated by pY in the cells, so that various vital activities of the cells are influenced, and the purpose of treating cancers is achieved.
3. Leukemia cell Membrane-penetrating peptide overview
Phage display technology developed by George p.smith was shared with the "enzyme directed evolution" technology in 2018 as a highly applied high-throughput screening technology. The technique can be generally divided into three steps: 1) Insertion of exogenous genes: inserting a specific exogenous gene into the phage protein gene using a gene recombination technique; 2) Amplifying and displaying the foreign protein: infecting the phage with exogenous genes on the surface of escherichia coli host bacteria, and expressing the exogenous genes so that the exogenous proteins can be displayed on the surface of the phage; 3) Biopanning: phage that bind to a particular target molecule are screened through a particular "bind-elute-bind" procedure to determine their polypeptide sequence. The technology has been successfully applied to polypeptide medicine research of various cancers, and leukemia cell targeting transmembrane polypeptide CAYHRRLRC is obtained by screening a random heptapeptide library based on phage display technology, and the sequence can be functionally divided into two parts: wherein the CAY sequence has the ability to specifically bind to leukemia cells, and the RLRRC sequence has the property of penetrating the cell membrane.
Disclosure of Invention
The invention solves the problem of providing an application of recombinant protein, namely chronic granulocytic leukemia cell penetrating peptide CR-SH2 super parent fusion protein (called CR-sSH for short below) and related expression vector thereof. The fusion protein CR-sSH can be competitively combined with protein containing tyrosine phosphorylation (pY) in chronic granulocytic leukemia cells, so as to block a plurality of signal paths mediated by endogenous SH2 structural domains/pY, inhibit the proliferation, movement and other vital activities of tumor cells, thereby achieving the anti-tumor effect and being applicable to the research and development of anti-chronic granulocytic leukemia drugs.
According to a first aspect of the present invention there is provided a fusion protein based on an SH2 superparent, said fusion protein comprising a chronic myelogenous leukemia cell-specific transmembrane peptide linked to said SH2 superparent, and further comprising an SH2 superparent.
Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 1.
According to another aspect of the present invention, there is provided a method for constructing an expression vector, comprising the steps of:
(1) Amplifying to obtain a gene segment for expressing SH2 super parent;
(2) Constructing the gene fragment obtained in the step (1) on an expression vector to obtain a recombinant plasmid containing the gene fragment of the SH2 super parent;
(3) And (3) taking the recombinant plasmid obtained in the step (2) as a template, and carrying out PCR amplification by using a forward primer shown in SEQ ID NO. 2 and a reverse primer shown in SEQ ID NO. 3 to obtain an expression vector containing the gene fragment of the SH2 super parent and the gene fragment of the chronic granulocytic leukemia cell specific transmembrane peptide.
Preferably, the expression vector of step (2) is pET-28a, pGEX-4T3, pETM or pETM.
According to another aspect of the invention, there is provided an expression vector constructed by the method.
According to another aspect of the present invention, there is provided a method for expressing and purifying an SH2 super-parent-based fusion protein, transforming the expression vector into competent cells, inducing the expression of a target protein, and purifying the target protein using agarose gel magnetic beads and imidazole eluate.
According to another aspect of the invention, there is provided the use of said SH 2-super-parent-based fusion protein for the preparation of a medicament for targeted treatment of chronic myeloid leukemia.
Preferably, the fusion protein is capable of specifically binding to phosphorylated tyrosine in chronic granulocytic leukemia cells, thereby inhibiting proliferation of chronic granulocytic leukemia cells.
In general, compared with the prior art, the above technical solution conceived by the present invention mainly has the following technical advantages:
The invention is based on the advantages of leukemia cell targeting transmembrane peptide and SH2 super-parent, prepares a fusion protein of leukemia cell targeting transmembrane peptide CAYHRLRRC-SH2 super-parent, namely CR-sSH2 fusion protein, and is used as a targeting candidate medicament for treating CML, the fusion protein has the function of specifically recognizing leukemia cells of leukemia cell targeting transmembrane peptide CAYHRLRRC, CAYHRLRRC polypeptide can help to introduce SH2 super-parent into cells, SH2 super-parent entering the cells can replace natural SH2 structural domain to combine with a plurality of tyrosine phosphorylation sites, and then signal paths related to cell proliferation and apoptosis are blocked. Meanwhile, the targeting advantage of specific recognition and entering into chronic granulocytic leukemia cells can effectively avoid damage to normal cells, and the capability of SH2 super-parent to block multiple channels enables the fusion protein to have multi-target property, so that the problem that traditional medicines are easy to generate drug resistance is solved. The fusion protein has low preparation cost, is easy to obtain, can greatly lighten the economic burden of patients, and has larger application value and wide application prospect.
