CN101265482A

CN101265482A - Recombination tumor chalone-tumor putrescence factor secretion type eukaryon expression vector and its preparation method and use

Info

Publication number: CN101265482A
Application number: CNA2008100250444A
Authority: CN
Inventors: 罗以勤; 赵亮; 姚丽娟
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-04-25
Filing date: 2008-04-25
Publication date: 2008-09-17

Abstract

The invention relates to a secretion type eucaryon expression vector for recombining tumor chalone-tumor necrosis factor and a preparation method and the application thereof, and discloses the secretion type eucaryon expression vector of the tumor chalone (tumstatin) and human tumor necrosis factor (TNF-Alpha) and the method of preparing fused protein. Tumstatin-TNF can respectively interact with an AlphavBeta3 receptor on the surface of a neovascularization endothelial cell and a tumor cell death receptor in a highly specific way. A fractional step cloning method is adopted to construct secretion type tumor chalone and the human tumor necrosis factor to syncretize a gene vector and the expression is performed in a eukaryotic cell. The tumor chalone is combined with the AlphavBeta3 receptor on the surface of tumor tissue neovascularization endothelial cell, so that a tumor tissue vascular endothelial cell and a tumor cell can be specifically killed, and the antitumor effect and the tumor angiopoiesis inhibiting effect can be achieved.

Description

Recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon and its production and use

One, technical field

The present invention relates to gene clone, gene recombination, the secreting, expressing of foreign gene in eukaryotic cell in medical biotechnology field, be particularly related to the method for preparing the fusion rotein tumor chalone-tumor putrescence factor with genetically engineered, secretion type eukaryon expression vector comprising fusion rotein, the host cell that contains this carrier, and some bioactivity research of this fusion rotein.

Two, background technology

Traditional treatments such as chemotherapy, radiotherapy and biotherapy all are at tumour cell itself.Yet solid tumor is different from hematological system tumor, must obtain nutrition from the host by blood vessel.The generation of noumenal tumour depends on the formation of the new vessel of tumour own, and tumor vascular regeneration is subjected to the regulation and control of numerous vascular stimulations and supressor.Along with the rapid progress of tumor vessel regeneration theory and corresponding basic clinical study, antineoplastic vascular generates treats an important content that has become in the combined therapy of tumour.Manually can extract angiostatins such as angiostatin, tumor chalone and be used for clinical treatment research.

The seventies in 20th century, it is to rely on blood vessel that American scholar Folkman has proposed tumor growth.This has established solid basis for the antitumor research of target vascular therapy.The development of solid tumor is divided into no blood vessel phase and blood vessel phase, and most human tumors are positioned at former position several months to several years and are in no blood vessel state, and in the no blood vessel stage, tumor tissues seldom surpasses 2～3mm.In case certain cell subset is transformed into short vasculogenesis phenotype in the tumor tissues, just begins vascularization.The vascularization process is complicated, and it needs the formation of endothelial tissue, mitotic division, pipeline formation and basilar membrane.The vascularization process is subjected to the adjusting of angiogenesis factor and supressor.The vascularization state shows that then the two equilibrium state of angiogenesis factor and supressor is broken.Entity tumor growth must rely on and continue and vasculogenesis (Angiogenesis) widely, and the tumour cell agglomerated masses is set up the blood supply system of self from host matrix, and the knurl body that has grown up to obtains new vascular components and also belongs to same process from the host.

Under the normal physiological state, the generation of blood vessel and supressor are in a kind of dynamic balance to blood vessel endothelium new life's adjusting, and vasculogenesis mechanism is in " closing " state.Under the pathological state, the generation of blood vessel and the acting in conjunction of supressor are in balance-loss state.Tumour develops into the wet face state of following neonate tumour blood vessel from the stationary state in blood vessel early stage, needs to break through the balance of these stimulating factors and supressor effect.Vasculogenesis stimulating factor effect in tumor tissues is in the rise state, and the Angiostatin effect is in the downward modulation state, and angiogenic action mechanism then is in " unlatching " state, neonate tumour blood vessel promptly occurs.Here it is so-called angiopoietic switch balance hypothesis.Neonatal blood vessels is confusing arrangement under the pathological state, and the new vessel wall is imperfect, has a large amount of cracks.This be because unstriated muscle, adventitial cell can not complete formation and arrange due to.Revascularization influences growth of tumor and transfer characteristics.This is because new vessel provides the basis that the pathological tissue nutrient guarantees its growing multiplication, simultaneously, the blood vessel of tumor neogenetic makes tumour cell directly communicate with the blood vessel recycle system of individuality, this also is the prerequisite that malignant tumour generation distant metastasis is sent out, and tumor vascular newborn quantity can be in order to judge the prognosis of tumour patient.

