CN106496329B - Fusion protein containing collagen binding structure domain - Google Patents

Abstract

The invention relates to a fusion protein with a recombinant collagen binding structure domain, and the amino acid sequence of the fusion protein is shown as SEQ ID NO: 2, respectively. The fusion protein is a solution preparation, can be used for preparing anti-tumor drugs, is administrated in a local intratumoral injection mode, and can provide a treatment dose for foot at one time. In the fusion protein, a local slow release system formed by the collagen binding structural domain CBD well avoids strong immune response caused by the diffusion of IFN-gamma to surrounding tissues. Meanwhile, repeated administration is not needed, so that the pain of a patient caused by the wound caused by the repeated administration and the huge treatment cost consumed by repeated administration are reduced, and the toxic and side effects caused by the frequent administration of a large dose are reduced.

Description

Fusion protein containing collagen binding structure domain

Technical Field

The invention relates to the field of biotechnology. In particular to a recombinant protein containing a Collagen Binding Domain (CBD) of matrix metalloproteinase (MMP2) and an active domain of gamma interferon (IFN-gamma) and a fusion protein thereof, in particular to a fusion protein with multiple activities.

Background

Tumors are one of the leading causes of disease and death worldwide. It seriously threatens the health of human beings and also attracts high attention of people. Over the past 20 years, a great deal of research has focused on the molecular events involved between the occurrence of tumors involved in tumor progression and signaling pathways. Studies have found that the molecular mechanisms of complex interactions between tumor cells and the tumor microenvironment play an important role in the progression of tumors. At the same time, over 40 years of research has shown that there is a large body of evidence to support extracellular matrix remodeling proteases, such as Matrix Metalloproteinases (MMPs), as regulators of changes in the microenvironment during major cancer progression. MMPs are also considered as a potential diagnostic and prognostic indicator of various different stages of tumor development. Based on the important role that MMPs play in the development of tumors, it is necessary to design and synthesize inhibitors or monoclonal antibodies that can inhibit the specific binding of the active site of certain MMPs without any cross-reactivity with other MMPs. Currently, various inhibitors of MMPs all exhibit varying degrees of antitumor activity in the experiments, but all fail in clinical trials due to poor therapeutic efficacy.

In order to overcome this problem of inhibitor design as much as possible, it is necessary to find a relevant substance acting at the substrate binding site, which is localized outside the active site, but is a molecule of the substance necessary for external localization during hydrolysis. There is a great deal of research evidence that specific foreign substance binding sites, located outside the catalytic sites, are essential for matrix metalloproteinases to localize the hydrolysis substance molecules. Therefore, substances that can block foreign substance-mediated interactions would be a better choice as highly selective MMP inhibitors. While the CBD of MMP2 fits well with the above-described features. There are studies reporting a number of features of the interaction of CBD with ligand binding that suggest that CBD is responsible for almost all of the properties of MMP2 binding to collagen. Increased gelatinase activity has been observed in a variety of pathological conditions including tumors, inflammation, infectious diseases, and degenerative diseases of cerebrovascular disease. Gelatinases are mainly classified into gelatinase a and gelatinase B, wherein gelatinase a mainly refers to MM2, and gelatinase B mainly refers to MMP 9. Although both gelatinases MMP-2 and MMP-9 have in common in many respects, only the CBD of MMP2 has been reported in the literature to be effective in binding to type IV collagen, and to bind to some natural and denatured collagen types, as well as elastin and heparin. Moreover, CBD-mediated binding of enzyme substrates and other extracellular matrix molecules is an essential element for the normal function of both MMP-2 and cell migration.

Therefore, if the same protein as CBD of MMP2 in vivo can be isolated and placed at a site rich in collagen in vivo and where pathological conditions such as tumor invasion and metastasis are caused by MMP upregulation, binding the same collagen binding site competitively with CBD in vivo, the upregulated MMP cannot cause the corresponding pathological condition and can inhibit the corresponding invasion and metastasis during tumor development. Moreover, if local stimulation is carried out by cytokines, on one hand, the local immune response of the tumor can be enhanced, and on the other hand, the effect of directly killing the tumor is achieved, so that the occurrence and the metastasis of the tumor are probably completely inhibited.

Interferon (IFN-. gamma.) is mainly produced by T cells and consists of a secretory peptide and an active domain, which is cleaved off during secretion out of the cells to form mature IFN-. gamma.. IFN-gamma has various anti-tumor effects of directly inhibiting tumor cell proliferation, increasing the expression of surface MHC antigens and tumor necrosis factors, resisting tumor angiogenesis and the like. Scientists began exploring the genetic engineering of interferons from the 70 s of the 20 th century. In 1993, the FDA approved the genetic engineering IFN-gamma to be put on the market. Along with the continuous marketing of the domestic gene engineering interferon, the clinical application is gradually increased, and a better curative effect is achieved. However, since IFN- γ has a small molecular weight and is easily filtered by glomeruli, it is generally administered systemically by intramuscular or subcutaneous injection, and has a short half-life in plasma, and requires a large dose of frequent administration, such as 2-3 times per week, and when IFN- γ is used to treat hepatitis, tumors, multiple sclerosis, rheumatoid diseases, the treatment cycle is often as long as several months to several years, such long-term frequent administration of large dose not only increases the pain and treatment cost of patients, but also is prone to produce toxic and side effects such as fever (which occurs in 90% of patients) mainly because IFN- γ is an important activator of mononuclear/macrophage, and it can induce and promote strong inflammatory response of local or systemic body, bone marrow suppression, weight loss, anorexia, etc., and severely restricts its application.

In conclusion, MMP2 can promote the generation and development of tumors, but CBD of MMP2 alone can bind to collagen in intercellular substance and can competitively inhibit the degradation of collagen by MMP 2; the active domain of IFN-gamma has multiple anti-tumor activities, but also causes severe toxic and side effects. If the two structures are fused together and the original functions and activities of the two structures are ensured to be kept in the fusion protein, the protein molecules can effectively inhibit the generation and the development of tumors. In the design and practice of fusion proteins, multiple technical hurdles are faced, including: 1. how to accurately find and locate the protein functional structural domain with medical value; 2. polypeptide fragments of different structural domains may mutually influence folding and space structures, finally result in the loss of functions and activities, how to ensure that two or more structural domains can keep respective original functions and activities is the key point for designing and preparing multifunctional fusion protein molecules; 3. after the structural domains with different protein values are combined, on the basis of giving play to respective original functions, how to realize the organic combination of functions and realize the cooperative reinforcement of the functions and the complementation of advantages and disadvantages, thereby achieving excellent treatment effect; and 4, due to correct design and appropriate experimental conditions, the stability and the solubility of the fusion protein after the expression are ensured, and the separation and the purification are easy. The existence of these technical bottlenecks makes the multifunctional fusion protein have serious difficulties from design, preparation to application.

The invention finds two potential anti-tumor active structural domains based on a large amount of scientific literature retrieval, reading and screening, designs and constructs a fusion protein coding sequence by utilizing a molecular biology technology, and adopts a prokaryotic expression technology and a protein affinity purification technology for expression and purification. By developing a large amount of software reasonable prediction and experimental tests, prokaryotic expression vectors are successfully designed and constructed, and experimental conditions capable of efficiently expressing and purifying soluble protein are explored.

Disclosure of Invention

The invention aims to provide a fusion protein with a recombinant collagen binding domain. On one hand, the collagen peptide can play a role of combining with collagen binding structural domains on extracellular matrix and basement membrane, thereby inhibiting the combination of the collagen binding site of MMP2 secreted by tumor cells and collagen and playing a role of inhibiting the invasion and metastasis of the tumor cells; meanwhile, the local IFN-gamma concentration of the tumor is enhanced, and the toxic and side effects of the recombinant IFN-gamma are reduced. On the other hand, the other component IFN-gamma in the fusion protein can be detained in the local part of the tumor, thereby not only exerting the direct tumor killing effect, but also enhancing the immune response of the local body of the tumor and reducing the immune escape of tumor cells.