Drawings
Fig. 1: sSH 2A structural schematic diagram. SH2 super parent (sSH 2), namely SH2 triple mutant (on the basis of natural SH2 domain to 8 th, 10 th and 15 th amino acid mutations, the corresponding amino acid from threonine mutation to valine, cysteine mutation to alanine, lysine mutation to leucine mutation.
Fig. 2: schematic of CR-sSH2 fusion protein anti-tumor pattern: the fusion protein of the leukemia cell targeting membrane-penetrating peptide CAYHRLRRC-SH2 super-parent, namely CR-sSH2 fusion protein, is used as a targeting candidate medicament for treating CML, has the function of specifically recognizing leukemia cells of the leukemia cell targeting membrane-penetrating peptide CAYHRLRRC, and CAYHRLRRC polypeptide can help to introduce SH2 super-parent into cells, and SH2 super-parent entering the cells can replace natural SH2 to combine with a plurality of tyrosine phosphorylation sites so as to block signal paths related to cell proliferation and apoptosis.
Fig. 3: construction of CR-sSH2 fusion proteins. A. And detecting the construction of the CR-sSH2 recombinant plasmid by nucleic acid electrophoresis. B. And detecting the pET-28a (+) vector by nucleic acid electrophoresis.
Fig. 4: expression and purification of CR-sSH2 fusion proteins. Coomassie blue staining demonstrated expression and purification of CR-sSH2 fusion proteins.
Fig. 5: the CR-sSH2 fusion protein can bind to pY in CML cells. In the chronic myelogenous leukemia cell lines K562, K562-r, CR-sSH2 bound more tyrosine phosphorylated protein than CR-wSH 2.
Fig. 6: CR-sSH2 fusion proteins are able to specifically recognize and efficiently enter CML cells. CR-sSH2 fusion protein can specifically identify and efficiently enter human chronic granulocytic leukemia cells rather than normal blood cells. CR-sSH2 fusion protein was unable to enter MCF7 and SW1990 cells. Scale bar:10 μm.
Fig. 7: CR-sSH2 fusion protein can significantly inhibit the proliferation capacity of CML cells. K562 cells were incubated with CR-sSH2 fusion protein at various concentrations (0.25,0.5,1,2,4,8 and 16. Mu.M); k562 cells were incubated with the fusion protein at different times (1, 2,4 and 8 h); k562-r cells were incubated with CR-sSH2 fusion protein at various concentrations (0.25,0.5,1,2,4,8 and 16. Mu.M); k562-r cells were incubated with fusion protein at different times (1, 2,4 and 8 h) (n=3, < P < 0.05); CR-sSH2 has no obvious inhibition to normal hematopoietic cells; the CR-sSH protein has an inhibiting effect on proliferation of primary cells of patients with chronic granulocytic leukemia. (n=6, < P < 0.05)
Fig. 8: CR-sSH2 fusion protein can induce K562 cells to generate apoptosis. A and B. Effects of CR-sSH2 on K562 apoptosis were detected using flow cytometry using an Annexin V-FITC/PI stain.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other. Various equivalents, substitutions, modifications or alterations are also possible in light of the ordinary skill and knowledge of those in the art without departing from the spirit and scope of the invention.
The invention relates to a construction of a recombinant sSH < 2 > vector, which uses the existing pETM-His-w/sSH < 2 > plasmid in a laboratory as a template, uses PCR technology to amplify and react to obtain sSH < 2 >, wSH < 2 >, CR-sSH < 2 > and CR-wSH < 2 > target fragments, uses double enzyme digestion to obtain a linearization vector pET-28a (+), respectively constructs a plurality of plasmids according to the homologous recombination principle, but not limited to the vector, including all empty vectors suitable for expression and purification, such as pGEX-4T2, pETM11 and the like, and can also be other possible expression vectors (but not limited to) plasmids or modified viruses (such as replication-defective retroviruses, adenoviruses and the like), as long as the vector is compatible with the host cells. Depending on the host cell selected and the expression vector used, other additional sequences (e.g., replication origin, additional DNA restriction endonuclease sites, enhancers, etc. sequences) may be introduced into the expression vector. In addition, the invention encompasses nucleic acid vectors comprising a monomer encoding at least one CR-sSH 2.
