CN106496329A - A kind of fusion protein containing collagen protein binding structural domain - Google Patents

Abstract

The present invention relates to a kind of fusion protein of the collagen protein binding structural domain with restructuring, its aminoacid sequence such as SEQ ID NO：Shown in 2.The fusion protein of the present invention is pharmaceutical solutionses, can be used to prepare antitumor drug, is administered by way of the intratumor injection of local, disposably gives foot therapy dosage.In the fusion protein of the present invention, the slow-released system of the local formed by collagen binding domain CBD avoids IFN γ well and is diffused into the strong immunne response caused by surrounding tissue.Simultaneously without the need for being repeatedly administered, this had both reduced the huge medical expense spent by the painful and repeated multiple times medication that the wound caused by multiple dosing is brought to patient, while reduce heavy dose of toxic and side effects frequently caused by medication again.

Description

A kind of fusion protein containing collagen protein binding structural domain

Technical field

The present invention relates to biological technical field.Specifically related to contain matrix metalloproteinase（MMP2）Collagen protein integrated structure Domain（collagen binding domain, CBD）And gamma interferon（IFN-γ）The recombiant protein of active structure domain and Its fusion protein, the fusion protein particularly with multiple activities.

Background technology

Tumor is one of first cause that world wide causes disease and death.It seriously threatens the health of the mankind, Cause the highest attention of people.Between past more than 20 years, substantial amounts of research focuses on involved tumor in tumour progression Generation and signal path between correlation molecule event.Between research discovery tumor cell and tumor microenvironment, complexity is mutual The molecular mechanism of effect is played an important role in the progress of tumor.40 years of researches show there is substantial amounts of evidence simultaneously Sertoli cell epimatrix remodeling proteins enzyme such as matrix metalloproteinase (matrix metalloproteinases, MMPs), be The moderator of micro-environmental variation in main cancer progression.MMPs is also considered as the one of multiple different tumor different stages of development The diagnosis of individual potentiality and prognostic indicator.Based on important function of the MMP played in tumor develops, design synthesizes energy Certain MMPs Active Site Specific is enough suppressed to combine and have not a particle of the inhibitor or list of cross reaction with other MMPs Clonal antibody just seems reasonably necessary.At present, the various inhibitors of MMP show different degrees of antitumor work in an experiment Property, but not good with therapeutic effect in clinical trial and count out.

In order to overcome this difficult problem of inhibitor design as much as possible, it is necessary to find one related in Binding Capacity position The material that point plays a role, it is positioned at outside avtive spot, is but positioning necessary material molecule in outside in hydrolytic process.And it is big Quantifier elimination evidence shows that specific exterior materials binding site is positioned at outside catalytic site, is that matrix metalloproteinase is fixed Position hydrolysis substance molecule is requisite.The material that therefore, it can block the interaction that exterior materials are mediated is selected as height The MMP inhibitor of selecting property will preferably be selected.And the CBD of MMP2 just conforms exactly to These characteristics.Studies have reported that title CBD with A large amount of features of the interaction of ligand binding show that CBD has almost been responsible for all characteristics that MMP2 is combined with collagen.And many Planting includes under pathological state that tumor, inflammation, infectious disease, the degenerative disorders of cerebrovascular disease find that gelatinase activity increases Plus.Gelatinase is broadly divided into gelatin enzyme A and Gelatinase B, and wherein gelatin enzyme A is primarily referred to as MM2, and Gelatinase B is primarily referred to as MMP9.Although MMP-2 and MMP-9 the two gelatinases have something in common in many aspects, there was only MMP2's according to the literature CBD is reported and effectively can be combined with IV Collagen Type VIs, and can be in conjunction with some natural and denatured collagen types, and elastic egg White and heparin.And, the combination zymolyte and other extracellular matrix molecules of CBD mediations are that MMP-2 and cell migration are sent out Wave the requisite key element of normal function.

Therefore, if out, the identical Protein Separation with the CBD of internal MMP2 can be placed in internal collagen content Position that is abundant and occurring to be raised the pathological condition for causing invasion and attack and transfer such as tumor by MMP, with internal CBD competitively In conjunction with identical collagen binding site, then the MMP of rise cannot cause corresponding pathological state, in the generating process of tumor In just can suppress to attack accordingly and transfer.If now in local, in addition cytokine is stimulated again, one side energy The immunne response for strengthening tumor by local has the effect of direct killing tumor simultaneously again, then the generation and transfer of tumor is just likely to It is totally constrained.

Interferon (IFN-γ) is mainly produced by T cell, and structure is made up of one section of secretion peptide and an active structure domain, Peptide is secreted during secreting out of cell to be cut away, form ripe IFN-γ.IFN-γ has Propagation, increases multiple antitumor actions such as surface MHC antigens and expression of tumor necrosis factor, Antineoplastic angiogenesis.20th century From the seventies, scientist starts the genetic engineering production for exploring interferon.1993, U.S. FDA ratified genetic engineering IFN-γ Put on market.As the continuous listing of domestic genetic engineering interferon, application clinically gradually increase, have received preferably Curative effect.But, due to IFN-γ molecular weight, easily by glomerular filtration, and during clinical practice, usually muscle and subcutaneous note The mode that penetrates, Formulations for systemic administration, and also plasma half-life is shorter, needs heavy dose of frequently medication, such as 2～3 times weekly, and IFN-γ During the diseases such as treatment hepatitis, tumor, multiple sclerosis, rheumatoid, treatment cycle is usually up to the several months to the several years, this long-term Heavy dose of frequently medication not only increases pain and the medical expense of patient, and is also easy to produce toxic and side effects as generated heat（90% disease People can occur）It is the important activator of Monocytes/Macrophages mainly due to IFN-γ, it can induce and promote body local Or caused by the strong inflammatory reaction of whole body, bone marrow depression, lose weight, anorexia etc., seriously constrain its application.

In sum, MMP2 can promote the occurrence and development of tumor, but individually the CBD of MMP2 can be in conjunction with iuntercellular The collagen protein of matter, while degraded that can be with competitive inhibition MMP2 to collagen protein；The active structure domain of IFN-γ has many Weight anti-tumor activity, but can also cause serious toxicity.If by both structure fusions together, and ensureing melting Keep each original function and activity, such protein molecular play the generation of tumor, development effectively in hop protein Inhibitory action.In the design of fusion protein and practice, Multiple techniques difficulty is faced, including：1. how accurately to find and position To the protein function domain for having medical value；2. the polypeptide fragment of different structure territory may influence each other and fold and empty Between structure, ultimately result in the forfeiture of function and activity, how to allow two or individual domain can keep each original function and Activity, is the key point for designing and preparing multi-functional fusion protein molecule；3. the domain combination of different protein values gets up Afterwards, on the basis of each original function is played, function organic assembling how is realized, realizes that function synergic is strengthened, excellent, inferior position Complementation, so that reach the therapeutic effect of brilliance；4. correct design and appropriate experiment condition, it is ensured that expressing fusion protein goes out To have stability and solubility afterwards, and it is easily isolated purification.The presence of these technical bottlenecks so that multi-functional fusion protein It is confronted with numerous difficulties from designing, being prepared in application process.

The present invention, based on the retrieval of substantial amounts of scientific literature, reads and screening, finds two potential anti-tumor activity structures Domain, using Protocols in Molecular Biology design construction fusion protein coded sequence, using prokaryotic expression technology and albumen affinity purification Technological expression and purification.Through carrying out substantial amounts of software reasonable prediction and experiment test, successful design and prokaryotic expression is constructed Carrier, finding out can high efficient expression and the experiment condition for being purified into soluble protein.