The angiogenesis inhibitor method, since tumor vessel formation and growth of tumor and transfer relationship are so close, the method that antineoplastic vascular generates can play anticancer effect naturally.This method more and more be experimental results show that it is feasible, can the working 5 different levels of anti-angiogenic generation: 1. stop the tumor cell secretion tumor angiogenesis factor (Tumorangiogenesis factors, TAFs); 2. block the effect of TAFs, promptly the antibody by anti-TAFs antibody or anti-TAFs acceptor neutralizes or blocks its biological effect; 3. anti-endothelial cell proliferation and migrating; 4. disturb the interaction of endotheliocyte and extracellular matrix (EMC), stop the formation of vasoganglion; 5. suppress the growth of vascular smooth muscle cell.The research of relevant anti-TAFs antibody neutralizing effect angiogenesis inhibitor is more, Gross etc. utilize the interior transplantation model of human colon carcinoma nude mouse to discover, using bFGF can make tumor growth accelerate, vessel density increases, if use anti-bFGF monoclonal antibody then can stop tumour in the intravital growth of nude mice simultaneously, VEGF antibody also has same effect.The problem of this method is that tumour cell can be secreted a series of TAFs, only suppresses indivedual factors and may be difficult to reach the ideal anticancer effect.Another approach of angiogenesis inhibitor is the propagation of anti-endotheliocyte and migrates, PF4, fumidil and human prolactin 16kD fragment all can anti-factors stimulated growth endothelial cell proliferation, the effect that thrombostondin (Thrombospondin), protamine and sulfuric acid chitin all have anti-endotheliocyte to move.Experiment in vitro has proved this point.

The medicament categories that suppresses angiogenic growth: 1. antibody class: the monoclonal antibody of the monoclonal antibody of neutralize VEGF, anti-new vessel endotheliocyte and genetic engineering antibody thereof, the anti-plain antibody etc. integrated cause apoptosis of vascular endothelial cell, destroy the formation of new vessel, tumor regression and prevention are shifted, to patients with terminal, in mammary cancer, colorectal carcinoma, lung cancer and melanoma, be confirmed especially.2. gene recombination class: angiostatin, endostatin, reorganization IL-12, interferon-' alpha ' 2a etc.3. chemosynthesis or extract class: prevent the Marimastat of basement membrane degradation and the non-protein formulation of Bartimastat synthetic, fumidil and derivative thereof, piperylene (PPS), Suramine,, picolinic acid (picolimic acid) etc.

At present, only the U.S. just has 36 kinds of angiogenesis inhibitors to enter the I～III clinical trial phase of oncotherapy.In February, 2004 world first inhibition tumor vessel medicine Avastin is through drugs approved by FDA listing (Folkman, 2004).In recent years, searching and research work that the researcher of China also begins to carry out angiogenesis inhibitor have obtained some progress.On September 12nd, 2005 recombinant human vascular endothelial inhibin injection liquid (trade(brand)name: the grace degree), be biological products one kind new medicine by State Food and Drug Administration's approval, this is the first in the world vascellum esoderma inhibin PTS, and the research of indication angiogenesis inhibitor has bright prospect.But, say that on the whole China is at the early-stage to the research of angiogenesis inhibitor, compared with developed countries, no matter still big distance is being arranged all aspect the application and development in fundamental research.Therefore, development has the antineoplastic vascular medicine of independent intellectual property right significant.

Tumstatin is a kind of new angiogenesis inhibitor of discovered in recent years, and Tumstatin is people's basilar membrane source collagen iv type angiogenesis inhibitor of latest find, and it suppresses vascular endothelial cell proliferation and causes the endothelial cell specific apoptosis.Tumstatin is incorporated into integrin alpha with non-RGD sequence _vβ ₃Acceptor also influences its activity, in conjunction with integrin alpha _vβ ₃It is most important that acceptor is brought into play anti-new vessel activity to tumstatin.Collagen iv is the main macromole of basement membrane of blood vessel, promotes the adhesion of cell, migration, differentiation and growth.Form 6 different α chains of nucleotide coding of collagen, promptly α 1～α 6, and form tripolymer with similar and different α chain, as (α 1) 2 α 2, and (α 3) 2 α 4, (α 3) 3, (α 4) 4 further form network and provide support for other macromole.Every α chain of collagen iv is made up of 3 parts: the 7S zone of N-end, 3 bursts of spiral zones of intermediary, the non-collegenous dormain territory 1 (NC1) of C-end.Tumstatin by 3 strands of spirals of collagen iv α 3 middle-of-chains zone C-hold 12 amino acid and non-collegenous dormain territory 1 (NC1) totally 244 amino acid form.The about 28kD of the relative molecular weight of Tumstatin, nucleotide sequence (738bp).