It is another object of the present invention to provide a DNA sequence encoding the fusion protein of the present invention. A DNA sequence of a fusion protein comprising a first region identical to the murine interferon (IFN- γ) amino acid mat _ peptide sequence and a second region identical to the Collagen Binding Domain (CBD) amino acid mat _ peptide sequence of human MMP 2. Wherein the first region is identical to the murine IFN-gamma amino acid mat _ peptide sequence and the second region is identical to the Collagen Binding Domain (CBD) amino acid mat _ peptide sequence of human MMP 2. The fusion protein without any connecting peptide added in the middle. As the design thought, the purified fusion protein is found to retain all the activities of IFN-gamma and all the characteristics of CBD domain in the later experimental exploration, and is the fusion protein in the true sense.

In the construction process of gene engineering bacteria or gene engineering cells, genes for expressing fusion proteins need to be obtained and amplified firstly, a PCR method is adopted, and an overlap extension PCR method is adopted. After obtaining a large amount of genes expressing fusion proteins, the expressed genes need to be transferred into a selected vector by using a proper vector so as to construct a recombinant expression vector.

Another object of the present invention is to provide a recombinant expression vector carrying a DNA sequence encoding the fusion protein of the present invention.

Another object of the present invention is to provide a host expressing a gene encoding the fusion protein of the present invention. The expression host selected by the invention is engineering bacterium BL 21.

The technical scheme provided by the invention is as follows: a fusion protein with a recombinant collagen binding domain, the amino acid sequence of which is as shown in SEQ ID NO: 2, respectively.

The fusion protein is a solution preparation, is administrated by a local intratumoral injection mode, and can be used for one-time foot treatment. In the fusion protein, a local slow release system formed by the collagen binding structural domain CBD well avoids strong immune response caused by the diffusion of IFN-gamma to surrounding tissues. Meanwhile, repeated administration is not needed, so that the pain of a patient caused by the wound caused by the repeated administration and the huge treatment cost consumed by repeated administration are reduced, and the toxic and side effects caused by the frequent administration of a large dose are reduced.

The fusion protein of the present invention includes a Collagen Binding Domain (CBD) of MMP2 of human origin and IFN-. gamma.of murine origin. Wherein the amino acid sequence encoded by the Collagen Binding Domain (CBD) of MMP2 of human origin is compared with the amino acid sequence of mouse, and the core domain of the CBD of human MMP-2 and the CBD core domain of mouse MMP2 are found to be unchanged.

In the fusion protein of the present invention, we removed the signal peptide portion of IFN-. gamma.and also removed other portions of the MMP2 protein molecule except for the Collagen Binding Domain (CBD).

In the fusion protein of the present invention, we located IFN- γ at the N-terminus of the fusion protein and the Collagen Binding Domain (CBD) of MMP2 at the C-terminus of the fusion protein. No linking peptide was introduced between IFN- γ and the Collagen Binding Domain (CBD) of MMP 2.

The invention also provides a DNA molecule for coding the fusion protein. The nucleotide sequence of the DNA molecule is shown as SEQ ID NO: 1 is shown.

Further, the present invention provides a vector comprising the above DNA molecule, and a host cell comprising the vector. The recombinant expression vector of the invention is PET-28(a) + plasmid.

The fusion protein can be used for preparing antitumor drugs.

The conversion method used by the invention is a YC conversion method, positive colonies are screened out through a resistance mark, then successfully converted bacteria are further identified through a bacteria liquid PCR or bacteria shaking quality improving plasmid enzyme cutting identification method, and finally sequence comparison is carried out through sequencing to find bacteria with consistent amino acid sequences for expression.

In the fusion protein, isopropyl thiogalactoside is adopted as an inducer to carry out induced expression of the protein, and simultaneously, a single CBD domain and IFN-gamma are expressed. In carrying out inducible expression of a protein, it is known that if the protein is present in the supernatant, it is easier to carry out a later purification experiment. Unfortunately, however, except that IFN-gamma expressed protein is present in the supernatant, the other two proteins expressed by us can be expressed by any method, for example, we have studied different induction times, different induction concentrations and different induction temperatures to induce and express proteins, and the expressed proteins are always present in the precipitate. This increases the difficulty of post purification.

In the fusion protein of the invention, the protein induced to be expressed is provided with 6 his tags, and the target protein is separated and purified by means of competitive affinity binding of 6 his and a nickel column during separation and purification.

In the fusion protein of the present invention, the protein induced to be expressed is secreted and exists in the form of inclusion body. Generally, the protein expressed in a secreted form has corresponding biological activity. Can be directly purified by a nickel column, dialyzed, freeze-dried and dissolved in PBS for use.

For the protein existing in the form of inclusion body, the protein with biological activity can be obtained by dialysis renaturation treatment after nickel column purification. In response, we have done a couple of efforts. Although we used various methods to make the protein present in the supernatant at the time of expression, the fact that it was present in the pellet was not reversible in the end. Therefore, we are ready for various kinds of work. Firstly, we consult many literature data about protein purification, analyze various factors that may affect the correct renaturation of expressed inclusion body protein, and conclude a set of own buffer solution system beneficial to protein renaturation. Secondly, for how to crack the extracted inclusion bodies, then pass through a nickel column and then carry out further dialysis renaturation, the inventor refers to the operation manual of fusion protein purification of Merck company, uses guanidine hydrochloride to denature the inclusion bodies, obtains inactive protein which we need through different PH gradients, then uses the renaturation buffer solution summarized by us to carry out a series of dialysis renaturation on the protein, and through a series of efforts and subsequent experimental identification, we successfully obtain active fusion protein.

The renaturation buffer system used in the invention is as follows:

(1) 6M Urea buffer: 0.1M NaH₂P04; 0.01M Tris-HCI, 6M Urea, pH: 7.2. dialyzing for 12-24 hours.

(2) Renaturation buffer solution: 20mM Tris, 2mM GSH, 5mM EDTA and 50mM Glycine, pH 8.0. Dialyzing for 24-48 h, and replacing the renaturation buffer solution once.

(3) 2M urea buffer: 0.1M NaH2P 04; 0.01M Tris-HCI, 2M Urea, pH: 7.2. dialyzed for 12 hours.

(4) 50mM Tris-HCl buffer: 50mM Tris, 200 mM NaCl, pH 7.4. Dialysis was performed for 24 hours with an intermediate change of fresh buffer.

All renaturation processes were carried out at 4 ℃.

The Collagen Binding Domain (CBD) of MMP2 was used for the first time for developing drugs for inhibiting tumor metastasis, and tests prove that the CBD can inhibit invasion and metastasis of tumor cells. In the fusion protein CBD-IFN-gamma, the functions of the CBD and the IFN-gamma for inhibiting the tumor metastasis play a synergistic role; on the other hand, the combination effect of the CBD and the collagen can combine the fusion protein CBD-IFN-gamma in the extracellular matrix of the tumor microenvironment for a long time, so that the fusion protein CBD-IFN-gamma is retained in the local part of the tumor at high concentration for a long time, namely IV collagen rich in tumor tissues is used as a natural slow release system of the fusion protein CBD-IFN-gamma. The method not only greatly enhances the antitumor activity of IFN-gamma, but also avoids toxic and side effects caused by frequent administration of large-dose IFN-gamma on the whole body, and simultaneously reduces the administration dose of IFN-gamma. The CBD-IFN-gamma between the two structural domains of the CBD-IFN-gamma can play a direct tumor cell killing role in the local part of the tumor, inhibit the generation of tumor blood vessels and promote the activity of T cells for killing the tumor cells.