Content of the invention

It is an object of the invention to provide a kind of fusion protein of the collagen protein binding structural domain with restructuring.On the one hand, It can play the effect combined with the collagen protein binding structural domain on extracellular matrix and basement membrane, so as to suppress tumor thin The collagen binding site of the MMP2 of intracrine and the combination of collagen, play a part of to suppress tumor cell invasion and transfer；Simultaneously Enhance the IFN-γ concentration of tumor by local again, alleviate the toxicity of restructuring IFN-γ.On the other hand, in the fusion protein Another composition IFN-γ can just be trapped in tumor by local, can both play direct Tumor cytotoxicity, can increase again The immunne response of strong tumor by local body, reduces the immunologic escape of tumor cell.

It is a further object to provide the DNA sequence of coding fusion protein of the present invention.Including with Mus interferon （IFN-γ）Aminoacid the firstth area of mat_peptide sequences identical and the collagen protein binding structural domain with people MMP2（CBD） The DNA sequence of the fusion protein in the secondth area of aminoacid mat_peptide sequences identical.Wherein the firstth area and Mus IFN-γ ammonia Base acid mat_peptide sequences are identical, the collagen protein binding structural domain of the secondth area and people MMP2（CBD）Aminoacid mat_ Peptide sequences are identical.Centre is not added with the fusion protein of any connection peptides.Exactly such mentality of designing, we are purified The fusion protein for going out in the Experimental Research in later stage, find it both remained IFN-γ institute active, remain CBD domains again All features, are fusion protein truly.

In the building process of genetic engineering bacterium or genetically engineered cell, it is necessary first to obtain and expand expressed fusion protein Gene, we use PCR methods, and employ Overlap extension PCR method.In the base for obtaining expressed fusion protein in a large number Need to proceed in selected carrier gene of expression so as to building recombinant expression carrier using suitable carrier because after.

It is a further object to provide carrying the recombinant expressed load of the DNA sequence of coding fusion protein of the present invention Body.

It is a further object to provide the host of expression fusion protein encoding gene of the present invention.In the present invention we From expressive host be engineering bacteria BL21.

The present invention provide technical scheme be：A kind of fusion protein of the collagen protein binding structural domain with restructuring, its Aminoacid sequence such as SEQ ID NO：Shown in 2.

The fusion protein of the present invention is pharmaceutical solutionses, is administered by way of the intratumor injection of local, disposably gives foot therapy Dosage.In the fusion protein of the present invention, the slow-released system of the local formed by collagen binding domain CBD is avoided well IFN-γ is diffused into the strong immunne response caused by surrounding tissue.Simultaneously without the need for being repeatedly administered, this both reduced The huge medical expense spent by painful and repeated multiple times medication that wound caused by multiple dosing is brought to patient, while Reduce heavy dose of toxic and side effects frequently caused by medication again.

The fusion protein of the present invention includes the collagen protein binding structural domain of the MMP2 of people source（CBD）And Mus source IFN-γ.The wherein collagen protein binding structural domain of the MMP2 of people source（CBD）Coded aminoacid sequence and the amino of Mus After acid sequence contrast, the CBD Core domains of the Core domain of the CBD of the MMP-2 of constructed people and the MMP2 of Mus are found Do not change.

In the fusion protein of the present invention, we eliminate the signal peptide moiety of IFN-γ, also go in MMP2 protein moleculars Except collagen protein binding structural domain（CBD）Outside other parts.

In the fusion protein of the present invention, IFN-γ is located at the N- ends of fusion protein, the collagen protein of MMP2 for we Binding structural domain（CBD）It is located at the C- ends of fusion protein.In IFN-γ and the collagen protein binding structural domain of MMP2（CBD）It Between and be not introduced into any connection peptides.

Present invention also offers the DNA molecular of the above-mentioned fusion protein of coding.The nucleotide sequence of the DNA molecular such as SE Q ID NO：Shown in 1.

Further, the invention provides the carrier comprising above-mentioned DNA molecular, and the host comprising the carrier is thin Born of the same parents.The recombinant expression carrier of the present invention is PET-28 (a)+plasmid.

Fusion protein of the present invention can be used to prepare antitumor drug.

Method for transformation used by the present invention is YC conversion methods, filters out positive bacterium colony by resistance marker, then passes through again Bacterium solution PCR shakes the method that bacterium proposes plasmid enzyme restriction identification, further the antibacterial of successful conversion is identified, eventually through survey Sequence, carries out the contrast of sequence, finds the antibacterial of consensus amino acid sequence, is expressed.

In the fusion protein of the present invention, we employ isopropylthiogalactoside and carry out albumen as derivant Abduction delivering, while also express single CBD domains and IFN-γ.In the abduction delivering for carrying out albumen, Wo Menzhi If road albumen is present in supernatant, the purification experiment for carrying out the later stage is just easier.But unfortunately, except IFN-γ institute table The protein for reaching is present in supernatant, and which kind of expression way no matter remaining the two kinds of albumen expressed by us take, and which kind of uses instead Method, such as we have probed into different induction times respectively, and different induced concentrations and different inducing temperatures are carried out to albumen Abduction delivering, expressed albumen are present in precipitation all the time.This adds increased the difficulty of later-period purification.

In the fusion protein of the present invention, the albumen of our institute's abduction deliverings be with 6 his labels, when isolating and purifying we Destination protein is isolated and purified out by the competition affine combination of 6 his and nickel post.

In the fusion protein of the present invention, our institute's abduction delivering albumen out have secreted also to have to forgive the bodily form Formula is present.Typically all there is corresponding biologic activity to the albumen that expresses with secreted form.Nickel post can directly be gone up Purification, lyophilizing after dialysis are used by being dissolved in PBS during use.

For the albumen existed with inclusion bodies, after ni-sepharose purification, need to also need dialysis renaturation to process can just obtain life The albumen of thing activity.In this regard, we have done effort.Although, when expression, we have used multiple methods so that albumen It is present in supernatant, but finally cannot all reverses the fact that be present in precipitation.Therefore, we have carried out various preparations.First First, we have consulted multiple documents and materials about protein purification, and analyzing to affect the inclusion body protein that expresses correct The various factors of renaturation, has summed up a set of Laemmli buffer system Laemmli for being conducive to protein renaturation of oneself.Secondly, for how by institute Then the inclusion body cracking of extraction crosses nickel post and then carries out further dialysis renaturation, and we with reference to the fusion egg of Merck ＆ Co., Inc. White purification process handbook, is carried out degeneration using guanidine hydrochloride to inclusion body, is obtained required for us by different PH gradients Do not have activated albumen, then, a series of dialysis renaturations have been carried out using the renaturation buffer that we have concluded that to albumen, pass through A series of effort and the identification of subsequent experimental, we have successfully been obtained activated fusion protein.

Renaturation buffer system employed in the present invention is as follows：

（1）6M urea buffer solutions：0.1M NaH₂P04；0.01M Tris-HCI, 6M carbamide, PH：7.2.Dialysis 12 hours～24 Hour.

（2）Renaturation buffer：20mMTris, 2mM GSH, 5mM EDTA and 50mM Glycine, PH8.0.Dialysis 24 hours～48 hours, the once new renaturation buffer of middle replacing.

（3）2M urea buffer solutions：0.1M NaH2P04；0.01M Tris-HCI, 2M carbamide, PH：7.2.Dialyse 12 little When.

（4）50mM Tris-HCl buffer：50mM Tris, 200 mM NaCl, PH:7.4.Dialysis 24 hours, centre is more Change once new buffer.

All renaturation processes are carried out under the conditions of 4 DEG C.

We are first by the collagen protein binding structural domain of MMP2（CBD）For developing the medicine for suppressing neoplasm metastasis, survey Examination proves that CBD can suppress the invasion and attack and transferance of tumor cell.In fusion protein CBD-IFN- γ, the suppression of CBD and IFN-γ The effect of neoplasm metastasis processed has played synergism；Another aspect CBD and the combination of collagen protein, again can be by fusion protein CBD-IFN- γ long-time combine in the extracellular matrix of tumor microenvironment, so as to long time high concentration be retained in tumor office Natural slow-released system of the IV collagens that portion, i.e. tumor tissues are rich in as fusion protein CBD-IFN- γ.This is not only greatly greatly The strong anti-tumor activity of IFN-γ, while also avoid the poison caused by the heavy dose of frequently medication of whole body of IFN-γ secondary, while Reduce IFN-γ dosage.Between two domains of CBD-IFN- γ, CBD-IFN- γ can be played directly in tumor by local Tumor cytotoxicity effect, suppress tumor-blood-vessel growth, promote killing tumor cell T cell activity.