The mechanism of action of Tumstatin and action pathway thereof, the effect of Tumstatin are the protein mRNA expression levels that does not change cell, be the protein synthesis that suppresses cell at protein level on the whole, and this effect are that specific effect is in vascular endothelial cell.Tumstatin is incorporated into integrin alpha with non-RGD sequence _vβ ₃And then by suppressing local adhesion kinase (focaladhesion kinase, be abbreviated as FAK) active approach, by suppressing phosphatidyl inose 3 (PI3) kinase activity approach, by suppressing the active and active approach of the plain target body (mTOR) of Mammals thunder handkerchief enzyme of protein serine/threonine (PKB/Akt), final by blocking-up eukaryotic translation initiation factor eukaryotic translation initiation factor (eIF4E) and its protein-bonded dependent protein synthesis of mRNA cap that dissociates and mediate endothelial cell specific, thus the propagation of inhibition endotheliocyte, the apoptosis of inducing endothelial cell, suppress the angiogenesis of tumour and suppress growth of tumor and transfer.Tumstatin effect characteristics and biological effect, Tumstatin is incorporated into integrin alpha in non-dependence RGD mode _vβ ₃, and interact by two different loci and its, a site is the tumstatin54-132 residue, participates in anti-new vessel and forms; And another site is the tumstatin197-215 amino-acid residue, suppresses tumor cell proliferation.Tumstatin performance biological effect district is positioned at 25 amino-acid residues (74-98).

A large amount of animal experiments shows that tumour necrosis factor (TNF) has powerful anti-tumor activity, but clinical effectiveness is undesirable, and reason is that toxic side effect is too big.The TNF anti-tumor activity relies on multiple effect, and it can trigger apoptosis and the necrosis that tumour cell and normal cell in the tumor microenvironment cause the destruction of related artery and damage, activation inflammation and immune response and cause tumour cell.Particularly aspect mediation tumor vessel damage, be subjected in the various kinds of cell that TNF influences endotheliocyte the most important.The tumor vessel damage is that TNF is to direct cytotoxicity of endotheliocyte and the result who causes endotheliocyte blood coagulation activity change (anti-freezing changes short coagulating into).But the normal blood vessels endotheliocyte also is subjected to the influence of TNF, and this is exactly ypotension and TNF toxicity and cause the reason of dose limitation probably.In blood vessel reparation and new vessel forming process, endotheliocyte raises the various kinds of cell surface molecular, and they can promote cell migration and propagation, α _vβ ₃Be exactly one of them, it in vivo in the new vessel forming process existence to endotheliocyte play crucial effects.Therefore, we select to use the blood-vessels target strategy to increase the selectivity of TNF to tumor vascular endothelial cell.

Three, summary of the invention

The present invention aims to provide a kind of by suppressing vasculogenesis with anti-tumor drug, and technical problem to be solved is with total length tumstatin gene and humanTNF-'s gene fusion.Make up secretor type reorganization pIRESneo3/sig-tumstatin-linker-TNF carrier for expression of eukaryon, with the PrimeSTAR of high-fidelity ^TMThe overlapping PCR of HS archaeal dna polymerase (SOEing), and VI type glue source α 3 chain N end signal peptides (sig) are added in each goal gene front, between tumstatin and TNF, increased simultaneously the connecting arm gene fragment, in the hope of can guarantee space conformation and the molecular activity of tumstatin and TNF in the fusion rotein during expressing protein in future.

Specifically:

An object of the present invention is to provide a kind of new have tumstatin and the bioactive medicine of TNF, it is the fusion rotein of tumstatin and TNF.

Another object of the present invention provide encoding said fusion protein DNA, contain the carrier of dna sequence dna and contain the host cell of this carrier.

A first aspect of the present invention, the secretor type fusion gene of a kind of tumstatin and TNF is provided, the technical scheme that adopts is, with human embryo kidney (HEK) 293 cells is material, extract total RNA, with the synthetic people tumstatin cDNA of RT-PCR method, this cDNA is cloned into the pGEM-T carrier obtains recombinant plasmid pGEM-T/tumstatin.Utilize PCR from pGEM-T/tumstatin and PBV220-TNF carrier, to amplify sig-tumstatin-linker and linker-TNF fragment respectively, and its fusion is obtained sig-tumstatin-linker-TNF.

A second aspect of the present invention provides the linker of the connecting arm gene fragment between a kind of tumstatin and the TNF fusion gene.

A third aspect of the present invention provides a kind of isolated DNA molecule, the secretor type fusion rotein that the invention of its code book is above-mentioned.

A fourth aspect of the present invention provides carrier that contains above-mentioned dna molecular and the host cell that contains above-mentioned carrier.The sig-tumstatin-linker-TNF fragment is inserted the plasmid of cutting pIRESneo3 through same enzyme after enzyme is cut, Transformed E .coliDH5 α, extracting plasmid and transfection CHO-K1 cell, secreting, expressing fusion rotein

A fifth aspect of the present invention provides the method for a kind of production fusion rotein of the present invention, and it comprises that the step is poly-: express under the condition of described fusion rotein being fit to, cultivate above-mentioned host cell, thereby give expression to described fusion rotein.

A sixth aspect of the present invention provides the purposes of fusion rotein of the present invention, i.e. application in the medicine of preparation treatment tumour.

The present invention has successfully made up expression moulding fusion rotein tumstatin-TNF and can make the tumstatin-TNF fusion rotein at eukaryotic expression system, just to extract and to separate, overcome the equal inclusion body form of fusion rotein in the prokaryotic expression, the connecting arm fragment among the fusion rotein tumstatin-TNF can guarantee space conformation and the molecular activity of tumstatin and TNF in the fusion rotein.The target ability of this fusion rotein is to utilize part tumstatin and acceptor α _vβ ₃Between the interaction of high special, killing tumor cells tissue blood vessel endotheliocyte and tumour cell have suppressed endothelial cells in tumor neogenetic blood vessels specifically, cause that simultaneously the apoptosis of tumour cell and necrosis have better action.