DNA coding sequence for fusion protein design:

ATG(Start code) GGCAGCAGC (1 # ligation sequence)CATCATCATCATCATCAC(His tag 1) AGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTC (2 # linker sequence)CACGGCACAGTCATTGAAAGCCTAGAAAGTCTGAATAACTATTTTAACTCAAGTGGCATAGATGTGGAA GAAAAGAGTCTCTTCTTGGATATCTGGAGGAACTGGCAAAAGGATGGTGACATGAAAATCCTGCAGAGCCAGATTA TCTCTTTCTACCTCAGACTCTTTGAAGTCTTGAAAGACAATCAGGCCATCAGCAACAACATAAGCGTCATTGAATC ACACCTGATTACTACCTTCTTCAGCAACAGCAAGGCGAAAAAGGATGCATTCATGAGTATTGCCAAGTTTGAGGTC AACAACCCACAGGTCCAGCGCCAAGCATTCAATGAGCTCATCCGAGTGGTCCACCAGCTGTTGCCGGAATCCAGCC TCAGGAAGCGGAAAAGGAGTCGCTGC(selected IFN active domain)GAAGGCCAAGTGGTCCGTGTGAAGTATGG GAACGCCGATGGGGAGTACTGCAAGTTCCCCTTCTTGTTCAATGGCAAGGAGTACAACAGCTGCACTGATACCGGC CGCAGCGATGGCTTCCTCTGGTGCTCCACCACCTACAACTTTGAGAAGGATGGCAAGTACGGCTTCTGTCCCCATG AAGCCCTGTTCACCATGGGCGGCAACGCTGAAGGACAGCCCTGCAAGTTTCCATTCCGCTTCCAGGGCACATCCTA TGACAGCTGCACCACTGAGGGCCGCACGGATGGCTACCGCTGGTGCGGCACCACTGAGGACTACGACCGCGACAAG AAGTATGGCTTCTGCCCTGAGACCGCCATGTCCACTGTTGGTGGGAACTCAGAAGGTGCCCCCTGTGTCTTCCCCT TCACTTTCCTGGGCAACAAATATGAGAGCTGCACCAGCGCCGGCCGCAGTGACGGAAAGATGTGGTGTGCGACCAC AGCCAACTACGATGATGACCGCAAGTGGGGCTTCTGCCCTGACCAAGGG(selected CBD sequence) AAGCTTGCGGCCGCACTCGAG (3 # linker sequence)CACCACCACCACCACCAC(His tag 2) TGA (stop code)

Polypeptide sequence of the fusion protein:

m (initiator methionine) -GSS (1 # linker) -HHHHHHHHHHHHHH (His tag 1) -SSGLVPRGSHMASMTGGQQMGR (2 # linker) -HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC (selected IFN-active domain) -EGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALFTMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQG (selected CBD sequence) -KLAAALE (3 # linker) -HHHHHHHHHHHH (His tag 2)

The scheme provided by the invention has the following advantages:

although expressed as inclusion bodies, the fusion protein of the present invention has biological activity through a series of dialysis renaturations, and is the sum of the respective biological activities of the two domains of the fusion protein.

The fusion protein in the invention mainly adopts intratumoral injection as a main treatment mode, the fusion protein is stored in freeze-dried powder after the concentration is measured, a certain amount of sterile PBS solution is added for dissolution when in use, and BSA is used after the concentration is measured again. The dose used can be calculated from the titer of the fusion protein relative to the commercial IFN-. gamma.and the single CBD of the fusion protein is used in the same number of moles relative to the IFN-. gamma. -CBD. All of them are administered once, and the nature of the binding CBD domain with affinity binding to extracellular matrix collagen forms a natural sustained release system in the tumor local.

The fusion protein and the derivatives thereof or the combination use of the fusion protein and other medicines have the characteristic of a collagen binding structural domain, so that the fusion protein has a better local slow-release effect, and the competitive inhibition effect of the fusion protein and MMP also prevents the tumor invasion and metastasis mediated by MMP so as to inhibit the distant metastasis of the tumor; simultaneously, IFN-gamma is used as a cell factor, not only has better functions of directly killing tumors and inhibiting angiogenesis, but also can better enhance local immune response of the tumors with the assistance of a CBD domain, promote the generation of in-vivo positive immune response and improve the killing effect of the tumors, and toxic and side effects on organisms possibly caused by over-strong immunity can be reduced or even eliminated because the toxic and side effects are retained in local tumors.

Drawings

FIG. 1 shows the restriction enzyme identification of the clone of the PCMV-Tag2A CBD plasmid and the clone of the PCMV-Tag2A IFN-. gamma.plasmid.

FIG. 2 shows the restriction enzyme identification of the plasmid PET-28(a) + CBD and the restriction enzyme identification of the plasmid PET-28(a) + IFN-. gamma..

FIG. 3 shows the identification of 1% DNA gel by means of DL5000 marker after cloning PET-28(a) + IFN-. gamma. -CBD plasmid and double digestion with ECOR I and Hind III.

FIG. 4 is an SDS-PAGE analysis of IPTG-induced expression of PET-28(a) + CBD, PET-28(a) + IFN-. gamma.and PET-28(a) + IFN-. gamma. -CBD in E.coli.

FIG. 5 is a SDS-PAGE analysis of purified PET-28(a) + IFN-. gamma., CBD, IFN-. gamma. -CBD.

FIG. 6 is a western blot analysis of his tag resistance of purified PET-28(a) + IFN-. gamma., CBD, IFN-. gamma. -CBD.

FIG. 7 is an analysis of IFN-. gamma.activity of the purified protein.

Fig. 8 left panel is a transwell migration experiment.

FIG. 9 shows that the fusion protein with IFN-gamma domain has obvious effect of killing cells through CCK8 detection.

FIG. 10 is a graph showing the determination of the amount of protein remaining in Matrigel gel, i.e., the amount of protein to which collagen can bind to the CBD domain, by densitometric analysis, and a graph showing the determination of the amount of fusion protein to which collagen can bind at various time points in Matrigel by means of his-tag western blot.

FIG. 11 shows the killing effect of the fusion protein on 4T-1 tumor cells as detected by CCK 8.

FIG. 12 is a graph of the antitumor effect of purified protein in breast cancer tumor mice.

FIG. 13 is a graph of the effect of purified protein on lung metastasis in breast cancer mice.

FIG. 14 is a statistical survival of melanoma mice, and IFN-. gamma. -CBD treatment was found to significantly prolong the survival of melanoma mice.

FIG. 15 is a schematic diagram of the design and operation of the fusion protein of the present invention.

Detailed Description

Referring to fig. 15, the protein structure of metallo-matrix protein-2 (MMP2) includes two parts, a Collagen Binding Domain (CBD) which functions to allow MMP2 to bind to the extracellular matrix, and a catalytic domain which functions to degrade the extracellular matrix. The protein sequence coded by gamma interferon (IFN-gamma) comprises a signal peptide with a secretion function and a mature IFN-gamma part with biological activity.

The invention utilizes a molecular biology method to construct a new artificial gene by the CBD of MMP2 and the bioactive structural domain of IFN-gamma, and the new artificial gene is connected to a prokaryotic carrier capable of expressing fusion protein, and the artificial fusion protein molecule CBD is induced, expressed and purified in escherichia coli: : IFN-gamma. Tests prove that the artificial protein molecule not only has the CBD function, namely, the artificial protein molecule is combined with tumor tissue extracellular matrix, but also has the IFN-gamma anti-tumor function, namely, the artificial protein molecule directly kills tumor cells, inhibits angiogenesis and activates and recruits lymphocytes.

According to our design, CBD allows the artificial fusion protein molecule CBD: : IFN-gamma can bind to tumor tissue extracellular matrix, and the following anti-tumor effects are achieved:

1. the fusion protein binds to collagen of extracellular matrix, competitively inhibits the binding of tumor MMP2 to and degrades the extracellular matrix, thereby inhibiting the metastasis of tumor cells;

2. CBD: : IFN-gamma can exist in the tumor for a long time and maintain local high concentration, thereby enhancing the action of the IFN-gamma for directly killing tumor cells, inhibiting angiogenesis and activating and recruiting anti-tumor lymphocytes;

3. in clinical tests, administration of low doses of soluble IFN- γ can lead to fever and systemic inflammatory responses, and high doses of IFN- γ can be lethal. CBD: : the collagen binding of IFN-gamma tumor tissue avoids the diffusion of active IFN-gamma, thereby reducing the systemic inflammatory response of IFN-gamma.

The present invention is further illustrated by the following specific examples.

Example 1: cloning of human CBD Gene and cloning of murine IFN-gamma Gene

The gene sequence of CBD was cloned from an existing human MMP 2.

Cloning of IFN- γ Gene:

(1) the LAase is used with < CTCGAG > Xhol and < GAATTC > EcoR1 as enzyme cutting sites, the primer is synthesized by Wuhan Pongsu biotechnology limited company, diluted with pure water to 10 μ M working solution concentration, and IFN- γ gene Sequence based on the primer design comes from NCBI Reference Sequence: NM-008337.3).

(2) Preparation of the template: extracting RNA from mouse cell, reverse transcribing to obtain cDNA as template for PCR reaction

(3) PCR reaction system: 50ul of reaction system contains: ddH₂O37.5 ul;10 × LA buffer 5 ul;10 mM dNTP 4ul; primer (F + R) 2ul, mcDNA 1ul, and LAase 0.5 ul. PCR reaction parameters: 1min at 95 ℃; 30s at 94 ℃, 30s at 57.9 ℃, 30s at 72 ℃ and 40 cycles. 5min at 72 ℃; infinity at 16 ℃.