The DNA encoding sequence of fusion protein design：

ATG（Initiation codon）GGCAGCAGC（1# catenation sequences）CATCATCATCATCATCAC（His labels 1） AGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATT C（2# catenation sequences）CACGGCACAGTCATTGAAAGCCTAGAAAGTCTGAATAACTATTTTAACTCAAGTGGCATAGATG TGGAAGAAAAGAGTCTCTTCTTGGATATCTGGAGGAACTGGCAAAAGGATGGTGACATGAAAATCCTGCAGAGCCAG ATTATCTCTTTCTACCTCAGACTCTTTGAAGTCTTGAAAGACAATCAGGCCATCAGCAACAACATAAGCGTCATTGA ATCACACCTGATTACTACCTTCTTCAGCAACAGCAAGGCGAAAAAGGATGCATTCATGAGTATTGCCAAGTTTGAGG TCAACAACCCACAGGTCCAGCGCCAAGCATTCAATGAGCTCATCCGAGTGGTCCACCAGCTGTTGCCGGAATCCAGC CTCAGGAAGCGGAAAAGGAGTCGCTGC（The IFN activity domain of selection）GAAGGCCAAGTGGTCCGTGTGAAGTATGG GAACGCCGATGGGGAGTACTGCAAGTTCCCCTTCTTGTTCAATGGCAAGGAGTACAACAGCTGCACTGATACCGGCC GCAGCGATGGCTTCCTCTGGTGCTCCACCACCTACAACTTTGAGAAGGATGGCAAGTACGGCTTCTGTCCCCATGAA GCCCTGTTCACCATGGGCGGCAACGCTGAAGGACAGCCCTGCAAGTTTCCATTCCGCTTCCAGGGCACATCCTATGA CAGCTGCACCACTGAGGGCCGCACGGATGGCTACCGCTGGTGCGGCACCACTGAGGACTACGACCGCGACAAGAAGT ATGGCTTCTGCCCTGAGACCGCCATGTCCACTGTTGGTGGGAACTCAGAAGGTGCCCCCTGTGTCTTCCCCTTCACT TTCCTGGGCAACAAATATGAGAGCTGCACCAGCGCCGGCCGCAGTGACGGAAAGATGTGGTGTGCGACCACAGCCAA CTACGATGATGACCGCAAGTGGGGCTTCTGCCCTGACCAAGGG（The CBD sequences of selection） AAGCTTGCGGCCGCACTCGAG（3# catenation sequences）CACCACCACCACCACCAC（His labels 2）TGA (termination codon)

The peptide sequence of fusion protein：

M（Initial methionine）-GSS（1# catenation sequences）-HHHHHH（His labels 1）-SSGLVPRGSHMASMTGGQQMGR （2# catenation sequences）-HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQA ISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC（Choose IFN activity domain）-EGQVVRVKYGNADGEYCKFPFLFNGKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEA LFTMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTTEDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTF LGNKYESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQG（The CBD sequences of selection）—KLAAALE（3# catenation sequences）— HHHHHH（His labels 2）

The scheme tool advantages below that the present invention is provided：

Although the fusion protein of the present invention is expressed in the form of inclusion body, but by a series of dialysis renaturation, It has biologic activity, and is the summation of the respective biologic activity of two domains of fusion protein.

Fusion protein in the present invention is mainly with intratumor injection as primary treatment regimen, fusion protein surveyed after concentration with Freeze-dried powder is stored, and adds a certain amount of aseptic PBS solution to be dissolved during use, and BSA is used after having surveyed concentration again.Use Dosage can be by fusion protein relative to commercialization IFN-γ potency calculate, and single fusion protein CBD is then with relative Use in the identical molal quantitys of IFN-γ-CBD.Once daily is, there is affine knot in conjunction with CBD domains and extracellular matrix collagen The characteristic of conjunction forms a natural slow-released system in tumor by local.

The fusion protein of the present invention and its derivant or its being used in combination with other drugs, have collagen binding domain This characteristic makes it have preferable local sustained release effect, and which also hampers the tumor that MMP is mediated with the competitive inhibitory effect of MMP Invasion and Metastasis so as to suppressing the metastasis of tumor；Used as cytokine, itself not only has preferably IFN-γ simultaneously Direct killing function of tumor and suppression angiogenesis function, and can preferably strengthen tumor by local under the assistance in CBD domains Immunne response, promote the generation of forward direction immunne response in vivo, improve the fragmentation effect of tumor, its own is too strong and right because of immunity Caused by body is possible, toxic and side effects also can be reduced because being trapped in tumor by local and even disappear.

Description of the drawings

Enzyme action identifications of the Fig. 1 for the clone of the clone and PCMV-Tag2A IFN-γ plasmids of PCMV-Tag2A CBD plasmids.

Fig. 2 is that the enzyme action identification of PET-28 (a)+CBD plasmids and the enzyme action of PET-28 (a)+IFN-γ plasmid are identified.

Fig. 3 be PET-28 (a)+IFN-γ-CBD plasmid clonings good after, through III double digestion of ECOR I and Hind after, race 1% DNA glue qualification figures, are DL5000 marker.

Fig. 4 is PET-28 (a)+CBD, and PET-28 (a)+IFN-γ and PET-28 (a)+IFN-γ-CBD are in escherichia coli The SDS-PAGE analyses of the abduction delivering of middle IPTG.

PET-28 (a)+IFN-γs of the Fig. 5 for purification, the SDS-PAGE analyses of CBD, IFN-γ-CBD.

PET-28 (a)+IFN-γs of the Fig. 6 for purification, the western blot of the his label resistances of CBD, IFN-γ-CBD Analysis.

IFN-γ activity analysiss of the Fig. 7 for the albumen of purification.

Fig. 8 left figures are transwell migration experiments.

Fig. 9 is by CCK8 detections it is evident that the fusion protein with IFN-γ domain has significantly kill cell Effect.

The upper figures of Figure 10 are to determine in matrigel glue collagen in residual protein sample, i.e. Matrigel by photodensitometry Caned protein content combined with CBD domains, figure below are to be western blot by his labels to determine in different time points The amount of the fusion protein that can be combined with collagen in matrigel.

Figure 11 is fragmentation effect of the fusion protein detected by CCK8 to 4T-1 tumor cells.

Figure 12 is antitumous effect of the albumen of purification in breast cancer tumour mice body.

Figure 13 is impact of the albumen of purification in breast cancer mouse body to Lung metastases.

Figure 14 is the Survival of the melanoma tumors mice of statistics, it is found that IFN-γ CBD treatments have been obviously prolonged black The life cycle of plain tumor mice.

Figure 15 is fusion protein of the present invention design and operation principle ideograph.

Specific embodiment

Referring to Figure 15, the protein structure of metal matrix albumen -2 (MMP2) includes two parts, collagen protein integrated structure Domain（CBD）And catalyst structure domain, it is to make MMP2 combine extracellular matrix that the former acts on, and the latter's effect is degradation of cell epimatrix. Gamma interferon（IFN-γ）Coding protein sequence includes the signal peptide with secretory function and the maturation with biologic activity Two parts of IFN-γ.

Method of the present invention using molecular biology, by the biological active domain of the CBD and IFN-γ of MMP2 together structure Build up a new artificial gene, and be connected to can be on the prokaryotic vector of expressed fusion protein, abduction delivering in escherichia coli Be purified into this artificial fused protein molecule CBD：：IFN-γ.By test, it was demonstrated that it is this artificial that we invent Protein molecular, not only has CBD functions, that is, combine tumor tissue cell's epimatrix, and there is IFN-γ anti-tumor function, i.e., Lymphocyte is recruited in direct killing tumor cell, suppression angiogenesis and activation.