Four, description of drawings

Fig. 1 is RT-PCR human cloning tumstatin gene fragments (738bp).

Fig. 2 is that pGEM-T/tumstatin recombinant plasmid pcr amplification and endonuclease bamhi identify that wherein 1 swimming lane is the amplified production electrophorogram, and the 2nd swimming lane is that enzyme is cut the product electrophorogram.

Fig. 3 is that PGR is an amplification sig-tumstatin-gene fragment product electrophorogram.

Fig. 4 is that PCR is an amplification sig-tumstatin-linker gene fragment product electrophorogram.

Fig. 5 is overlap amplification splicing method (SOEing) amplification sig-tumstatin-linker-TNF gene fragment product electrophorogram.

Fig. 6 is the pcr amplification and the endonuclease bamhi product electrophorogram of pIRESneo3/sig-tumstatin-linker-TNF plasmid, wherein M is Mark (1Kb), 1 swimming lane is a pIRESneo3/sig-TNF recombinant plasmid Nhe I/BamH I endonuclease bamhi, 2 swimming lanes are pcr amplification sig-TNF gene fragments, 3 swimming lanes are pcr amplification linker-TNF gene fragments, 4 swimming lanes are pcr amplification sig-tumstatin-linker gene fragments, 5 swimming lanes are pcr amplification sig-tumstatin-linker-TNF gene fragments, and 6 swimming lanes are pIRESneo3/sig-tumstatin-linker-TNF recombinant plasmid Nhe I/BamH I endonuclease bamhis.

Fig. 7 is a pcr amplification sig-TNF gene fragment product electrophorogram, and wherein M is Mark, and the 1st, 2,3 swimming lanes are the sig-TNF fragment, and the 4th swimming lane is the TNF+ fragment, and the 5th swimming lane is the sig+ fragment.

Fig. 8 is pIRESneo3/sig-tumstatin and pIRESneo3/sig-TNF recombinant plasmid pcr amplification and endonuclease bamhi product electrophorogram, wherein M is Mark (1Kb), the 2nd swimming lane is a pIRESneo3/sig-tumstatin double digestion fragment, the 3rd swimming lane is the pIRESneo3/sig-tumstatin pcr amplified fragment, the 5th swimming lane is reorganization pIRESneo3/sig-TNF plasmid pcr amplified fragment for reorganization pIRESneo3/sig-TNF plasmid double digestion fragment, the 6th swimming lane.

Fig. 9 is pcr amplification sig, sig-tumstatin gene fragment product electrophorogram, and wherein M is Mark, and the 1st, 3 swimming lanes are about 820bp sig-tumstatin fragment, and the 6th swimming lane is about 100bp sig fragment.

Figure 10 is pcr amplification sig-EGFP, sig-tumstatin-linker-EGFP gene fragment product electrophorogram, and wherein M is Mark, and 1 swimming lane is the sig-tumstatin-linker-EGFP fragment, and 2 swimming lanes are the sig-EGFP fragment,

Figure 11 is pIRESneo3/sig-tumstatin-linker-EGFP recombinant plasmid pcr amplification and endonuclease bamhi product electrophorogram, wherein M is Mark, 2 swimming lanes are recombinant plasmid double digestion (Nhe I+BamH I) fragment, 3 swimming lanes are sig-tumstatin-linker-EGFP pcr amplified fragment (about 1500bp), 4 swimming lanes are the sig-tumstatin-linker pcr amplified fragment, and 5 swimming lanes are the pcr amplified fragment of EGFP.

Figure 12 is pIRESneo3/sig-EGFP recombinant plasmid pcr amplification and endonuclease bamhi product electrophorogram, wherein M is Mark, 2 swimming lanes are recombinant plasmid double digestion (Nhe I+BamH I), 3 swimming lanes are the sig-EGFP pcr amplified fragment, 4 swimming lanes are the sig pcr amplified fragment, 5 swimming lanes are the EGFP pcr amplified fragment

Figure 13 is that the Chinese hamster ovary celI RT-PCR of transfection pIRESneo3/sig-tumstatin-linker-TNF recombinant plasmid identifies the product electrophorogram, and wherein as seen 1 swimming lane expects the dna fragmentation (about 1300bp) of size, and M is Mark,

Figure 14 is that the proteic Western blot of tumstatin-linker-TNF and sig-TNF identifies, wherein 1,3 bands are sig-TNF, and 2,4 bands are tumstatin-linker-TNF.

Figure 15 is tumstatin, tumstatin-TNF, TNF inducing endothelial cell apoptosis, wherein the black line is that PBS is a blank, red line is a tumstatin inducing endothelial cell apoptosis, and green line is a TNF inducing endothelial cell apoptosis, and blue line is a tumstatin-TNF inducing endothelial cell apoptosis.