(4) After PCR, 2ul of PCR product + 2ul of 10 × loading buffer was taken for 1% agarose gel electrophoresis detection.

(5) After the size of the band is consistent with the target, DNA gel cutting recovery and gel recovery kit are carried out, and after the gel is recovered, the concentration of the contained DNA (Nanodrop 1000) is measured and then connected with the PCMV vector.

(6) Connecting the PCMV carrier with the gel-cutting recovery product, wherein the connecting system is as follows: PCMV 3ul, IFN-gamma 14ul, 10 XT 4buffer 2ul, T4 DNA ligase 1ul, 20ul total system. The connection reaction conditions are as follows: 20 ℃ and 2h.

(7) Conversion by YC method after ligation, carried out under aseptic conditions (alcohol burner, sterile EP tube, tip), as follows: the 50ul system contains: ligation product 20 ul; 5 XKCM 10 ul; ddH2O (sterile) 20 ul. After the addition, mix well, add one competent cell (stored at-80 ℃ C., on pre-ice), mix well EP tube after the addition, stand on ice for 20 min, stand at room temperature for 10min, add 500ul LB (sterile, non-resistant). Shaking on a shaker for 50 min (200 r,37 deg.C), taking out, 3000 r × 5min, discarding most of supernatant, blowing, sucking into a sterile gun head, driving into kan + resistant plate, uniformly coating the whole plate with an alcohol-burnt and air-dried coating rod, placing in 37 deg.C incubator, inverting the plate after one hour, and culturing at 37 deg.C incubator overnight (no more than 16 h).

(8) And selecting single colony from the colony in the transformation plate, adding 100ulkan + resistant culture medium, shaking for 1h, and then carrying out bacteria liquid PCR. The PCR reaction system is as follows: 20ul of the system contained: ddH2O 8 ul; 2 × mix10 ul; IFN-gamma primer (F + R) 1ul, bacterial liquid 1 ul. Reaction conditions are as follows: at 95 deg.C for 5, at 94 deg.C for 30s, at 57.9 deg.C for 30s, and at 72 deg.C for 30s, for 40 cycles. 5min at 72 ℃; infinity at 16 ℃.

(9) Identification of transformation results: after the PCR is finished, DNA glue is used for electrophoresis, the cell which is consistent with the size of a target band and has higher brightness is selected for bacteria shaking, the cell is preserved and then sequenced, the sequencing company is Wuhan engine biotechnology limited, and the sequencing primer is a universal primer and is provided by the company. After sequencing, blast on pubMed shows that the gene sequence is completely correct, namely taking out the bacterial liquid of the preserved colony PCR, inoculating, shaking (preserving) the bacterial liquid and extracting the plasmid.

FIG. 1 shows the restriction enzyme identification of the clone of the PCMV-Tag2A CBD plasmid and the clone of the PCMV-Tag2A IFN-gamma plasmid. After the 1A is the PCMV-Tag2A CBD plasmid, the plasmid is cloned and then runs 1% of DNA gel identification chart, the M band represents DL2000 marker, the a band represents constructed PCMV-Tag2A CBD plasmid is subjected to EcoRI and Hind III double enzyme digestion, and CBDs of PCMV-Tag2A and hMMP2 are cut out respectively. FIG. 1B shows the identification chart of 1% DNA gel after cloning pCMV-tag2A IFN-gamma plasmid, wherein M band represents DL5000 marker, and B band represents constructed pCMV-tag2 AIFN-gamma plasmid, which is digested by EcoRI and XholI, to cut out PCMV-tag2A and mouse IFN-gamma.

Example 2 was carried out: the cloned CBD of human MMP2 and the CDS region sig peptide of the DNA sequence of mouse IFN-gamma are inserted between two enzyme cutting sites EcoR1 and Hind III of an expression vector PET28a (+). The method of fusion recombination is adopted.

(1) First, primers for fusion recombination are designed.

(2) Fragment PCR, fragment PCR reaction system: 50ul reaction System, ddH₂O37.5 ul;10 × LA buffer 5 ul;10 mM dNTP 4ul; primer (F + R) 2ul, plasmid 1ul, LA enzyme 0.5 ul. PCR reaction parameters: 5min at 95 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 40 cycles. 5min at 72 ℃; infinity at 16 ℃. After PCR cloning, 2ul of PCR product + 2ul of 10 × loading buffer was subjected to 1% agarose gel electrophoresis.

(3) Vector linearized PCR reaction system: 50ul of reaction system contains: ddH₂O 33ul;10 XKOD buff 5 ul; 2mM dNTP 5 ul; 25Mm MgSO 4: 3ul; primer (F + R) 2ul, plasmid 1ul, KOD enzyme 1 ul. PCR reaction parameters: 1min at 95 ℃; 30s at 95 ℃, 30s at 66 ℃ and 3min at 68 ℃ for 35 cycles. 10min at 68 ℃; infinity at 16 ℃.

(4) After PCR cloning, 2ul of PCR product + 2ul of 10 × loading buffer was taken for 1% agarose gel electrophoresis detection.

(5) After the electrophoresis determines that the size of the strip is correct, carrying out precipitation recovery, and specifically operating as follows: the PCR product + ddH2O filled 100 ul; +250ul absolute ethanol; + 10ul (3M pH5.2) NaAc. Mixing at-20 deg.C for 30 min. Take out and centrifuge for 12,000r × 10 min. Removing supernatant, adding 500ml of 75% ethanol into the precipitate, shaking gently, mixing, centrifuging again, and centrifuging for 12,000r × 5 min; this was repeated twice, the whole supernatant was discarded, the remaining supernatant was not aspirated, centrifugation was performed again for 12,000 r.times.5 min, water was aspirated, air-dried at room temperature (5-15 min), 20ul ddH20 was added, and DNA concentration was measured by Nanodrop.

(6) The enzyme digestion of DPn1, the product recovered from the precipitation is digested with DPn1, and the enzyme digestion system of DPn1 is as follows: fragment (b): 20 ul; PET28a (+) Hind III-EcoR 1: 18.8 ul;10 × buff T: 3ul; DPn1 enzyme: 1ul, supplement 30ul of the system with ddH20, and digest at 37 ℃. And (4) after enzyme digestion is finished.

(7) And (6) cutting the gel, recovering the gel, performing DNA gel electrophoresis on all the enzyme digestion products, observing the size of a strip after electrophoresis to a certain extent, and cutting the gel under an ultraviolet instrument. And (3) putting the cut target gel block into an EP (EP) tube, weighing, and then recovering the DNA gel.

(8) Performing fusion PCR on the gel cutting recovery product, wherein the fusion PCR reaction system is as follows: ddH 20: 24ul PET28a (+) H-E: 5 ul; homo CBD: 7 ul; 2mM dNTP: 5 ul;10 XKOD buff: 5 ul; 25mM MgSO 4: 3ul; KOD enzyme: 1ul, 50ul system. PCR reaction parameters: 1min at 95 ℃; 30s at 95 ℃, 30s at 66 ℃ and 3min at 68 ℃ for 35 cycles. 10min at 68 ℃; infinity at 16 ℃. After the fusion PCR is completed, the PCR product is transformed, and the transformation operation method is the same as above.

(9) Identification of transformation results: the colony grown after transformation is picked up by a sterile small gun and put into a culture medium with a certain amount of Kan + and the bacteria is shaken over night, plasmid is extracted, a plasmid extraction instruction (Beijing Jiang alliance international biological gene science and technology Co., Ltd.) is extracted, and then enzyme digestion identification is carried out, wherein an enzyme digestion system is as follows: 20ul system, plasmid: 16 ul;10 × buff M: 2ul; EcoR1: 1ul; hind III 1ul; 20ul system, and the enzyme digestion is carried out for 2h at 37 ℃. And (3) carrying out DNA gel identification after enzyme digestion is finished, and sending the DNA gel identification to a company for sequencing identification after the identification result shows that the size of the band is correct, and identifying that the individual base mutation is found but the coded amino acid sequence is not changed, thus completing the construction.