According to our design, CBD can allow this artificial fusion protein molecule CBD：：IFN-γ can be in conjunction with tumor Tissue extracelluar matrix, realizes following antitumous effect：

1. fusion protein is combined with the collagen protein of extracellular matrix, competitive inhibition tumor MMP2 combine and degradation of cell outside Substrate, so that suppress Nasopharyngeal neoplasms；

2. CBD：：IFN-γ can be present in tumor long-time, and maintain high local concentrations, directly kill so as to strengthen IFN-γ The effect for hindering tumor cell, the effect and activation that suppress angiogenesis and the effect for recruiting antineoplastic lymphocyte；

3. the solvable IFN-γ of low dosage in clinical trial, is given, the inflammatory reaction that generates heat with system, high agent can be caused The IFN-γ of amount can be lethal.CBD：：The collagen protein of IFN-γ tumor tissues is combined, it is to avoid the diffusion of activated IFN-γ, So as to reduce the systemic inflammatory responses of IFN-γ.

With reference to specific embodiment, the present invention will be further described.

Embodiment 1：The clone and the clone of Mus IFN-γ gene of people's CBD genes

The gene order of CBD is cloned from existing people MMP2.

The clone of IFN-γ gene：

（1）. adopt LA enzymes with<CTCGAG>Xhol with<GAATTC>EcoR1 is restriction enzyme site, and primer holds up section's biology skill by Wuhan Art company limited synthesizes, the working solution concentration for being diluted to 10 μM with pure water, and the IFN-γ gene order of design of primers institute foundation is come Source NCBI Reference Sequence: NM_008337.3）.

(2). the preparation of template：RNA is extracted in mouse cell, and then reverse transcription obtains the template that cDNA reacts as PCR

(3) .PCR reaction systems：Contain in 50ul reaction systems：ddH₂O 37.5ul；10×LA buffer 5ul； 10mMdNTP 4ul；Primer（F+R）2ul;mcDNA 1ul;LA enzyme 0.5ul.PCR response parameters：95 ℃ 1 min；94 ℃ 30 s, 57.9 DEG C of 30 s, 72 DEG C of 30 s, 40 circulations.72 ℃ 5 min；16 ℃ ∞.

(4) after the completion of .PCR, 2ul PCR product+2ul 10 × loading buffer are taken, the agarose for carrying out 1% coagulates Gel electrophoresis are detected.

(5). after stripe size is consistent with target, carries out DNA and cut glue reclaim, glue reclaim test kit measures institute after glue reclaim Contain DNA concentration（Nanodrop 1000）, then it is attached with PCMV carriers.

(6) .PCMV carriers are attached with glue reclaim product is cut, and linked system is as follows：PCMV 3ul; IFN-γ14ul; 10×T4buffer 2ul;T4 DNA ligase 1ul, common 20ul systems.Coupled reaction condition：20 DEG C, 2h.

(7). YC method conversions are carried out after connecting, and conversion is aseptically carried out（Alcohol burner, aseptic EP pipes, pipette tips）, behaviour Make as follows：50ul systems contain：Connection product 20ul；5×KCM 10ul；DdH2O (aseptic) 20ul.After adding, mix, then plus Enter a competent cell（- 80 DEG C preservation, with preposition on ice）, after adding, EP pipes are mixed, 20 min, then room temperature is placed on ice 10 min are placed, 500ul LB (aseptic, non-resistant) are added.After shaking 50 min (200 r, 37 DEG C) on shaking table, take out, 3000 r × 5 min, after discarding more than half supernatant, dispel, and suck aseptic pipette tips, squeeze in kan+ resistance plates, burnt with ethanol The whole plate of the spreading rod uniform application crossing and dry, puts 37 DEG C of incubators, after a hour is inverted plate, 37 DEG C of incubators trainings Support overnight（It is less than 16h）.

(8). to converting the bacterium colony in flat board, single bacterium colony is chosen, add 100ulkan+ resistance culture bases, after shaking bacterium 1h, carry out Bacterium solution PCR.PCR reaction systems are as follows：20ul systems contain：ddH2O 8ul；2×mix 10ul；IFN-γ primer（F+R） 1ul;Bacterium solution 1ul.Reaction condition：95 ℃ 5 ;94 DEG C of 30 s, 57.9 DEG C of 30 s, 72 DEG C of 30 s, 40 circulations. 72 ℃ 5 min；16 ℃ ∞.

(9). the identification of conversion results：After the completion of PCR, electrophoresis is carried out with DNA glue, select in the same size with purpose band And brightness the higher person carries out shaking bacterium, sequencing after protecting bacterium, is sent, sequencing company is Wuhan Qing Ke Bioisystech Co., Ltd, sequencing is drawn Thing is universal primer, is provided by company.After sequencing, on PubMed, blast once, it is found that gene order is completely correct, will The bacterium solution of protected bacterium colony PCR is taken out, and connects bacterium, shakes bacterium（Protect bacterium）Upgrading grain.

Enzyme action mirror of the accompanying drawing 1 for the clone of the clone and PCMV-Tag2A IFN-γ plasmids of PCMV-Tag2A CBD plasmids Fixed.Wherein 1A be PCMV-Tag2A CBD plasmid clonings good after, run 1% DNA glue qualification figures, M bars swimming with representative is DL2000 marker, a swimming band representative is the PCMV-Tag2A CBD plasmids that build through III double digestion of EcoR I and Hind Afterwards, the CBD of PCMV-Tag2A and hMMP2 has been cut out respectively.Accompanying drawing 1B be pCMV-tag2A IFN-γs plasmid cloning good after, race 1% DNA glue qualification figures, the swimming of M bars are DL5000 marker with represented, and it is the pCMV-tag2A for building that b swims with representative After IFN-γ plasmid is through I enzyme action of EcoR I and Xhol, PCMV-tag2A and mouse IFN-γs have been cut out respectively.

Embodiment 2：CDS areas sig by the CBD of the MMP2 of the people for cloning and the DNA sequence of Mus IFN-γ Peptide is inserted between III two restriction enzyme sites of EcoR1 and Hind of expression vector PET28a (+).Using fusion restructuring Method.

(1). the primer of design fusion restructuring first.

(2). the PCR of fragment, fragment PCR reaction system：50ul reaction systems, ddH₂O 37.5ul；10×LA buffer 5ul；10mMdNTP 4ul；Primer（F+R）2ul;Plasmid 1ul;LA enzyme 0.5ul.PCR response parameters：95℃5min；94℃ 30s, 55 DEG C of 30s, 72 DEG C of 30s, 40 circulations.72℃5 min；16℃∞.After the completion of PCR clone PCRs, 2ul PCR products are taken + 2ul 10 × loading buffer, carry out 1% agarose gel electrophoresiies detection.

(3). the PCR reaction systems of vector linearization：Contain in 50ul reaction systems：ddH₂O 33ul；10×KOD buff 5ul；2mMdNTP 5ul；25Mm MgSO4：3ul；Primer（F+R）2ul;Plasmid 1ul;KOD enzyme 1ul.PCR reacts Parameter：95℃1min；95 DEG C of 30s, 66 DEG C of 30s, 68 DEG C of 3 min, 35 circulations.68℃10min；16℃ ∞.

(4), after the completion of .PCR clone PCRs, 2ulPCR product+2ul 10 × loading buffer is taken, 1% fine jade is carried out Sepharose electrophoresis detection.

(5). after electrophoresis determines that stripe size is correct, precipitation recovery is carried out, concrete operations are as follows：PCR primer+ddH2O Polishing 100ul；+ 250ul dehydrated alcohol；+ 10ul(3M PH5.2) NaAc.After mixing, -20 DEG C, 30 min.Take out, be centrifuged 12,000r×10min.Supernatant is abandoned, in precipitation, 75% ethanol of 500ml is added, light rolling mixes, recentrifuge, 12,000 r × 5min；Repeat this operation twice, abandon whole supernatants, residue does not exhaust supernatant, and recentrifuge 12,000r × 5min blot water, then (5～15min) is dried in room temperature, add 20ul ddH20, measure DNA concentration with Nanodrop.