Figure 16 is that tumstatin-linker-TNF endotheliocyte tubular structure forms test, and A is that 5 μ g/ml BSA endotheliocyte tubular structures form test, and B is that tumstatin-linker-TNF endotheliocyte tubular structure forms test.

Figure 17 is an inhibition test in tumstatin, the tumstatin-TNF body.

Five, embodiment

(1) experiment material

1.1 bacterial strain, cell and plasmid: bacillus coli DH 5 alpha, PBV220-TNF plasmid vector, human embryonic kidney cell line 293 cells are this chamber and preserve, pGEM-T is available from Promega company, plasmid pIRESneo3 is available from Clontech company, human embryonic kidney cell line 293 cells are cultivated to contain 1640 of 10% newborn calf serum, the CHO-K1 cell is cultivated protein electrophoresis instrument (Phamarcia Hoefer Biotech.Ltd.) with the DMEM that contains 10% foetal calf serum.

1.2 main agents in a small amount plasmid extraction test kit, DNA glue reclaims test kit, cell total rna extraction agent box available from magnificent Shun bio-engineering corporation; RT-PCR test kit, PCR test kit, various restriction enzyme, DNA mark and ligase enzyme are available from TaKaRa company.Trizol, Superscript II ThermoScript II, G418 and lipofectamine lipofectamine ^TM2000 available from Invitrogen company.Primer is synthetic by Shanghai Sangon company.Foetal calf serum is available from folium ilicis chinensis company.

(2) method

The preparation of 1 fresh competence bacterium

(1) frozen E.coli DH5 α is planted on the LB solid medium 37 ℃ of overnight incubation;

(2) the single clone of picking is inoculated in the 4mL LB liquid nutrient medium, and 37 ℃ of 180rpm shaking culture are spent the night;

(3) next day bacterium liquid was transferred in fresh LB liquid nutrient medium by 1: 100,37 ℃ of 250rpm shaking culture 2-4h make OD ₆₀₀Value reaches 0.4;

(4) ice bath 10min makes culture be cooled to 0 ℃;

(5) bacterium liquid 1.5mL is moved in the Eppendorf pipe of precooling 4100rpm, 4 ℃ of centrifugal 5min;

(6) abandon supernatant, the Eppendorf pipe is inverted on the filter paper behind the 1min, bacterium is suspended in again the 0.1moL/L CaCl of 1mL ice precooling ₂In, gently behind the mixing, ice bath 30min;

(7) 4100rpm, 4 ℃ of centrifugal 5min

(8) abandon supernatant, the Eppendorf pipe is inverted on the filter paper behind the 1min, add the 0.1moL/LCaCl of 100 μ l ice precooling in every pipe ₂, resuspended bacterium preserves standby in the ice bath.

2 design of primers design two primers according to the people tumstatin encoding sequence among the GenBank.

3.1 RT-PCR human cloning tumstatin total length encoding gene: to logarithmic phase, counting, centrifugal collecting cell extract total RNA (pressing the operation of RNA extraction agent box specification sheets) with human embryonic kidney cell line 293 cell cultures.With Oligo (dT) is synthetic cDNA first chain of primer, and getting 2 μ l reverse transcription products is template, carries out conventional pcr amplification tumstatin gene with primer p1 and primer p2.

10×Taq?buffer+KCl 5μl

2mM?dNTPs 5μl

p1(20mM) 1.5μl

p2(20mM) 1.5μl

Taq enzyme (5.0U/ μ l) 1.25 μ l

25mM?MgCl ₂ 24μl

tumstatin?cDNA 2μl

ddH ₂O 29.75μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 94 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 50s (sex change); 55 ℃, 50s (annealing); 72 ℃, 60s (amplification) carries out 30 circulations, extends 10min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, dna fragmentation (about 738bp) Fig. 1 of visible expection size.

3.2pGEM-T/tumstatin structure: the Tumstatin gene clone is to the pGEM-T carrier.

Ligase enzyme Buffer 5 μ l

PGEM-T carrier 1 μ l

Pcr amplification tumstatin cDNA product 3 μ l

T4 dna ligase 1 μ l

Amount to 10 μ l

Abundant mixing, 16 ℃ of ligations are spent the night

3.2.1 pGEM-T/tumstatin transformed competence colibacillus bacterium E.coli DH5 α:

(1) in every pipe 100 μ l competence bacteriums, adds the above-mentioned connection product of 10 μ l, mixing gently, ice bath 30min;

(2) pipe is put into 42 ℃ circulator bath, heat-shocked 90s;

(3) fast pipe is transferred to 1-2min in the ice bath;

(4) every pipe adds 37 ℃ of LB liquid nutrient medium 500 μ l, 37 ℃ of 150rpm shaking culture 30min;

(5) the centrifugal 5min of 4100rpm;

(6) abandon supernatant, add the fresh LB liquid nutrient medium of 200 μ l, and coat LB penbritin selectivity solid medium, leave standstill 10min;

(7) be inverted plate and in 37 ℃ of constant incubators, cultivate 12-16h;

3.2.2 pGEM-T/tumstatin recombinant plasmid pcr amplification is identified:

Bacterium liquid fragmentation pattern: single clone is inoculated in the 4mL LB amicillin resistance selected liq substratum on the picking LB penbritin solid medium, and 37 ℃ of 180rpm shaking culture are spent the night; Get bacterium liquid 30 μ l, the centrifugal 1min of 12000rpm abandons supernatant, adds 10 μ l ddH ₂O, 99 ℃ of 10min.Pcr amplification identifies that method is the same.1 swimming lane among Fig. 2.