FIG. 2 shows the restriction enzyme identification of the plasmid PET-28(a) + CBD and the restriction enzyme identification of the plasmid PET-28(a) + IFN-. gamma.. FIG. 2A shows the identification of 1% DNA gel after cloning the plasmid PET-28(a) + CBD, wherein M band represents DL5000 marker, and c band represents constructed plasmid PET-28(a) + CBD, which are cleaved by ECOR I and Hind III enzyme, respectively to obtain PET-28(a) + and CBD. FIG. 2B is an identification chart of 1% DNA gel after cloning of PET-28(a) + IFN-. gamma.plasmid, wherein M band represents DL5000 marker, and d band represents constructed PET-28(a) + CBD plasmid, which is cleaved by ECOR I and Hind III enzyme, to respectively cut out PET-28(a) + and IFN-. gamma..

Example 3 of implementation: inserting sig peptide of CDS region of IFN-. gamma.DNA sequence of cloned mouse into PET-28(a) + upstream of CDS region of DNA sequence of CBD of human MMP2 in CBD

The operation method is still fusion recombination, PET-28(a) + CBD is used as a vector, PET-28(a) + IFN-gamma is used as a fragment, and the operation is the same as the construction of PET-28(a) + CBD and PET-28(a) + IFN-gamma.

FIG. 3 shows the identification of 1% DNA gel after cloning the PET-28(a) + IFN-. gamma. -CBD plasmid, wherein the M band represents DL5000 marker, and the e band represents constructed PET-28(a) + IFN-. gamma. -CBD plasmid, after being cleaved by ECOR I and Hind III, from which PET-28(a) + and IFN-. gamma. -CBD were cut, respectively.

Example 4 of implementation: SDS-PAGE analysis of IPTG-induced expression of PET-28(a) + CBD, PET-28(a) + IFN-gamma-CBD in E.coli

Only PET-28(a) + IFN-. gamma.is present in the supernatant, and the remainder is present in the precipitate, as will be mainly described below by way of example of inducible expression of PET-28(a) + CBD.

Before induction, plasmids of PET28a (+) CBD, PET28a (+) IFN-gamma and PET28a (+) IFN-gamma-CBD which are constructed before are used; respectively transforming expression competence BL21, then picking single colony to shake bacteria, preserving bacteria, and performing induced expression of protein.

Exploration of IPTG Induction time

(1) Adding 5mL Kan + liquid culture medium into 8 tubes, and adding 0.5 mL of bacterial liquid obtained by shaking the preserved PET28a (+) CBD BL21 overnight, and shaking for about 1.5h until OD of the bacterial liquid is about 0.8.

(2) Numbering 0,1,2, 3,4,5,6 and 7 on the tube respectively; and 100mM IPTG 50ul was added to each of the cells, i.e., IPTG concentration of 1mM,37 ℃ and 200r, followed by shaking.

(3) Taking out test tubes with corresponding numbers after 0,1,2, 3,4,5,6,7 and h respectively, centrifuging for 4000r multiplied by 10min, discarding supernatant, centrifuging again until the supernatant can not be absorbed, and storing at 4 ℃.

(4) After all tubes were collected, 1mL of PBS was added to resuspend the tubes, centrifuge 4000 r.times.10 min, repeat, aspirate the supernatant and store at-20 ℃.

(5) The next day, the pellet was removed and placed on ice, 500ul PBS was added to each pellet, 6ul PMSF (final concentration of 1 mM) was added, and ultrasound (125W, 50% power, 3S supra, 5S stop) was repeated (20 times or so) on ice until completely transparent.

(6) Centrifugation, 12,000r × 10min, pipetted supernatant into a new EP tube until no supernatant was aspirated into the pellet (ice left throughout the run).

(7) The protein concentration in the supernatant was measured (BCA protein concentration assay), and protein sampling was performed based on the measured protein concentration by selecting the total amount of protein with the minimum concentration of 20 ul.

(8) And (3) running the SDS-PAGE glue by using 70V voltage, and stopping the voltage when the bromophenol blue runs to the boundary line between the concentrated glue and the separation glue and is changed to 90V voltage and 1 cm from the bottom of the glass plate.

(9) The gel block was taken out and placed in a large glass jar and Coomassie brilliant blue dye solution was poured in and dyed overnight (shaking, sealing with a plastic bag).

(10) The next day, the staining solution was poured off, and a destaining solution was added to perform destaining (shaking)Shaking), taking out after the block is decolorized to be transparent, taking a picture, and soaking the gel in ddH₂And (4) in O.

(11) The protein expression reaches the peak value after 3h of induction from the running fruit, and the protein expression does not increase after the time is prolonged, and the protein exists in the form of inclusion bodies, and the size of the expressed protein is not different from the pre-estimated size. As shown in fig. 4A.

Exploration of IPTG Induction concentrations

The specific operation is as above, only when the bacterial liquid OD is approximately equal to 0.8, IPTG 1 (0 ul), 2 (2.5 ul), 3 (5 ul), 4 (10 ul), 5 (20 ul), 6 (30 ul), 7 (40 ul) and 8 (50 ul) are respectively added; and collecting samples after 3 h. Similarly to the search for IPTG induction time, it was found that the protein was present in inclusion bodies, and when the amount of IPTG was 5. mu.l, that is, the concentration of IPTG was 0.1mM, the amount of protein expressed was the largest, and further, the concentration of IPTG was increased, and the amount of protein expressed was not increased, which is not shown in the figure.

Exploration of IPTG Induction temperature

The specific operation is as above, only when the bacterial liquid OD is approximately equal to 0.8, the test tubes are taken out, the numbers are respectively 0,4h,8h and 20h, 5ul IPTG is respectively added in addition to the number 0, the temperature is 25 ℃,200 r, the test tubes with the relevant numbers are respectively taken out at the corresponding time points (for example, the test tube with the number 4h is taken out after 4 h), and 0 and 4h are taken out simultaneously. In the same manner as the search for the IPTG induction time, it was similarly found that the protein was present in the form of inclusion bodies even when the expression was induced at a low temperature, and the size of the expressed protein was not different from that estimated in advance. That is, the above induced expression indicates that the protein is present in the precipitate and is present in the form of inclusion bodies. The figure is omitted.

And 4, the optimal conditions of PET-28(a) + IFN-gamma induction expression are adopted for the induction expression of PET-28(a) + CBD, the concentration of the selected IPTG is 0.1mM, the induction time is explored, the induction time is respectively selected to be 0,1,2 and 4 hours, the protein is found to be expressed in the supernatant after induction, the amount is not low, and the protein expression amount reaches the peak value at 2 hours. As shown in fig. 4B.

The optimal conditions of PET-28(a) + IFN-gamma-CBD induction expression are also adopted for the induction expression of PET-28(a) + CBD, the concentration of the selected IPTG is 0.1mM, the induction time is explored, 0,1,2 and 4 hours are respectively selected, no protein expression is found in supernatant after induction, and only a large amount of protein expression exists in precipitates, namely the protein exists in inclusion bodies and reaches the peak value when the protein expression is 2 hours, as shown in figure 4C.

Example 5 was carried out: nickel column purification of fusion protein:

1. purification of IFN-gamma protein by nickel column under natural condition

(1) 50ul of each of the previously sterilized PET28a (+) IFN-. gamma.BL 21 was inoculated into two tubes. 5mL Kan + liquid medium was added to each tube, followed by shaking overnight.

(2) Preparing 1L Kan + liquid culture medium, adding the above shake bacteria liquid (10 mL), 200r,37 deg.C, shaking for about 4h until OD is about 0.8

(3) After 1mL of IPTG is added for induction for 2h, the mixture is taken out and placed in a 50 mL centrifuge tube for centrifugation, 10000 r multiplied by 5min, the part which is not separated is placed on ice, after all the parts are separated, the separation is carried out again, the supernatant is discarded, and the mixture is stored at the temperature of minus 20 ℃.

(4) The suspension was precipitated and suspended in 2 mL of PBS, centrifuged at 10000 r.times.5 min, and the supernatant was discarded and washed again until no supernatant was aspirated.

(5) Adding 1 XNi-NTA combined buffer solution into the precipitate for re-suspension, merging into a tube, adding 400ul PMSF after complementing 20 mL, carrying out ultrasonic treatment on ice (125W, 50% power, super 3S, stop 5S) until the bottom is transparent, then carrying out high-speed centrifugation for 10000 r × 5min, absorbing supernatant into a clean centrifugal tube, namely protein filtrate, and filtering the protein solution into a new centrifugal tube by using a 0.45 μm filter.