(6). DPn1 enzyme action, precipitation recovery product DPn1 is carried out enzyme action, DPn1 enzyme action systems are as follows：Fragment： 20ul；PET28a (+) HindⅢ- EcoR1：18.8ul；10×buff T：3ul；DPn1 enzymes：1ul, is supplied with ddH20 30ul systems, 37 DEG C of enzyme action.After the completion of enzyme action.

(7). glue reclaim is cut, whole digestion products DNA gel electrophoresis is carried out, after electrophoresis to a certain degree observes stripe size afterwards Carry out cutting glue under ultraviolet device.The target blob of viscose for cutting is put in EP pipes, DNA glue reclaims after weighing, are carried out.

(8). fusion DNA vaccine, will cut glue reclaim product carries out fusion DNA vaccine, and fusion DNA vaccine reaction system is as follows：ddH20： 24ul PET28a (+)H-E：5ul；Homo CBD：7ul；2mMdNTP：5ul；10×KOD buff：5ul；25mM MgSO4： 3ul；KOD enzymes：1ul, 50ul system.PCR response parameters：95℃1min；95 DEG C of 30s, 66 DEG C of 30s, 68 DEG C of 3min, 35 are followed Ring.68℃10min；16℃ ∞.After the completion of fusion DNA vaccine, PCR primer is converted, conversion operation method is ibid.

(9). the identification of conversion results：The bacterium colony grown after conversion is risen with aseptic lancet choicest and is put into added with a certain amount of In Kan culture medium, bacterium is shaken overnight, upgrading grain, plasmid extraction description（The limited public affairs of Beijing village ally border biological gene science and technology Department）, enzyme action identification is then done, enzyme action system is as follows：20ul systems, plasmid：16ul；10×buff M:2ul；EcoR1:1ul； HindⅢ:1ul；20ul systems, 37 DEG C of enzyme action 2h.DNA glue identifications are carried out after the completion of enzyme action, and qualification result is just finding stripe size Really, company is therefore sent to carry out sequencing identification, identification finds that the aminoacid sequence that Individual base is mutated but encodes is constant, i.e. this structure Complete.

Accompanying drawing 2 is that the enzyme action identification of PET-28 (a)+CBD plasmids and the enzyme action of PET-28 (a)+IFN-γ plasmid are identified. Wherein accompanying drawing 2A be PET-28 (a)+CBD plasmid clonings good after, run 1% DNA glue qualification figures, wherein M bars swimming with representative is DL5000 marker, c swimming band representative be PET-28 (a)+CBD plasmids that build through III double digestion of ECOR I and Hind after, PET-28 (a)+and CBD has been cut out respectively.Accompanying drawing 2B be PET-28 (a)+IFN-γ plasmid cloning good after, run 1% DNA glue reflect Scheme calmly, wherein M bars swimming is DL5000 marker with represented, and it is PET-28 (a)+CBD plasmids for building that d swims with representative After through III double digestion of ECOR I and Hind, PET-28 (a)+and IFN-γ has been cut out respectively.

Embodiment 3：The sig peptide in the CDS areas of the IFN-γ DNA sequence of the mouse for cloning are inserted into The upstream in the CDS areas of the DNA sequence of the CBD of the MMP2 of people in PET-28 (a)+CBD

Operational approach is still fusion restructuring, and, used as carrier, PET-28 (a)+IFN-γ is used as fragment, behaviour for PET-28 (a)+CBD Make same PET-28 (a)+CBD, the structure of PET-28 (a)+IFN-γ.

Fig. 3 be PET-28 (a)+IFN-γ-CBD plasmid clonings good after, run 1% DNA glue qualification figures, wherein M bars swim band That represented is DL5000 marker, and the swimming band representative of e bars is PET-28 (a)+IFN-γ-CBD plasmids for building through ECOR I After III double digestions of Hind, PET-28 (a)+and IFN-γ-CBD has been cut out respectively.

Embodiment 4：PET-28 (a)+CBD, PET-28 (a)+IFN-γ, PET-28 (a)+IFN-γ-CBD is big The SDS-PAGE analyses of the abduction delivering of IPTG in enterobacteria

Wherein only have PET-28 (a)+IFN-γ to be present in supernatant, remaining is present in precipitation, is mainly with PET- below Illustrate as a example by the abduction delivering of 28 (a)+CBD.

First by PET28a (+) CBD constructed before before induction, PET28a (+) IFN-γ, PET28a (+) IFN-γ- The plasmid of CBD；Translation table reaches competence BL21 respectively, and then picking single bacterium colony shakes bacterium, protects bacterium, carries out the induction table of albumen Reach.

The exploration of 1.IPTG induction times

(1). take 8 test tubes and be separately added into 5mL Kan fluid mediums, and protected bacterium PET28a (+) CBD BL21 are shaken bacterium mistake The bacterium solution of night gained respectively adds 0.5 mL, shakes 1.5h or so, to bacterium solution OD ≈ 0.8.

(2). numbering 0,1,2,3,4,5,6,7 respectively on pipe again；And respectively add 100mM IPTG 50ul, i.e. IPTG concentration For 1mM, 37 DEG C, 200r shakes bacterium.

(3). again respectively 0, take out the test tube of reference numeral after 1,2,3,4,5,6,7, h respectively, centrifugation 4000r × 10min, abandons supernatant, and recentrifuge, till inhaling less than supernatant, is preserved to 4 DEG C.

(4). after all of test tube is all gathered, 1 mL PBS are resuspended for each addition, are centrifuged 4000r × 10min, repeat Once, after exhausting supernatant, -20 DEG C of preservations are put.

(5). the precipitation taking-up of above-mentioned bacterium solution was put on ice in second day, each addition 500ul PBS add 6ul PMSF（Eventually Concentration is 1mM）, then put ultrasound on ice（125W, 50% power, super 3S stop 5S）Repeat（20 times or so）Extremely fully transparent.

(6). centrifugation, 12,000r × 10min, draw supernatant into new EP pipes, are inhaled less than supernatant completely into precipitating （In operation, ice is put all the time）.

(7). measure protein concentration in supernatant（BCA determination of protein concentration methods）, according to the protein concentration that measures, select minimum dense The Tot Prot of degree 20ul carries out albumen sample preparation for standard.

(8). SDS-PAGE glue is run, is first run with 70V voltages, treated that bromophenol blue is gone to concentrate at glue and separation gel demarcation line, change Run with 90V voltages, stop voltage when away from glass plate bottom 1.

(9). taking-up blob of viscose is put（Shaking, is sealed with plastic bag Mouthful）.

(10). dyeing liquor is outwelled within second day, adds destaining solution to be decolourized（Shaking）, treat that blob of viscose decolourizes to transparent, take out, Take pictures, glue can be steeped in ddH₂In O.

(11). expressing quantity peaking when caning be found that induction 3h from the cementing fruit of race, then extend the time, expressing quantity Do not increase, and albumen is with inclusion bodies presence, the albumen size for giving expression to and the simultaneously zero difference that pre-estimates.Such as Accompanying drawing 4A.

The exploration of 2.IPTG induced concentrations

Concrete operations as above, simply as bacterium solution OD ≈ 0.8, are separately added into IPTG 1（0ul）, 2（2.5ul）, 3（5ul）, 4 （10ul）, 5（20ul）, 6（30ul）, 7（40ul）, 8（50ul）；Sample is received after 3h.Other are identical with the exploration of IPTG induction times, Similarly find that albumen is present in inclusion body, when it is that IPTG concentration is 0.1mM for 5ul to add IPTG amounts, expressed Protein content at most, is further added by IPTG concentration, and expressing quantity does not increase, and figure is omited.