3.2.3 pGEM-T/tumstatin recombinant plasmid Bam H I+HindIII double digestion is identified: picking pcr amplification positive bacteria is inoculated in the 4mL LB amicillin resistance selected liq substratum, and 37 ℃ of 180rpm shaking culture are spent the night.Extracting plasmid (pressing the operation of test kit process specifications).The positive bacteria plasmid advances a double digestion to be identified

BamH?I Buffer 5μl

100×BSA 0.5μl

BamH?I 1μl

pGEM-T/tumstatin 10μl

ddH ₂O 33.5μl

Amount to 50 μ l

37 ℃ of water-bath 40min add again

HindIII?Buffer 5μl

HindIII 1μl

37 ℃ of water-bath 40min, enzyme cut capable 0.8% agarose gel electrophoresis of product, 2 swimming lanes among Fig. 2.。

The structure of 4 reorganization pIRESneo3/sig-tumstatin-linker-TNF plasmids

4.1PCR amplification sig-tumstatin-linker gene fragment: with p3 and p5 primer is that template is carried out pcr amplification with pGEM-T/tumstatin.

10×Taq?buffer+KCl 5μl

2mM?dNTPs 5μl

p3(20mM) 1.5μl

p5(20mM) 1.5μl

Taq enzyme (5.0U/ μ l) 1.25 μ l

25mM?MgCl ₂ 4μl

pGEM-T/tumstatin 1.25μl

ddH ₂O 31.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 95 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (sex change); 55 ℃, 30s (annealing); 72 ℃, 60s (amplification) carries out 30 circulations, extends 5min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, dna fragmentation (about 740bp) Fig. 3 of visible expection size.

It as template, is primer with p4 and p6 again that the purified recovery test kit of pcr amplification product reclaims (pressing the process specifications operation), and row second is taken turns pcr amplification, and amplified production is the sig-tumstatin-linker that carries Nhe I+BamH I restriction enzyme site.

10×Taq?buffer+KCl 5μl

2mM?dNTPs 5μl

p4(20mM) 1.5μl

p6(20mM) 1.5μl

Taq enzyme (5.0U/ μ l) 1.25 μ l

25mM?MgCl ₂ 4μl

First round pcr amplification product 1.25 μ l

ddH ₂O 31.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 95 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (sex change); 55 ℃, 30s (annealing); 72 ℃, 60s (amplification) carries out 30 circulations, extends 5min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, dna fragmentation (about 840bp) Fig. 4 of visible expection size.The purified recovery test kit of pcr amplification product reclaims (pressing the process specifications operation).

4.2 pcr amplification linker-TNF fragment: with p7 and p8 primer is the linker-TNF fragment of template amplification belt lacing with PBV220-TNF

10×Taq?buffer+KCl 5μl

2mM?dNTPs 5μl

p7(20mM) 1.5μl

p8(20mM) 1.5μl

Taq enzyme (5.0U/ μ l) 1.25 μ l

25mM?MgCl ₂ 4μl

PBV220-TNF 1.25μl

ddH ₂O 31.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 95 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (sex change); 55 ℃, 30s (annealing); 72 ℃, 60s (amplification) carries out 30 circulations, extends 5min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, 3 swimming lanes among Fig. 6.。The purified recovery test kit of pcr amplification product reclaims (pressing the process specifications operation).

4.3 overlap amplification splicing method (SOEing) amplification sig-tumstatin-linker-TNF: the PrimeSTAR that uses high-fidelity again ^TMHS archaeal dna polymerase p4 and p8 primer are template overlap amplification splicing method (SOEing) amplification with sig-tumstatin-linker fragment and linker-TNF fragment.

5×PrimeSTAR?buffer+MgCl ₂ 10μl

2.5mM?dNTPs 4μl

p4(20mM) 0.5μl

p8(20mM) 0.5μl

Sig-tumstatin-linker fragment 1 μ l

Linker-TNF fragment 1 μ l

The PrimeSTAR of high-fidelity ^TMHS archaeal dna polymerase (2.5U/ μ l) 0.5 μ l

ddH ₂O 32.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 98 ℃ of reaction 2min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 98 ℃, 10s (sex change); 68 ℃, 4min (annealing, amplification) carries out 30 circulations, extends 5min in 64 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, dna fragmentation (about 1300bp) Fig. 5 of visible expection size, the purified recovery test kit of pcr amplification product reclaim (pressing the process specifications operation).