(6) The gravity column was set up, 1.5mL of matrix (Ni-NTA his Bind Resin from Novagen, shanghai jade bob biotechnology, ltd.) was added, 1 xni-NTA binding buffer was filtered through a 0.22 μm filter and added to the column, the lower end of the column was closed, the column was washed out of alcohol (2 column length volumes) and after dropping, protein filtrate was added, and the effluent was collected with a clean tube (repeatable) and stored at 4 ℃ for SDS-PAGE electrophoretic analysis. Using 10mL of 1 xNi-NTA rinsing buffer filtered by a 0.22 mu m filter to rinse for 2 times, collecting rinsing components for SDS-PAGE electrophoretic analysis, gradually dripping a certain amount of 1 xNi-NTA eluting buffer filtered by the 0.22 mu m filter to elute target proteins, using a 1.5mL EP tube to collect effluent, using Nanodrop 1000 to measure protein concentration while collecting, stopping dripping the eluting buffer when the displayed value is negative, collecting eluting components for SDS-PAGE electrophoretic analysis.

(7) SDS-PAGE electrophoretic analysis shows that the protein is purified, and the purity is more than or equal to 90 percent, namely the purification is finished.

(8) Dialyzed against 50mM Tris-HCl buffer at 4 ℃ overnight. Measuring the concentration of the protein, subpackaging and freeze-drying into powder. Storing at-80 deg.C.

2. Nickel column purification of Inclusion body (insoluble protein) CBD

(1) 50ul of each of the previously sterilized PET28a (+) CBD BL21 was inoculated into two tubes. 10mL Kan + liquid medium was added to each tube, followed by shaking overnight.

(2) Preparing 1L Kan + liquid culture medium, adding the above shake bacteria liquid (20 mL), 200r,37 deg.C, shaking for about 2h until OD is about 0.8,

(3) after 1mL of IPTG is added for induction for 2h, the mixture is taken out and placed in a 50 mL centrifuge tube for centrifugation, 10000 r multiplied by 5min, the part which is not separated is placed on ice, after all the parts are separated, the separation is carried out again, the supernatant is discarded, and the mixture is stored at the temperature of minus 20 ℃.

(4) The suspension was precipitated and suspended in 2 mL of PBS, centrifuged at 10000 r.times.5 min, and the supernatant was discarded and washed again until no supernatant was aspirated. Adding PBS for resuspension, merging into a tube, adding 400ul of PMSF after complementing 20 mL, carrying out ultrasonic treatment on ice (125W, 50% power, super 3S, stopping 5S) until the bottom is transparent, then carrying out high-speed centrifugation for 10000 r multiplied by 5min, removing supernatant by suction, adding 20 mL of denatured lysis/binding buffer solution and 50ul of beta-mercaptoethanol, then adding 400ul of PMSF, and shaking on ice overnight until the solution is fully dissolved (namely the dissolved protein solution).

(5) After the gravity column was set up, 1.5mL of matrix (adsorption column) was added, denatured lysis/binding buffer was filtered through a 0.22 μm filter and added to the column, the lower end of the column was removed and the column was capped, and after the completion of the elution of alcohol (2 column length volume), a proteolytic solution was added and the effluent was collected in a clean tube (reproducible), and stored at 4 ℃ for SDS-PAGE electrophoretic analysis. Gradually dripping a certain amount of the denatured washing buffer solution filtered by the 0.22 μm filter to elute the target protein, collecting the effluent with a 1.5mL EP tube while measuring the protein concentration with the Nanodrop 1000, stopping dripping the elution buffer solution when the display value is negative, and collecting the eluted fraction for SDS-PAGE electrophoretic analysis.

(6) Since guanidine hydrochloride-containing samples form a precipitate when treated with SDS, the samples must be diluted with water (1: 6 dilution) or dialyzed or precipitated with TCA to remove guanidine hydrochloride before SDS-PAGE is performed.

(7) SDS-PAGE electrophoretic analysis shows that the protein is purified, and the purity is more than or equal to 90 percent, namely the purification is finished.

(8) The recombinant protein eluate containing 8M urea was placed in a dialysis bag and dialyzed overnight at 4 ℃ in 6M urea buffer.

(9) Then dialyzed in a reconstitution Buffer at 4 ℃ for 24h and replaced with a disposable reconstitution Buffer.

(10) Dialyzed in 2M urea buffer at 4 ℃ overnight.

(11) Dialyzed against 50mM Tris-HCl Buffer at 4 ℃ overnight to exchange Buffer. Measuring the concentration of the protein, subpackaging and freeze-drying into powder. Storage at-80 deg.C

The IFN-gamma-CBD nickel column purification method is the same as that of the CBD.

(purification of the desired solution under native conditions: 1 XNi-NTA binding buffer: 50mM NaH₂PO4, pH8.0, 300mM NaCl, 10mM imidazole. 1 × Ni-NTA rinsing buffer: 50mM NaH₂P04, pH8.0, 300mM NaCI, 20mM imidazole. 1 × Ni-NTA elution buffer: 50mM NaH₂PO4, pH8.0, 300mM NaCI, 250mM imidazole.

Purification of the desired solution under denaturing conditions: denaturing lysis/binding buffer: 6M GuHCI; 0.1M NaH₂P04; 0.01M Tris-HCI, pH 8.0. Denatured rinse buffer: 8M urea; 0.1M NaH₂P04; 0.01M Tris-HCI, pH 6.3. Denaturing elution buffer: 8M urea; 0.1M NaH₂P04；0.01M Tris-HCI，The pH was 4.5. Renaturation Buffer: 20mM Tris, pH8.0, 2mM GSH, 5mM EDTA and 50mM Glycine.

Treatment of the dialysis bag: cut appropriate length dialysis bags, place in solution 1 (0.5M EDTA with 8% NaHCO3, pH 8.0) and solution 2 (0.5M EDTA, pH 8.0) respectively, boil for 10min, and then rinse thoroughly with deionized water for use.

The figure is omitted.

Example 6 of implementation: molecular weight size and His resistance identification of fusion protein

First, the experimental procedure

1. Dissolving lyophilized protein powder IFN-gamma, CBD, IFN-gamma-CBD with a certain amount of PBS

2. The concentration is measured, and a certain amount of each protein is taken, two protein samples are respectively prepared, SDS-PAGE gel is run, one protein sample is stained by Coomassie brilliant blue, and the other protein sample is used for detecting the his resistance by western blot.

Second, experimental analysis

FIG. 5 shows purified PET-28(a) + IFN-. gamma., CBD and IFN-. gamma. -CBD run on the same 12% SDS-PAGE gel stained with Coomassie Brilliant blue, M representing a protein marker whose molecular weight is indicated, and 1,2, and 3 representing IFN-. gamma., CBD and IFN-. gamma. -CBD, respectively. It can be seen from the figure that both IFN-gamma purified by supernatant method and CBD and IFN-gamma CBD purified in precipitate form and renatured by dialysis have higher purity.

FIG. 6 shows a western blot with his resistance, where M represents protein marker, 1,2, and 3 represent IFN-. gamma.CBD, and IFN-. gamma.CBD, respectively. As can be seen from the figure, all the purified proteins carry his tags, and relatively no hetero-proteins carrying his tags exist.

Example 7 detection of IFN-. gamma.Immunity in fusion proteins

First, the experimental procedure

1.4T-1 cells are uniformly spread in a plurality of 90 cm dishes after being overgrown.

2. When the cell density in the dish is 80% -90%, adding BSA, IFN-gamma, CBD and IFN-gamma-CBD respectively; 1000 ng/ml.

3. After the protein is stimulated for 15min, cells are scraped by using cells, the cells are centrifuged for 2000 r multiplied by 5min, the supernatant is taken, a certain amount (200 ul) of PBS and protease inhibitor (1.5 ul) are added, ice ultrasound is carried out (30W, 3S and 5 times), the supernatant is centrifuged for 12000 r multiplied by 10min again, and the protein concentration in the supernatant is measured by a BCA method.

4. Selecting 80ug of each protein as total amount, preparing two samples, running SDS-PAGE gel, making BSA, IFN-gamma, CBD, IFN-gamma-CBD, and detecting Stat1 and beta-actin respectively; PStat1, β -actin.