The exploration of 3.IPTG inducing temperatures

Concrete operations as above, simply as bacterium solution OD ≈ 0.8, are taken out, respectively 0,4h, 8h, 20h of numbering, add in addition to numbering 0 and respectively Enter 5ul IPTG, 25 DEG C, 200 r. simultaneously take out correlation number test tube in corresponding time point respectively（As taken out numbering 4h after 4 h Test tube）, 0 with while 4h take out.Other are identical with the exploration of IPTG induction times, even similarly finding at low temperature Abduction delivering is carried out, albumen is still with inclusion bodies presence, the albumen size for giving expression to and the simultaneously zero difference that pre-estimates. I.e. above abduction delivering all illustrates that albumen is present in precipitation, exists with inclusion bodies.Figure is omited.

The abduction delivering of 4.PET-28 (a)+IFN-γ employs the optimal conditionss of PET-28 (a)+CBD abduction deliverings, The IPTG concentration of selection is 0.1mM, induction time is explored, 0,1,2,4h for selecting respectively, finds albumen after induction There is expression in supernatant, and amount is not low, in 2h, expressing quantity has reached peak value.Such as accompanying drawing 4B.

The abduction delivering of 5.PET-28 (a)+IFN-γ-CBD equally employs PET-28 (a)+CBD abduction deliverings Optimal conditionss, the IPTG concentration of selection is 0.1mM, and induction time is explored, and 0,1,2,4h for selecting respectively, after induction It was found that protein free expression in supernatant, have in only precipitating the expression of a large amount of albumen, i.e. albumen be present in inclusion body and And during 2h, expressing quantity reaches peak value, such as accompanying drawing 4C.

Embodiment 5：The ni-sepharose purification of fusion protein：

1. ni-sepharose purification IFN-γ albumen under natural endowment

(1). protected bacterium PET28a (+) IFN-γ BL21 will respectively take 50ul and be inoculated in two test tubes before.Per each addition 5 ML Kan fluid mediums, then shake bacterium overnight.

(2). 1L Kan fluid mediums are prepared, by above-mentioned shaken bacterium solution（10 mL）Add, 200r, 37 DEG C, shake 4 h left The right side, to OD ≈ 0.8

(3). add 1 mL IPTG induce 2 h after, take out be placed in 50 mL centrifuge tubes be centrifuged, 10000 r × 5min, not from Be partially disposed on ice, all after good, then from most supernatant once, is abandoned, put -20 DEG C of preservations.

(4). bacterium solution precipitation is taken out, respectively plus 2 mL PBS, blow outstanding after, be centrifuged 10000 r × 5min, abandon most supernatant, then Wash once, to it can not inhale supernatant.

(5). add 1 × Ni-NTA combination buffers resuspended in precipitation, be incorporated in a pipe, after supplying 20 mL, add 400ul PMSF, ultrasonic on ice（125W, 50% power, super 3S stop 5S）Surpass bottom transparent when, then 10000 r of high speed centrifugation × 5min, it is albumen filtrate to draw supernatant in clean centrifuge tube, with 0.45 μm of filter filtrate protein liquid in new centrifuge tube In.

(6). gravity post is placed, 1.5 mL substrate are added（Ni-NTA his Bind Resin are purchased from Novagen Shanghai Yu Bo bio tech ltd）, then filtered in 1 × Ni-NTA combination buffers addition post with 0.22 μm of filter, remove post Lower end closed lid, washes out the ethanol in post（2 times of column length volumes）After dripping off, albumen filtrate is added, and is received with clean pipe Collection effluent（Repeatable）, 4 DEG C preserve for SDS-PAGE electrophoretic analysiss.With 10ml with 0.22 μm of filter filtered 1 × Ni-NTA wash buffers are rinsed 2 times, collect rinse component, for SDS-PAGE electrophoretic analysiss. with a certain amount of Filtered 1 × Ni-NTA the elution buffers of 0.22 μm of filter gradually Deca eluting destination protein, is received with 1.5 mL EP pipes Collection effluent, surveys protein concentration when collecting with Nanodrop 1000, when value to be shown is for bearing, stops Deca elution buffer, Elution fraction is collected, for SDS-PAGE electrophoretic analysiss.

(7) .SDS-PAGE electrophoretic analysiss find that albumen is purified out, and purity >=90%, i.e. purification are completed.

(8). use 50mM Tris-HCl buffer, 4 DEG C of dialysed overnights again.Albumen is surveyed subpackage after concentration and is lyophilized into powder End.- 80 DEG C of preservations.

2. inclusion body（Insolubility albumen）The ni-sepharose purification of CBD

(1). protected bacterium PET28a (+) CBD BL21 will respectively take 50ul and be inoculated in two test tubes before.Per each addition 10mL Kan fluid mediums, then shake bacterium overnight.

(2). 1L Kan fluid mediums are prepared, by above-mentioned shaken bacterium solution（20 mL）Add, 200r, 37 DEG C, shake 2h left The right side, to OD ≈ 0.8,

(3). add 1 mL IPTG induce 2 h after, take out be placed in 50 mL centrifuge tubes be centrifuged, 10000 r × 5min, not from Be partially disposed on ice, all after good, then from most supernatant once, is abandoned, put -20 DEG C of preservations.

(4). bacterium solution precipitation is taken out, respectively plus 2 mL PBS, blow outstanding after, be centrifuged 10000 r × 5min, abandon most supernatant, then Wash once, to it can not inhale supernatant.Plus PBS is resuspended, it is incorporated in a pipe, after supplying 20 mL, adds 400ul PMSF, on ice Ultrasound（125W, 50% power, super 3S stop 5S）Surpass bottom transparent when, then 10000 r of high speed centrifugation × 5min inhales and abandons supernatant Afterwards, 20 mL denatured lysis/combination buffer, 50ul beta -mercaptoethanols is added to add 400ul PMSF, shake on ice overnight To fully dissolving（As soluble protein liquid）.

(5). after placing gravity post, add 1.5 mL substrate（Adsorption column）, then split with 0.22 μm of filter filtration degeneration Solution/combination buffer is added in post, removes post lower end closed lid, washes out the ethanol in post（2 times of column length volumes）After dripping off, plus Enter protein dissolution liquid, and effluent is collected with clean pipe（Repeatable）, 4 DEG C preserve for SDS-PAGE electrophoretic analysiss. Rinsed 2 times with 0.22 μm of filtered degeneration wash buffer of filter with 10ml, collect rinse component, for SDS-PAGE Electrophoretic analysiss. with the filtered denaturing elution buffer of a certain amount of 0.22 μm of filter gradually Deca eluting destination protein, use 1.5 Effluent collected by mL EP pipes, surveys protein concentration when collecting with Nanodrop 1000, when value to be shown is for bearing, stops Deca Elution buffer, collects elution fraction, for SDS-PAGE electrophoretic analysiss.

(6). as the sample of hydrochloric guanidine can form precipitation with SDS when being processed, carrying out SDS-PAGE electrophoresis Before, sample must dilute with water（ 1:6 dilutions）, or dialyse or guanidine hydrochloride is removed using TCA precipitations.

(7) .SDS-PAGE electrophoretic analysiss find that albumen is purified out, and purity >=90%, i.e. purification are completed.

(8). the recombiant protein eluent containing 8M carbamide is put in bag filter, in 6M urea buffer solutions, 4 DEG C, thoroughly Analysis is overnight.

(9). and then in renaturation Buffer, 4 DEG C, dialyse 24h, and changes renaturation Buffer.

(10). in 2M urea buffer solutions, 4 DEG C of dialysed overnights.

(11). in 50mMTris-HCl buffer, 4 DEG C of dialysed overnights are exchanging Buffer.Albumen is surveyed subpackage after concentration It is lyophilized into powder.- 80 DEG C of preservations

The same CBD of the ni-sepharose purification method of IFN-γ-CBD.