4.4 sig-tumstatin-linker-TNF and pIRESneo3 double digestion:

Buffer2 5μl

100×BSA 0.5μl

Nhe?I 1μl

Sig-tumstatin-linker-TNF fragment (or pIRESneo3) 15 μ l

ddH ₂O 28.5μl

Amount to 50 μ l

37 ℃ of water-bath 40min add again

BamH?I Buffer 5μl

BamH?I 1μl

37 ℃ of water-bath 40min, enzyme cut capable 0.8% agarose gel electrophoresis of product, and corresponding product sig-tumstatin-linker-TNF fragment and pIRESneo3 are reclaimed in rubber tapping.

4.5 the structure of reorganization pIRESneo3/sig-tumstatin-linker-TNF plasmid: endonuclease bamhi sig-tumstatin-linker-TNF and pIRESneo3 ligation.

Ligase enzyme Buffer 1 μ l

Enzyme is cut sig-tumstatin-linker-TNF fragment 3 μ l

Enzyme is cut pIRESneo3 1 μ l

T ₄Dna ligase 1 μ l

ddH ₂O 4μl

Amount to 10 μ l

Abundant mixing, 16 ℃ of ligations are spent the night.

4.6 endonuclease bamhi sig-tumstatin-linker-TNF is connected the transformed competence colibacillus bacterium E.coli DH5 α of product with pIRESneo3:

(2) pipe is put into 42 ℃ circulator bath, heat-shocked 90s;

(3) fast pipe is transferred to 1-2min in the ice bath;

(4) every pipe adds 37 ℃ of LB liquid nutrient medium 500l, 37 ℃ of 150rpm shaking culture 30min;

(5) the centrifugal 5min of 4100rpm;

(6) abandon supernatant, add the fresh LB liquid nutrient medium of 200 μ l, and coat LB kalamycin resistance selectivity solid medium, leave standstill 10min;

(7) be inverted plate and in 37 ℃ of constant incubators, cultivate 12-16h;

4.7 E.coli DH5 α recombinant plasmid pIRESneo3/sig-tumstatin-linker-TNF pcr amplification is identified:

Bacterium liquid fragmentation pattern: single clone is inoculated in the 4mL LB amicillin resistance selected liq substratum on the picking LB kalamycin resistance selectivity solid medium, and 37 ℃ of 180rpm shaking culture are spent the night; Get bacterium liquid 30 μ l, the centrifugal 1min of 12000rpm abandons supernatant, adds 14.7 μ l ddH ₂O, 99 ℃ of 10min.

5×PrimeSTAR?buffer+MgCl ₂ 25μl

2mM?dNTPs 2μl

p4(20mM) 0.5μl

p8(20mM) 0.5μl

The PrimeSTAR of high-fidelity ^TMHS archaeal dna polymerase (2.5U/ μ l) 0.3 μ l

25mM?MgCl ₂ 22μl

Above-mentioned bacterial lysate 14.7 μ l

Amount to 25 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 98 ℃ of reaction 2min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 98 ℃, 10s (sex change); 68 ℃, 4min (annealing, amplification) carries out 30 circulations, extends 5min in 64 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, positive bacteria as seen expect the size dna fragmentation (about 1300bp) Fig. 6.

The picking positive bacteria is inoculated in the 4mL LB amicillin resistance selected liq substratum, and 37 ℃ of 180rpm shaking culture are spent the night.Extracting plasmid (pressing the operation of test kit process specifications).The positive bacteria plasmid advances a double digestion and identifies that method is the same, Fig. 6.M is Mark (1Kb), 1 swimming lane is recombinant plasmid pIRESneo3/sig-TNF double digestion (Nhe I+BamH I) product, 2 swimming lanes are the sig-TNF pcr amplified fragment, 3 swimming lanes are the linker-TNF pcr amplified fragment, 4 swimming lanes are sig-tumstatin-linker PCR, and 5 swimming lanes are the sig-tumstatin-linker-TNF pcr amplified fragment.6 swimming lanes are recombinant plasmid pIRESneo3/sig-tumstatin-linker-TNF double digestion (Nhe I+BamH I) product.

The structure of 5 pIRESneo3/sig-TNF plasmids

5.1 with p9 and p10 primer is the sig+ fragment of template amplification and TNF band overlap with pGEM-T/sig-tumstatin-linker,

10×Taq?buffer+KCl 5μl

2mM?dNTPs 5μl

p9(20mM) 1.5μl

p10(20mM) 1.5μl

Taq enzyme (5.0U/ μ l) 1.25 μ l

25mM?MgCl ₂ 4μl

pGEM-T/sig-tumstatin-linker 1.25μl

ddH ₂O 30.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 95 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (sex change); 55 ℃, 30s (annealing); 72 ℃, 60s (amplification) carries out 30 circulations, extends 5min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, the purified recovery test kit of pcr amplification product reclaim (pressing the process specifications operation).

5.2 with p11 and p8 primer is the TNF+ fragment of template amplification and sig band overlap with PBV220-TNF: method is the same.