The CR-sSH2 is connected with an empty vector pET-28a (+) through a molecular cloning means of homologous recombination, and a prokaryotic expression vector pET-28a (+) -CR-sSH2 is constructed. Not limited to this vector, but includes all empty vectors suitable for expression purification, the vector constructed is Plasmid-CR-sSH2.
The CR-sSH2 is prepared by inserting CAYHRRLRC into pET-28a (+) -sSH2 to construct pET-28a (+) -CR-sSH2, and the purified protein CR-sSH can be directly added into a cell culture medium to co-culture with cells, enter the cells by utilizing the penetrating effect of CR, and further inhibit the proliferation of the cells. The use of cell penetrating peptides is not limited to CR alone, or any other carrier material that facilitates sSH's 2 delivery to chronic myeloid leukemia cells.
SH2 super parent (sSH 2), namely SH2 triple mutant (mutation of amino acid at 8 th, 10 th and 15 th positions is carried out on the basis of natural SH2 domain, corresponding amino acid is respectively changed from threonine to valine, cysteine to alanine, lysine to leucine, and figure 1), has better binding capacity to protein or polypeptide containing pY compared with SH2 domain (wSH 2) or antibody against pY in natural state. Based on the characteristics, SH2 super parents can combine a large number of different pY proteins, so that various signal paths in cancer cells are blocked, various vital activities such as proliferation, apoptosis and the like of the cells are influenced, and the cancer cells are effectively killed. The gene sequence for expressing SH2 super parent (sSH 2) is shown as SEQ ID NO. 4.
The invention utilizes the advantages of the leukemia cell targeting membrane penetrating peptide and the SH2 super parent to prepare a 'leukemia cell targeting membrane penetrating peptide CAYHRLRRC-SH2 super parent' fusion protein, namely CR-sSH2 fusion protein, which is used as a targeting candidate medicament for treating CML, has the function of specifically recognizing leukemia cells of the leukemia cell targeting membrane penetrating peptide CAYHRLRRC, CAYHRLRRC polypeptide can also help the SH2 super parent to enter cells, and the SH2 super parent entering cells can replace a natural SH2 domain to combine with a plurality of tyrosine phosphorylation sites (pY), so as to block signal paths related to functions of cell proliferation, apoptosis and the like (figure 2). The targeting advantage of specific recognition and entering into chronic granulocytic leukemia cells can effectively avoid damage to normal cells, and the capability of SH2 super parent to block multiple channels enables the fusion protein to have multi-target property, so that the problem that traditional medicines are easy to generate drug resistance is avoided. Cloning the gene fragment corresponding to the CR-sSH2 fusion protein into the expression vector pET-28a (+) is not limited to the use of the vector, as long as the expression vector is easy to express and purify the target protein, and the expression vector used herein has an HIS tag, so that the purification and detection of the protein are facilitated. The CR-sSH2 fusion protein is expressed in the escherichia coli BL21, can be specifically combined with tyrosine residue phosphorylation residue of protein, has strong affinity of sSH protein and pY, and can specifically identify chronic granulocytic leukemia cells. If CR-sSH2 is introduced into a pY-overactivated chronic granulocytic leukemia cell, sSH2 will replace the endogenous SH2 domain in the cancer cell, and will competitively bind to the pY protein, thereby blocking multiple signaling pathways involved in the endogenous pY protein/SH 2 domain, as shown in FIG. 2.