（Solution needed for purification under natural endowment：1 × Ni-NTA combination buffers： 50mM NaH₂PO4, PH 8.0, 300mM NaCl, 10mM imidazoles.1 × Ni-NTA wash buffers： 50mM NaH₂P04, PH 8.0,300mM NaCI, 20mM imidazoles.1 × Ni-NTA elution buffers： 50mM NaH₂PO4, PH8.0,300mM NaCI, 250mM imidazoles.

Solution needed for purification under Denaturing：Denatured lysis/combination buffer：6M GuHCI； 0.1M NaH₂P04； 0.01M Tris-HCI, PH8.0.Degeneration wash buffer：8M carbamide；0.1M NaH₂P04；0.01M Tris-HCI, PH6.3. Denaturing elution buffer：8M carbamide；0.1M NaH₂P04；0.01M Tris-HCI, PH4.5.Renaturation Buffer：20mM Tris, PH8.0, 2mM GSH, 5mM EDTA and 50mM Glycine.

The process of bag filter：The bag filter of clip appropriate length, is respectively placed in solution 1（0.5M containing 8%NaHCO3 EDTA, PH8.0）With solution 2（0.5MEDTA, PH8.0）In respectively boil 10min, then deionized water thoroughly cleaning, standby.

Figure is omited.

Embodiment 6：Fusion protein molecule amount size and His Resistance Identifications

One. experimentation

1., by the protein powder IFN-γ of lyophilizing, CBD, IFN-γ-CBD are dissolved with a certain amount of PBS

2. concentration is measured, respectively takes a certain amount of, made two parts of albumen samples respectively, run SDS-PAGE glue, portion does coomassie brilliant blue staining, Portion does western blot detection his resistances.

Two. experimental analysiss

PET-28 (a)+IFN-γ of the accompanying drawing 5 for purification, CBD and IFN-γ-CBD run the SDS-PAGE in same 12% On glue, through the result of coomassie brilliant blue staining, that M is represented is albumen marker, and the molecular size range of representative is marked, 1,2, 3 represent IFN-γ, CBD, IFN-γ-CBD respectively.Whether the IFN- being purified into by the method for supernatant as seen from the figure γ, or in the form of precipitating purification again through the CBD and IFN-γ CBD of dialysis renaturation, be respectively provided with higher purity.

By the western blot that make of his resistances, that M is represented is albumen marker to accompanying drawing 6, and 1,2,3 represents respectively IFN-γ, CBD, IFN-γ-CBD.As seen from the figure, purified albumen carries his labels, and relative without with his The foreign protein of label is present.

Embodiment 7:IFN-γ immunocompetence detection in fusion protein

One. experimentation

1.4T-1 cells are uniformly layered on after covering with multiple 90 wares.

2., when in ware, cell density length is to 80%-90%, BSA, IFN-γ, CBD, IFN-γ-CBD are respectively added； 1000ng/ml.

3. after above-mentioned albumen stimulates 15 min, scraping cells are scraped with cell, be centrifuged 2000 r × 5min, take supernatant, add A certain amount of（200ul）PBS and protease inhibitor（1.5ul cooktail）Afterwards, ultrasonic on ice（30W, 3S, 5 time）, again from 12000 r of the heart × 10min takes supernatant, and BCA methods measure protein concentration in supernatant.

4. each albumen selects 80ug as total amount, respectively two parts of samples of system, runs SDS-PAGE glue, is BSA, IFN-γ, CBD, IFN-γ-CBD, detects Stat1, β-actin respectively；PStat1, β-actin.

Two. experimental analysiss

Accompanying drawing 7 is to detect the situation of IFN-γ this domain to STAT1 phosphorylations, cell line used by western blot For 4T-1 breast cancer cell lines, in figure, BSA is present as negative control protein, and IFN-γ and CBD are also the right of fusion protein According to.CBD does not have as seen from the figure stimulates the activity of STAT1 phosphorylations, and IFN-γ has the work for stimulating STAT1 phosphorylations Property, and after connecting collagen binding domain CBD, this stimulation STAT1 phosphorylations are yet suffered from, i.e., fusion protein of the invention The immunocompetence that should have IFN-γ corresponding.

Embodiment 8：In fusion protein, CBD suppresses migratory activity detection, Transwell migration to test

One. experimentation

1. prepare before testing：Small one and large one each ice chest, the Matrigel of subpackage（It is diluted on ice, not so can solidifies）, One block of plate has 12 cells；There are four albumen to need to do（BSA, IFN-γ, CBD, IFN-γ-CBD）.

2. the mixed liquor of Matrigel and albumen is prepared, by Matrigel：1640（0%FBS）Press 1:2 dilutions are 10ul 1640 culture medium of Matrigel+20ul, therefore 3 cells（Amount with 4 cells, in case not enough）Total amount is 66.7ul Matrigel+133.3ul 1640EP pipes 4,（The wherein each 10ug of albumen, after albumen volume is with PBS polishings, then plus 1640 and supplies 160ul）It is placed on ice, fully mixes, notes trying not to produce bubble, and glue can not overdo, and can not spray ethanol.

3. 3 cells of each albumen sample respectively add the above-mentioned mixed liquors of 50ul, note necessarily to produce bubble, can at pipette tips Remain a part of liquid, it is to avoid produce bubble, and vacantly add against bottom during Deca, shake up, fully cover cell bottom, place 5h in incubator, after glue fully solidifies, inoculates cell.

4. prepare cell suspension, first use trypsin digestion cell 2-5 minutes, plus containing 10% blood serum medium terminate digestion after from The heart abandons supernatant, with 10% blood serum medium re-suspended cell is contained, counts under mirror, adjusts cell number, and each cell inoculation is 1 × 10⁵ Individual.

5. inoculating cell, adds 200ul cell suspension, lower room to add and contain 10%FBS in upper room（Serum）Culture medium, per Hole adds 500ul.It is put in incubator, after 3 h, uses serum-free medium culture 48h instead.

6. carry out cell violet staining, take pictures, in another round, add 4% paraformaldehydes of 500ul to fix 30 min Afterwards, 30min, then an addition PBS 1ml/ hole in 24 new orifice plates are dyeed with 0.1% violet staining liquid of 500ul, will Transwell cells are transferred in the hole containing PBS, cleaning, Transwell rooms are transferred in new PBS and are gently rocked, and washing is thin Born of the same parents.

7. the cell of upper interior is wiped with cotton balls, notes softly, cell back-off being air-dried 30min, being taken pictures under the microscope, Count the total cellular score gone through in each cell.

Two. experimental analysiss

8 left side of accompanying drawing is that the ratio for shooting under the microscope is more typical in Transwell migration experiments, crystallized purple dyeing, Past 4T-1 cells are migrated in Transwell cells dorsal surface.Right figure is the result for quantifying, and has counted that whole cell is all to be worn Past cell quantity.

BSA significantly can be reached a conclusion by us are schemed as normal control, for BSA, three kinds of albumen IFN- γ, CBD, IFN-γ-CBD are respectively provided with the activity for suppressing 4T-1 cell migration.Wherein, IFN-γ-CBD has compared to other albumen There is collagen binding domain CBD to suppress cell migration for the effect of higher suppression cell migration, i.e., fusion protein of the invention Activity.

Embodiment 9：Direct killing Activity determination of the fusion protein to tumor cell, mainly CCK8 are detected

One. experimentation

1. first paving matrigel in 96 orifice plates, Matrigel：1640（0%FBS）Press 1:2 dilutions, 4 secondary orifices, with 5 times of volumes, Plus 4 holes.

2., per hole 20ul, protein B SA, IFN-γ, each 1ug of CBD, IFN-γ-CBD are added per hole.37 DEG C put 5h.

3. 2000/100 μ l of 4T-1 cells are spread, in being uniformly layered on added with 96 orifice plate of matrigel glue. while it is thin to spread 4T-1 2000/100 μ l of born of the same parents, are uniformly layered on not plus in 96 orifice plate of blank of matrigel glue.