Second, experimental analysis

FIG. 7 shows the phosphorylation of STAT1 by IFN-. gamma.domain detected by western blot using 4T-1 breast cancer cell line, BSA as negative control protein, and IFN-. gamma.and CBD as fusion protein controls. It can be seen from the figure that the CBD has no activity of stimulating phosphorylation of STAT1, and IFN- γ has an activity of stimulating phosphorylation of STAT1, and after attaching the collagen binding domain CBD, this stimulation of phosphorylation of STAT1 still exists, i.e. the fusion protein of the present invention should have immune activity corresponding to IFN- γ.

Example 8 was carried out: detection of CBD migration inhibition activity in fusion protein, Transwell migration experiment

First, the experimental procedure

1. Preparation before experiment: one ice box each for one big and one small, matrix (diluted on ice, but not solidified), dispensed, one plate with 12 cells; there are four proteins to do (BSA, IFN-. gamma., CBD, IFN-. gamma. -CBD).

2. Preparing a mixture of Matrigel and protein, mixing the Matrigel: 1640 (0% FBS) diluted 1:2, i.e. 10ul Matrigel +20ul 1640 medium, so that the total amount of 3 cells (4 cells were allocated, in case of insufficient amount) was 66.7ul Matrigel +133.3ul 1640EP tubes 4 (wherein each 10ug of protein was filled with PBS and then 1640 was added to 160 ul) and placed on ice, and mixed well, taking care that bubbles were not generated as much as possible, and the gel could not be over-fired, nor sprayed with alcohol.

3. 50ul of the above-mentioned mixed liquid is respectively added to 3 cells of every protein appearance, notices must not produce the bubble, and rifle head department can remain some liquid, avoids producing the bubble, and during the dropwise add unsettled the adding to the bottom, shakes evenly, fully covers the cell bottom, places the incubator incasement for 5h, waits to glue fully to solidify the back, reseedes the cell.

4. Preparing cell suspension, digesting cells with pancreatin for 2-5 min, adding 10% serum culture medium to stop digestion, centrifuging, discarding supernatant, re-suspending cells with 10% serum culture medium, counting under the mirror, adjusting cell number, inoculating to 1 × 10 in each chamber⁵And (4) respectively.

5. Cells were seeded, 200ul of cell suspension was added to the upper chamber, and 500ul of medium containing 10% FBS (serum) was added to the lower chamber, per well. Placing into an incubator, and culturing for 48h by using a serum-free culture medium after 3 h.

6. And (3) performing cell crystal violet staining and photographing, adding 500ul of 4% paraformaldehyde into the other row of wells, fixing for 30min, staining for 30min by using 500ul of 0.1% crystal violet staining solution, adding 1 ml/well of PBS into a new 24-well plate, transferring the Transwell chamber into a well containing the PBS, cleaning, transferring the Transwell chamber into new PBS, slightly shaking, and washing the cells.

7. The cells in the upper chamber were wiped off with a cotton ball, gently, the chamber was left upside down and air dried for 30min, and the total number of cells passing through each chamber was counted by taking a picture under a microscope.

Second, experimental analysis

FIG. 8 shows on the left side 4T-1 cells which were stained with crystal violet and migrated on the dorsal side of a Transwell chamber, which is typical of a Transwell migration experiment, taken under a microscope. The right panel is the quantification result, counting the number of cells that have passed through the entire chamber.

As a normal control, we can clearly conclude from the figure that all three proteins IFN-. gamma.CBD, IFN-. gamma.CBD have the activity of inhibiting migration of 4T-1 cells relative to BSA. Among them, IFN-. gamma. -CBD has a stronger effect of inhibiting cell migration than other proteins, i.e., the fusion protein of the present invention has the activity of the collagen binding domain CBD to inhibit cell migration.

Example 9 was carried out: the direct killing activity of the fusion protein to tumor cells is detected, mainly by detecting CCK8

First, the experimental procedure

1. First, Matrigel was plated in 96-well plates, Matrigel: 1640 (0% FBS) diluted 1:2, 4 secondary wells, 5 volumes, add 4 wells.

2. 20ul of protein BSA, IFN-. gamma.CBD, IFN-. gamma. -CBD were added to each well at1 ug. Standing at 37 deg.C for 5 h.

3. The 4T-1 cells were plated at 2000/100. mu.l and uniformly plated in 96-well plates with matrigel gel while the 4T-1 cells were plated at 2000/100. mu.l and uniformly plated in blank 96-well plates without matrigel gel.

After 4.48 hours, CCK8 detection is carried out, 10ul of CCK8 reagent is added into each hole, and after 2 hours, the absorbance value at 450nm is detected.

Second, experimental analysis

The main objective of this experiment was to detect the direct killing activity of IFN-. gamma.on tumor cells, with CCK8 (detection of cell viability) as the detection index, where BSA was used as a control protein. As shown in the attached figure 9, the simple IFN-gamma has the effect of directly killing the tumor cells to reduce the cell viability, the CBD does not have the effect, and when the IFN-gamma and the CBD are fused together, the fusion protein still has the effect of killing the tumor cells to reduce the activity of the tumor cells.

Example 10 of implementation: fusion protein Release assay in Matrigel

First, the experimental procedure

1. 15 centrifugal tubes of 15ml are respectively taken and evenly divided into 3 groups, each group is marked with three groups of IFN-gamma, CBD and IFN-gamma-CBD, and each group is marked with 0,1, 3,7 and 14 days.

2. The following system was prepared, each tube was filled with 100ul of the corresponding protein and Matrigel. Each tube is as follows: (protein 5ug + PBS 50 ul: Matrigel =1: 1), each was prepared in 6-fold volume system and then mixed well.

3. 100ul of the above mixture was put into each tube, and the gel was placed at 37 ℃ for 5 hours, taking care that no air bubbles were generated.

4. After the gel was completely solidified, 10ml of PBS was added to each gel except for 0 group (stored directly in a refrigerator at-20 ℃ C.), and the gel was placed in a 37 ℃ incubator.

5. Sucking out supernatant in 1,3,7, and 14 days, storing in 4 deg.C refrigerator, sucking out supernatant in precipitate, and storing at-20 deg.C.

6. After all samples are collected, dissolving the gel block, preparing a sample (adding 25ul of 5 xSDS Loading buffer, boiling for 5min in a metal bath, after the gel block is completely dissolved into a liquid state, centrifuging, throwing the sol solution to the bottom of a tube, sucking the sol solution into a 1.5ml centrifugal tube for preparing the sample) to prepare a western blot, and detecting the binding capacity of the fusion protein to the collagen by detecting his resistance.

Second, experimental analysis

The upper graph in fig. 10 is a graph in which the residual amount of proteins in Matrigel at different time points is determined by using his tags as western blots, and the lower graph is a graph in which the residual protein sample in Matrigel, namely the sustained release effect of various proteins in collagen in Matrigel, is determined by performing densitometry analysis by lowering bands of the western blots of the graphs.

It can be seen from the figure that the CBD alone and the fusion protein IFN- γ -CBD of the invention have a certain sustained release function in matrigel, indicating that the purified protein with CBD domain has the ability to bind to collagen, although matrigel is not pure collagen. Of course, the longer time-release effect of IFN-. gamma. -CBD than CBD may also be related to the molecular weight and retention effect present.

EXAMPLE 11 IFN-. gamma. -CBD has both the binding activity of CBD to collagen and the IFN-. gamma.Activity

First, the experimental procedure

1. First, Matrigel was plated in 96-well plates, Matrigel: 1640 (0% FBS) diluted at 1:2, 4 sub-wells, 5 volumes, 4 wells, 4ug of each protein, 20ul per well, 5h at 37 ℃.

2. After the gel is completely solidified, adding PBS for soaking, and changing the solution every 8h for 3 days.

3. Then, 2000T-1 cells/100. mu.l were initially plated and uniformly plated in a 96-well plate containing Matrigel.

4. After culturing for 48 hours, carrying out CCK8 detection, adding 10ul of CCK8 detection reagent, continuing to incubate for 2 hours, and measuring the absorbance value at 450 nm.