The penetrating peptide composed of CAYHRLRRC sequences has the characteristic of directly targeting chronic granulocytic leukemia cell membrane into cells, and in order to facilitate the introduction of sSH protein into cancer cells, recombinant plasmid expression CR-sSH2 fusion protein is constructed by utilizing a genetic engineering method, and the membrane penetrating capability and anti-tumor effect of the penetrating peptide are studied. The transmembrane peptide used herein is CAYHRLRRC sequences, but is not limited to this transmembrane peptide, any other carrier material that facilitates delivery of sSH2 to cells or tissues. The experimental result shows that CR-sSH2 can penetrate and enter cancer cells, sSH2 replaces an endogenous SH2 structural domain in the cancer cells and performs competitive binding with pY protein, so that a plurality of signal paths related to the endogenous pY protein/SH 2 structural domain are blocked, proliferation and invasion of tumor cells are inhibited, and the anti-tumor effect is obvious.
Example 1: construction of pET-28a-CR-sSH2 fusion gene expression vector
Cloning of 1.1wSH2 (SH 2 Domain) and sSH2 (SH 2 super parent) genes of interest
The existing pETM-His-wSH 2 and pETM-His-sSH plasmids in the laboratory are used as templates, and PCR technology is adopted to amplify wSH and sSH2 target fragments respectively. The primer sequences are shown in Table I. The specific PCR reaction system is as follows:
the PCR instrument reaction procedure was as follows:
And (3) carrying out agarose gel electrophoresis on the obtained PCR product to identify whether the size is correct, and carrying out gel cutting recovery on the strip at the correct position and then purifying by using a kit for later use.
1.2 Construction of pET-28a-w/sSH2-His recombinant plasmid vector construction
Selecting a pET-28a (+) expression vector as a background vector according to an experimental design, and selecting two sites of NcoI and XhoI on the vector for double digestion, wherein the reaction system is as follows:
And (3) carrying out low-speed centrifugal mixing on the mixed product, rapidly placing the product in a water bath kettle at 37 ℃ for enzyme digestion reaction after finishing, and recovering the same cut rubber of the product obtained after 2 hours and purifying for later use.
The target fragments wSH and sSH2 amplified by PCR are respectively inserted into a double-digested pET-28a (+) vector, and a one-step cloning kit is used for constructing a pET-28 a-w/sSH-His recombinant plasmid according to the experimental principle of homologous recombination, wherein the system is as follows:
if other proper expression vectors are used, corresponding connection sites can be selected according to the specific conditions of restriction enzyme sites and the basic principle of primer design, corresponding target fragment amplification primers can be designed, and the reaction system of homologous recombination can be adjusted. The principle used is consistent, but there may be minor differences in the amount of material used in the implementation. All vectors suitable for expression purification, such as pGEX-4T3, pETM30, pETM11, etc., may be other possible expression vectors (but are not limited to) plasmids or modified viruses (e.g.replication defective retroviruses, adenoviruses, etc.), as long as the vector is compatible with the host cell used.
After the connection reaction is completed, the DH5 alpha competent cells are transformed by the connection products, after the connection products are cultured overnight, monoclonal is selected, the culture is carried out by shaking, the bacterial liquid PCR preliminary identification is carried out, and then the sequencing verification is carried out; the successfully constructed recombinant plasmid was designated pET-28a-w/sSH2-His (recombinase, ligation reagents were purchased from Nanjinopran Corp.).
If other suitable vectors are used, the successful vector names are the corresponding Plasmid-wSH and Plasmid-sSH2 or are named according to their specific vector names.
1.3 Construction of prokaryotic expression vector of CR-sSH2 fusion Gene
The existing research shows that the membrane penetrating peptide with the sequence CAYHRLRRC can directly penetrate through the cell membrane of chronic granulocytic leukemia cells to enter the cells, and in order to facilitate the introduction of sSH protein into living cells, a genetic engineering method is utilized to construct recombinant plasmid to express pET-28a-CR-w/sSH2-His. The pETM-w/sSH 2-His plasmid which is successfully constructed in the experiment is used as a template, the PCR related primers are designed as shown in the table I, and CR sequences are added in front of the designed forward primers because CR-wSH and CR-sSH target fragments are required to be amplified respectively, and the PCR reaction system is as follows:
the PCR instrument reaction procedure was as follows:
and after the PCR program is finished, taking out the PCR tube, adding a 6×loading buffer, performing agarose gel electrophoresis to check the position of a product band, and cutting gel and recovering the obtained CR-w/sSH2 target fragment to obtain a purified product. The empty vector and homologous recombination are the same as before.