CCK8 detections are done after 4.48 hours, add CCK8 reagent 10ul per hole, after 2 h, absorbance at detection 450nm.

Two. experimental analysiss

The main purpose of this experiment is the direct killing activity for detecting IFN-γ to tumor cell, and Testing index is CCK8（Cell The detection of vigor）, wherein BSA is used as reference protein.By accompanying drawing 9 it can be seen that simple IFN-γ has directly kill tumor The effect of cell, make cell viability decline, CBD does not have this effect, when IFN-γ and CBD are merged, i.e., this Bright fusion protein still has the effect of killing tumor cell, causes the decline of activity of tumor cells.

Embodiment 10：Fusion protein release experiment in Matrigel

One. experimentation

1. take 15 15ml centrifuge tubes respectively, be equally divided into 3 groups, be respectively labeled as IFN-γ, three groups of CBD, IFN-γ-CBD;Per Group labelling 0 again, 1,3,7,14 day.

2. system is formulated as follows, often the corresponding albumen of pipe subpackage and each 100ul of Matrigel.As follows per body system：（Albumen 5ug+PBS are 50ul: Matrigel=1:1）, respectively the system with 6 times of volumes, then mixes.

3. above-mentioned mixed liquor 100ul is respectively taken in each pipe, be careful not to produce bubble, glue be put into 37 DEG C, 5h.

4. after glue solidifies completely, 10mlPBS is added respectively, numbering is 0 group（- 20 DEG C Refrigerator stores are directly put）Except, put Enter in 37 DEG C of incubators.

5. respectively 1, supernatant was suctioned out in 3,7,14 days, be put into 4 DEG C of Refrigerator stores, supernatant exhausts rearmounted -20 DEG C in precipitation Preserve.

6. after whole samples all collect neat after, blob of viscose is dissolved, sample preparation (plus 25ul 5 × SDS Loading are carried out Buffer, boils 5min in metal bath, treats that blob of viscose is completely dissolved as liquid, is centrifuged and for sol solutionses to be thrown to ttom of pipe, sucks 1.5ml Sample preparation in centrifuge tube) western blot are, by detecting binding ability of the his resistances come detection fusion albumen to collagen.

Two. experimental analysiss

10 upper figure of accompanying drawing is to be western blot by his labels to determine the albumen in different time point matrigel Surpluses, figure below is to carry out photodensitometry by the band of the upper figure western blot of drop come residual in the matrigel that determines Stay albumen sample, i.e., the slow release effect that various albumen can be reached in the collagen in Matrigel.

Individually the fusion protein IFN-γ-CBD of CBD and the present invention has necessarily in matrigel as seen from the figure Slow-release function, illustrate that the purified albumen with CBD domains is that have the ability combined with collagen, although matrigel is not pure Collagen.Certainly IFN-γ-CBD than CBD have the longer time slow release effect may also with molecular weight big, the retention effect for existing Relevant.

Embodiment 11:IFN-γ-CBD had both had the activity combined by CBD with collagen while having IFN-γ activity

One. experimentation

1. first paving matrigel in 96 orifice plates, Matrigel：1640（0%FBS）Press 1:2 dilutions, 4 secondary orifices, with 5 times of volumes, Plus 4 holes. every kind of Tot Prot 4ug, per hole 20ul, 37 DEG C put 5h.

2., after the perfectly sound solidification of glue, PBS immersions are added, a not good liquor is changed every 8h, is changed 3 days.

3. then start to spread 2000/100 μ l of 4T-1 cells, be uniformly layered in 96 orifice plates added with Matrigel.

4. CCK8 detections are done after cultivating 48 hours, after adding CCK8 detectable 10ul, are continued incubation 2h, are surveyed at 450nm Absorbance.

Two. experimental analysiss

Figure 12 is fragmentation effect of the fusion protein detected by CCK8 to 4T-1 tumor cells.BSA is present as reference protein, Used as positive control, experiment before has shown that IFN-γ and IFN-γ-CBD have directly drop to IFN-γ without PBS washings The effect of low 4T-1 cell viabilities.This experiment is primarily intended to prove that the fusion protein of the present invention is strictly the fusion of two kinds of albumen Rather than the mixture of two kinds of simple albumen.Analyzed by accompanying drawing 12 and can be obtained, the fusion protein of the present invention is IFN-γ and CBD Fusions rather than mixture.

Embodiment 12:Inhibition of the fusion protein of the present invention to the growth and transfer of 4T-1 malignant breast carcinomas.

Balb/c female mices, 56 week old, purchased from Beijing HFK Bio-Technology Co., Ltd., 4% chloral hydrate After anesthesia.100ul 1 × 10 is injected in second breast pad of each mice⁷Individual 4T-1 cells/ml.Implantation tumor 6 days Afterwards, tumor size is surveyed, is grouped according to tumor size difference, it is ensured that every group of tumor size is quite, larger mutual with compared with little tumour Benefit is divided into 5 groups：

The active level of the fusion protein done with us by the therapeutic dose of each albumen as reference, with IFN-γ 10000U/ Mice, IFN-γ-CBD therapeutic doses using with IFN-γ with isoreactivity as reference, and CBD then with it with IFN-γ-CBD point The difference of son amount size is calculated and is obtained.After treatment, a tumor size was surveyed per three days, by sacrifice after 32 days, Complete tumor tissues and pulmonary is taken out, tumor tissues directly take out and are put in paraformaldehyde, after lung tissue is taken out, first count and turn The size of stove is moved, paraformaldehyde is then placed in, 4 DEG C of preservations.Figure 12 shows that the growth curve of each group mice, Figure 13 show respectively The Pulmonary metastasis focuses quantity of group mice.

Fusion protein of the invention is compared with matched group as seen from Figure 12, with significantly suppressing tumour growth Effect.As seen from Figure 13, fusion protein of the invention has the effect for significantly suppressing neoplasm metastasis compared with matched group, also Really, the number of pulmonary's metastasis is significantly inhibited.* represents P<0.01. simultaneously compared with matched group, even if it can be found that Will etc.

The IFN-γ and CBD of activity is added together treatment, can not play the suppression growth and metastasis of tumours same as treatment group, Embody the superiority that two kinds of structures are merged by the fusion protein of the present invention with engineered method.

Embodiment 13：The fusion protein of the present invention has the effect for extending the life span of melanoma tumors well.

One. experimentation

6～8 week old of male Mus of C57BL/6, purchased from Beijing China Fukang biotechnology share

Company limited.After 4% chloral hydrate anesthesia, in the dorsal sc injection 2.5 × 10 of every mice⁴B16-OVA：P2 tumors Cell.5 groups are randomly divided into after 5 days, are respectively provided with as follows：

The active level of the fusion protein done with us by the therapeutic dose of each albumen as reference, with IFN-γ 10000U/ Mice, IFN-γ-CBD therapeutic doses using with IFN-γ with isoreactivity as reference, and CBD then with it with IFN-γ-CBD point The difference of son amount size is calculated and is obtained.After treatment, the Survival of mice is observed daily.And make a record, whole process It has been continued for more than 80 days.Figure 14 shows the survival curve of each group mice.

As seen from Figure 14 for matched group, the fusion protein of the present invention has Life span effect, and the IFN-γ of isoreactivity and CBD are added together treatment, can not play and prolong as treatment group is same The effect of the life span of bearing tumor.

Sequence table

<110>Wuhan Union Hospital

<120>A kind of fusion protein containing collagen protein binding structural 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 kind of fusion protein containing collagen protein binding structural domain, its aminoacid sequence such as SEQ ID NO：2 institutes Show.

2. a kind of coding claim 1 described in fusion protein DNA molecular.

3. DNA molecular according to claim 1, it is characterised in that：The nucleotide sequence of the DNA molecular such as SEQ I D NO：Shown in 1.

4. a kind of carrier comprising DNA molecular described in Claims 2 or 3.

5. application of the fusion protein described in claim 1 in antitumor drug is prepared.