Second, experimental analysis

FIG. 12 shows the killing effect of the fusion protein on 4T-1 tumor cells as detected by CCK 8. BSA was present as a control protein and IFN- γ not washed with PBS was used as a positive control, and previous experiments have shown that IFN- γ and IFN- γ -CBD have the effect of directly reducing 4T-1 cell viability. This experiment is intended primarily to demonstrate that the fusion protein of the invention is indeed a fusion of two proteins rather than a mixture of two proteins alone. From the analysis of FIG. 12, the fusion protein of the present invention is a fusion of IFN-. gamma.and CBD rather than a mixture.

EXAMPLE 12 inhibitory Effect of the fusion protein of the present invention on the growth and metastasis of 4T-1 malignant breast cancer.

Balb/c female mice, 5-6 weeks old, purchased from Beijing Huafukang Biotech GmbH, 4% chloral hydrate, post anesthesia. 100ul of 1X 10 injections were injected into the second breast pad of each mouse⁷4T-1 cells/ml. After planting tumors for 6 days, measuring the sizes of the tumors, grouping according to the difference of the sizes of the tumors, ensuring that the sizes of the tumors in each group are equivalent, and complementarily dividing the larger tumors and the smaller tumors into 5 groups:

the therapeutic dose of each protein is calculated by taking the activity quantification of the fusion protein made by us as a reference, taking IFN-gamma 10000U/mouse, taking the same activity of IFN-gamma-CBD as the therapeutic dose of IFN-gamma-CBD as a reference, and taking the difference of the molecular weight of the CBD and the molecular weight of the IFN-gamma-CBD as a reference. After treatment, tumor size was measured every three days until 32 days later, the mice were sacrificed, the intact tumor tissue and lungs were taken out, the tumor tissue was directly taken out and placed in paraformaldehyde, after the lung tissue was taken out, the size of the metastases was counted first, then paraformaldehyde was placed, and stored at 4 ℃. Fig. 12 shows the growth curve of each group of mice, and fig. 13 shows the number of lung metastases of each group of mice.

As can be seen from FIG. 12, the fusion protein of the present invention has a more significant effect of inhibiting tumor growth compared to the control group. As can be seen from FIG. 13, the fusion protein of the present invention also has significant effect of inhibiting tumor metastasis compared to the control group, and the number of lung metastases is significantly inhibited. P <0.01. also, when compared with the control group, it was found that even if they were equal

The combination of IFN-gamma and CBD can not inhibit the growth and metastasis of tumor, which reflects the superiority of the fusion protein of the invention to fuse two structures by genetic engineering method.

Example 13: the fusion protein has the effect of prolonging the survival time of the melanoma.

First, the experimental procedure

C57BL/6 male mouse 6-8 weeks old, purchased from Beijing Hua Fukang biotech shares

A limited company. After 4% chloral hydrate anesthesia, 2.5X 10 injections were subcutaneously injected into the back of each mouse⁴B16-OVA: p2 tumor cells. After 5 days, the samples were randomly divided into 5 groups, which were set as follows:

the therapeutic dose of each protein is calculated by taking the activity quantification of the fusion protein made by us as a reference, taking IFN-gamma 10000U/mouse, taking the same activity of IFN-gamma-CBD as the therapeutic dose of IFN-gamma-CBD as a reference, and taking the difference of the molecular weight of the CBD and the molecular weight of the IFN-gamma-CBD as a reference. After treatment, mice were observed daily for survival. And recording is carried out, and the whole process lasts for more than 80 days. Fig. 14 shows survival curves for each group of mice.

It can be seen from FIG. 14 that the fusion protein of the present invention has a significant effect of prolonging the survival time of the tumor mice compared to the control group, and the combined treatment of IFN-. gamma.and CBD with equal activity does not have the same effect of prolonging the survival time of the tumor as the treatment group.

Sequence listing

<110> affiliated cooperation hospital of college of Tongji medical college of Huazhong university of science and technology

<120> a fusion protein comprising a collagen binding domain

<160>2

<210>1

<211>1083bp

<212>DNA

<220>

<221>CDS

<400>1

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat

atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcca cggcacagtc

attgaaagcc tagaaagtct gaataactat tttaactcaa gtggcataga tgtggaagaa

aagagtctct tcttggatat ctggaggaac tggcaaaagg atggtgacat gaaaatcctg

cagagccaga ttatctcttt ctacctcaga ctctttgaag tcttgaaaga caatcaggcc

atcagcaaca acataagcgt cattgaatca cacctgatta ctaccttctt cagcaacagc

aaggcgaaaa aggatgcatt catgagtatt gccaagtttg aggtcaacaa cccacaggtc

cagcgccaag cattcaatga gctcatccga gtggtccacc agctgttgcc ggaatccagc

ctcaggaagc ggaaaaggag tcgctgcgaa ggccaagtgg tccgtgtgaa gtatgggaac

gccgatgggg agtactgcaa gttccccttc ttgttcaatg gcaaggagta caacagctgc

actgataccg gccgcagcga tggcttcctc tggtgctcca ccacctacaa ctttgagaag

gatggcaagt acggcttctg tccccatgaa gccctgttca ccatgggcgg caacgctgaa

ggacagccct gcaagtttcc attccgcttc cagggcacat cctatgacag ctgcaccact

gagggccgca cggatggcta ccgctggtgc ggcaccactg aggactacga ccgcgacaag

aagtatggct tctgccctga gaccgccatg tccactgttg gtgggaactc agaaggtgcc

ccctgtgtct tccccttcac tttcctgggc aacaaatatg agagctgcac cagcgccggc

cgcagtgacg gaaagatgtg gtgtgcgacc acagccaact acgatgatga ccgcaagtgg

ggcttctgcc ctgaccaagg gaagcttgcg gccgcactcg agcaccacca ccaccaccac

tga

<210>2

<211>360

<212>PRT

<220>

<221>MUTAGEN

<400>2

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

Gly Ser Glu Phe His Gly Thr Val Ile Glu Ser Leu Glu Ser Leu Asn

Asn Tyr Phe Asn Ser Ser Gly Ile Asp Val Glu Glu Lys Ser Leu Phe

Leu Asp Ile Trp Arg Asn Trp Gln Lys Asp Gly Asp Met Lys Ile Leu

Gln Ser Gln Ile Ile Ser Phe Tyr Leu Arg Leu Phe Glu Val Leu Lys

Asp Asn Gln Ala Ile Ser Asn Asn Ile Ser Val Ile Glu Ser His Leu

Ile Thr Thr Phe Phe Ser Asn Ser Lys Ala Lys Lys Asp Ala Phe Met

Ser Ile Ala Lys Phe Glu Val Asn Asn Pro Gln Val Gln Arg Gln Ala

Phe Asn Glu Leu Ile Arg Val Val His Gln Leu Leu Pro Glu Ser Ser

Leu Arg Lys Arg Lys Arg Ser Arg Cys Glu Gly Gln Val Val Arg Val

Lys Tyr Gly Asn Ala Asp Gly Glu Tyr Cys Lys Phe Pro Phe Leu Phe

Asn Gly Lys Glu Tyr Asn Ser Cys Thr Asp Thr Gly Arg Ser Asp Gly

Phe Leu Trp Cys Ser Thr Thr Tyr Asn Phe Glu Lys Asp Gly Lys Tyr

Gly Phe Cys Pro His Glu Ala Leu Phe Thr Met Gly Gly Asn Ala Glu

Gly Gln Pro Cys Lys Phe Pro Phe Arg Phe Gln Gly Thr Ser Tyr Asp

Ser Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr

Thr Glu Asp Tyr Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro Glu Thr

Ala Met Ser Thr Val Gly Gly Asn Ser Glu Gly Ala Pro Cys Val Phe

Pro Phe Thr Phe Leu Gly Asn Lys Tyr Glu Ser Cys Thr Ser Ala Gly

Arg Ser Asp Gly Lys Met Trp Cys Ala Thr Thr Ala Asn Tyr Asp Asp

Asp Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly Lys Leu Ala Ala Ala

Leu Glu His His His His His His

Claims (5)

1. A fusion protein containing a collagen binding domain, the amino acid sequence of which is as shown in SEQ ID NO: 2, respectively.

2. A DNA molecule encoding the fusion protein of claim 1.

3. The DNA molecule of claim 1, wherein: the nucleotide sequence of the DNA molecule is shown as SEQ ID NO: 1 is shown.

4. A vector comprising the DNA molecule of claim 2 or 3.

5. Use of the fusion protein of claim 1 for the preparation of a medicament against breast cancer or melanoma.