The nucleotide sequence of the target gene CR-w/sSH-His was amplified by PCR, and the pET-28 a-CR-w/sSH-His recombinant plasmid was successfully constructed. As shown in fig. 3.
The membrane-penetrating peptide used herein is CAYHRLRRC, but is not limited to this, and may be a TAT protein transduction domain, and if a TAT protein transduction domain is used, it is necessary to add a nucleotide sequence of TAT in designing a primer. And the adjustment of the dosage of the recombination reaction and the ligation reaction is carried out according to the corresponding principles. If any other carrier material is used that facilitates delivery of sSH protein to cells or tissues, this is required in accordance with the experimental principles of the corresponding field and is readily accomplished by one of ordinary skill in the art.
Table I recombinant plasmid and corresponding primer sequence
The underlined part is the CR base sequence.
Example 2: expression and purification of CR-w/sSH-His fusion proteins
E.coli BL21 (DE 3) competent cells were transformed with pET-28 a-CR-w/sSH-His, cultured overnight, selected for monoclonal, cultured by shaking, absorbance was measured, IPTG was added at 0.02% of the total volume until OD600nm reached 0.6-0.8, and induced to express at 16℃for 18h. His-tagged CR-w/sSH protein purification was performed using Ni-NTA agarose gel beads and imidazole eluents of different concentrations. The coomassie blue staining results of the proteins before and after purification are shown in fig. 4. CR-w/sSH-His fusion proteins are shown in Table II.
Amino acid sequence of epi-two fusion protein
The single underlined part is the CR sequence; the double underlined part is the w/sSH2 sequence, the three amino acids in bold italics are the mutated amino acids of sSH; the curve is underlined with His tag.
Example 3: CR-w/sSH2-His fusion protein specifically enters chronic granulocytic leukemia cells CR-w/sSH-His fusion protein can penetrate into K562 cells, and specifically bind with PY protein to inhibit proliferation of cells.
1.1PULL DOWN experiments demonstrated that CR-sSH2-His was stronger than CR-wSH2-His bound PY protein.
K562 chronic myelogenous leukemia cells and its imatinib-resistant strain, K562-r, were seeded at a cell density of 1X 10 6 cells per well in 6cm cell culture dishes (3 dishes each) in DMEM (high sugar) containing 10% fetal bovine serum and incubated in a carbon dioxide incubator (37 ℃). When the cell density is about 90% in the next day, total cell proteins are extracted, CR-sSH2-His, CR-wSH2-His and CR-His (control group) are respectively added into whole cell lysates of 3 groups of cells according to the final concentration of the fusion protein of 2 mu M, corresponding volumes of Ni-NTA agarose gel magnetic beads are incubated for 3 hours at 4 ℃, centrifugation is carried out, the supernatant is discarded, and a proper amount of PBS and protein loading buffer are added into the Ni-NTA agarose gel magnetic beads for boiling denaturation, and then protein immunoblotting experiment detection is carried out. As shown in fig. 5.
Anti-His antibodies used in immunoblot detection were purchased from CST, anti-PY antibodies were purchased from Abcam.
1.2 Immunofluorescence staining experiments prove that the mutant protein can specifically identify and enter cancer cells.
1.2.1K 562 and K562-r cells were seeded at 1X 10 6 cells per well in 6cm cell culture dishes (3 dishes each) in DMEM (high sugar) containing 10% fetal bovine serum and cultured in a carbon dioxide incubator (37 ℃). The next day when the cells grew to 80%, CR-sSH2-His, CR-wSH2-His and CR-His were added to 3 dishes of cells, respectively, at a final concentration of 2. Mu.M of fusion protein (control). After further culturing in the incubator for 6 hours, the cells were subjected to immunofluorescent staining and observed under a fluorescent microscope. When the His antibody is used for detection, CR-sSH-His can penetrate cell membranes and enter K562 and K562-r cells efficiently. As shown in fig. 6.
1.2.2 Selecting Molt-4 and CCRF-CEM cell lines, co-incubating with 2. Mu.M concentration of CR-sSH-His protein in a 37℃incubator for 6 hours, and performing immunofluorescent staining, the result shows that CR-sSH-2-His can efficiently penetrate cell membranes and enter target cells. The results are shown in FIG. 6.
1.2.3 Selection of Primary cells from chronic granulocyte patients experiments were performed with blood cells from healthy volunteers, incubated with 2. Mu.M concentration of CR-sSH-His fusion protein in a 37℃incubator for 6h, immunofluorescence staining observations showed that CR-sSH-His could penetrate cell membranes efficiently and into Primary cells from chronic granulocyte patients, rarely into normal blood cells. The results are shown in FIG. 6.
1.2.4 Selection of human breast cancer cell line MCF7 and human pancreatic cancer cell line SW1990 were incubated with 2. Mu.M concentration of CR-sSH-His protein in a 37℃incubator for 6h and immunofluorescent staining, which showed little entry of CR-sSH-His into both cell lines. The results are shown in FIG. 6.
Anti-His antibodies used in performing immunofluorescent staining were purchased from CST.
1.3CCK8 cell proliferation inhibition experiment detects that the fusion protein CR-sSH can inhibit proliferation of chronic granulocytic leukemia cells, and has obvious anti-tumor effect.
CR-sSH2-His can inhibit K562 and K562-r cell proliferation: the K562 and K562-r cells are inoculated into a 96-well plate, the number of cells in each well is ensured to be about 5000, and 3 auxiliary wells are arranged. After overnight incubation in a carbon dioxide cell incubator (37 ℃) 2 days after inoculation, CR-sSH2-His was added directly to the cell culture medium, using increasing concentrations from 0. Mu.M to 16. Mu.M, treatment time was 24h, 10. Mu.l CCK8 reagent was added per well, absorbance was measured at A=480 nm, histograms were drawn according to the corresponding values, and cell proliferation of K562 and K562-r cells was inhibited, with significant differences from concentrations of 1. Mu.M to 16. Mu.M. The experimental results are shown in FIG. 7.
CR-sSH2-His can inhibit primary cell proliferation in patients with chronic myelogenous leukemia: six peripheral blood samples from chronic myelogenous leukemia patients were collected, incubated with CR-sSH2-His protein for 24h with different concentration gradients (0.5,1,2,4,8 and 16. Mu.M), and the results showed that: the cell viability gradually decreased with increasing concentration of CR-sSH-His protein, indicating that the CR-sSH-His protein has an inhibitory effect on proliferation of primary cells in patients with chronic myelogenous leukemia. The experimental results are shown in FIG. 7.
1.3.3CR-sSH-His can inhibit proliferation of normal human peripheral blood mononuclear cells: normal human peripheral blood mononuclear cells are separated and planted in a 96-well plate, cells are incubated with fusion protein CR-sSH-His with different concentrations (0.5,1,2,4,8 and 16 mu M) for 24 hours, and the experimental result shows that the fusion protein CR-sSH-His has no obvious inhibition effect on normal hematopoietic cells within the experimental concentration range (0-16 mu M). The experimental results are shown in FIG. 7.
1.4CR-sSH2 can induce apoptosis in chronic myelogenous leukemia cells.
K562 cells in the logarithmic growth phase are counted and then incubated with 2 mu M of CR-sSH2-His, treated for 6, 12 and 24 hours respectively, and treated by an Annexin V-FITC/PI double-staining method, and apoptosis of the cells is detected by a flow cytometer. The results showed that the proportion of CR-sSH-His induced apoptosis in K562 cells increased with increasing treatment time. The experimental results are shown in FIG. 8.
Test examples
The CR-sSH2 fusion protein provided by the invention is used as a targeting candidate drug for treating CML, has the function of specifically recognizing leukemia cells of leukemia cell targeting transmembrane peptide CAYHRLRRC, and CAYHRLRRC polypeptide can help to introduce SH2 super-parent into cells, and SH2 super-parent entering the cells can replace natural SH2 to combine with a plurality of tyrosine phosphorylation sites, so that signal paths related to cell proliferation and apoptosis are blocked, and life processes such as proliferation, movement and apoptosis of tumor cells are influenced. The fusion protein has low preparation cost and easy acquisition, and can greatly reduce the economic burden of patients. Meanwhile, the targeting advantage of specific recognition and entering leukemia cells can effectively avoid damage to normal cells, and the capability of SH2 super-parent body to block multiple channels enables the fusion protein to have multi-target property, so that the problem that the traditional medicine is easy to generate drug resistance is avoided.
All vectors suitable for expression purification, such as pGEX-4T3, pETM30, pETM11, etc., may be other possible expression vectors (but are not limited to) plasmids or modified viruses (e.g.replication defective retroviruses, adenoviruses, etc.), as long as the vector is compatible with the host cell used. Depending on the host cell selected and the expression vector used, other additional sequences (e.g., replication origin, additional DNA restriction endonuclease sites, enhancers, etc. sequences) may be introduced into the expression vector.
To facilitate the introduction of sSH proteins into cells, the reported leukemia cell-targeting transmembrane polypeptide CAYHRRLRC is incorporated into a drug delivery system. The sequence of the targeting transmembrane peptide can be functionally divided into two parts: wherein the CAY sequence has the ability to specifically bind to leukemia cells, and the RLRRC sequence has the property of penetrating the cell membrane. Experimental results show that the CR-sSH2 fusion protein can penetrate and enter K562 cells to replace the combination of endogenous PY/SH2 structural domains in cancer cells, block a plurality of signal paths involved in the protein, inhibit proliferation of tumor cells, induce apoptosis and have obvious anti-tumor effect.
The membrane-penetrating peptide used herein is CAYHRRLRC, but is not limited to this, and may be a TAT protein transduction domain, and if a TAT protein transduction domain is used, it is necessary to add a nucleotide sequence of TAT in designing a primer. And the adjustment of the dosage of the recombination reaction and the ligation reaction is carried out according to the corresponding principles. If liposomes, or nanoparticles, or any other carrier material that facilitates delivery of sSH's 2 to cells or tissues are used, they need to be in accordance with the experimental principles of the corresponding field and are readily accomplished by one of ordinary skill in the art.
CR-w/sSH-His fusion protein can penetrate into K562 cells, and can specifically bind with PY protein to inhibit proliferation of cells. The PULL DOWN experiment demonstrated that CR-sSH2-His has a stronger affinity than CR-wSH2-His for binding to PY protein (FIG. 5). Immunofluorescent staining experiments demonstrated that the CR-sSH2-His protein was able to specifically recognize and enter chronic granulocytic leukemia cells (FIG. 6). CCK8 cell proliferation inhibition experiments detect that fusion protein CR-sSH2 can inhibit proliferation of chronic granulocytic leukemia cells, and has obvious anti-CML effect (figure 7). An Annexin V-FITC/PI staining experiment proves that CR-sSH2 can induce K562 cells to undergo apoptosis (FIG. 8).
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (7)
1. A fusion protein based on an SH2 super-parent, wherein the fusion protein comprises a chronic granulocytic leukemia cell-specific transmembrane peptide, and further comprises an SH2 super-parent, and the chronic granulocytic leukemia cell-specific transmembrane peptide is linked to the SH2 super-parent;
the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1.
2. The construction method of the expression vector is characterized by comprising the following steps:
(1) Amplifying to obtain a gene fragment expressing SH2 super-parent as shown in SEQ ID NO. 4;
(2) Constructing the gene fragment obtained in the step (1) on an expression vector to obtain a recombinant plasmid containing the gene fragment of the SH2 super parent;
(3) And (3) taking the recombinant plasmid obtained in the step (2) as a template, and carrying out PCR amplification by using a forward primer shown in SEQ ID NO. 2 and a reverse primer shown in SEQ ID NO. 3 to obtain an expression vector containing the gene fragment of the SH2 super parent and the gene fragment of the chronic granulocytic leukemia cell specific transmembrane peptide.
3. The method of claim 2, wherein the expression vector in step (2) is pET-28a, pGEX-4T3, pETM or pETM.
4. An expression vector constructed by the method of claim 2 or 3.
5. A method for expressing and purifying fusion protein based on SH2 super parent is characterized in that the expression vector of claim 4 is transformed into competent cells, target protein expression is induced, and target protein purification is carried out by agarose gel magnetic beads and imidazole eluent.
6. Use of an SH 2-superparent-based fusion protein according to claim 1 for the preparation of a medicament for targeted treatment of chronic granulocytic leukemia.
7. The use of claim 6, wherein the fusion protein specifically binds to phosphorylated tyrosine in chronic granulocytic leukemia cells, thereby inhibiting proliferation of chronic granulocytic leukemia cells.
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