5.3 with p9 and p8 is primer, is that template overlap amplification splicing method (SOEing) amplification obtains sig-TNF with sig+ fragment and TNF+ fragment:

10×Taq?buffer+KCl 5μl

2mM?dNTPs 5μl

p9(20mM) 1.5μl

p8(20mM) 1.5μl

Taq enzyme (5.0U/ μ l) 1.25 μ l

25mM?MgCl ₂ 3.75μl

Sig+ fragment 2 μ l

TNF+ fragment 1.25 μ l

ddH ₂O 28.75μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 95 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (sex change); 55 ℃, 30s (annealing); 72 ℃, 60s (amplification) carries out 30 circulations, extends 5min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, the purified recovery test kit of pcr amplification product reclaim (pressing the process specifications operation).The results are shown in Figure 7, the 1,2,3 swimming lanes are the sig-TNF fragment; The 4th swimming lane is the TNF+ fragment; The 5th swimming lane is the sig+ fragment; The 6th swimming lane is Mark.

5.4 the structure of reorganization pIRESneo3/sig-TNF plasmid: sig-TNF is through Nhe I+BamH I double digestion, same enzyme is cut the pIRESneo3 plasmid, and method is the same.

Utilize the T4DNA ligase enzyme that enzyme is cut the sig-TNF fragment and cut the pIRESneo3 plasmid vector with enzyme and be connected, method of attachment is the same.

5.5 E.coli DH5 α recombinant plasmid pcr amplification is identified: connect product Transformed E .coli DH5 α, carry out pcr amplification with Taq archaeal dna polymerase and sig-TNF primer.Choose and severally detect the male bacterium colony, prepare plasmid in a small amount through clone PCR and Nhe I+BamH I double digestion.Method is the same.The results are shown in Figure 8.The 4th is Mark; The 5th swimming lane is reorganization pIRESneo3/sig-TNF plasmid double digestion fragment; The 6th swimming lane is reorganization pIRESneo3/sig-TNF plasmid pcr amplified fragment.

6.1 the amplification of sig-tumstatin fragment PCR: with p9 and p12 is primer, is template amplification with pGEM-T/sig-tumstatin-linker,

10×Taq?buffer+KCl 5μl

2mM?dNTPs 5μl

p9(20mM) 1.5μl

p12(20mM) 1.5μl

Taq enzyme (5.0U/ μ l) 1.25 μ l

25mM?MgCl ₂ 4μl

pGEM-T/sig-tumstatin-linker 1.25μl

ddH ₂O 31.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 95 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (sex change); 55 ℃, 30s (annealing); 72 ℃, 60s (amplification) carries out 30 circulations, extends 5min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, the purified recovery test kit of pcr amplification product reclaim (pressing the process specifications operation), and the dna fragmentation of visible expection size the results are shown in Figure 9.1st, 3 swimming lanes are about 820bp sig-tumstatin fragment; The 5th swimming lane is Mark; The 6th swimming lane is about 100bp sig fragment.

6.2 the structure of recombinant plasmid pIRESneo3/sig-tumstatin: sig-tumstatin is through Nhe I+BamH I double digestion, same enzyme is cut the pIRESneo3 plasmid, and method is the same.

Utilize the T4DNA ligase enzyme that enzyme is cut the sig-tumstatin fragment and cut the pIRESneo3 plasmid vector with enzyme and be connected, method of attachment is the same.

6.3 the E.coli DH5 α of recombinant plasmid pIRESneo3/sig-tumstatin transforms and pcr amplification is identified: connect product Transformed E .coli DH5 α, carry out pcr amplification with Taq archaeal dna polymerase and sig-tumstatin primer.Choose and severally detect the male bacterium colony, prepare plasmid in a small amount through clone PCR and Nhe I+BamH I double digestion.Method is the same.The results are shown in Figure 8.The 1st is Mark, and the 2nd swimming lane is a pIRESneo3/sig-tumstatin double digestion fragment; The 3rd swimming lane is the pIRESneo3/sig-tumstatin pcr amplified fragment.

7 sig-tumstatin-linker-EGFP and sig-EGFP fragment.

7.1 the sig-tumstatin-linker fragment amplification of overlap is arranged with EGFP.With p9 and p13 is primer, and pGEM-T/sig-tumstatin-linker is a template, product called after a.

7.2 the EGFP fragment amplification of overlap is arranged with sig-tumstatin-linker.With p14 and p15 is primer, pEGFP-C ₂Be template, product called after b.

7.3 the sig fragment amplification of overlap is arranged with EGFP.With p9 and p16 is primer, and pGEM-T/sig-tumstatin-linker is a template, product called after c.

7.4 the EGFP fragment amplification of overlap is arranged with sig.With p17 and p15 is primer, pEGFP-C ₂Be template, product called after d.

5×PrimeSTAR?buffer+MgCl ₂ 10μl

2.5mM?dNTPs 4μl

Upstream primer (20mM) 0.5 μ l

Downstream primer (20mM) 0.5 μ l

PrimeSTAR ^TMHS archaeal dna polymerase (2.5U/ μ l) 0.5 μ l

Template 1 μ l

ddH ₂O 33.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 98 ℃ of reaction 2min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 98 ℃, 10s (sex change); 68 ℃, 2min (annealing, amplification) carries out 30 circulations, extends 5min in 68 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, corresponding product a, b, c, d are reclaimed in rubber tapping.

7.5 sig-tumstatin-linker-EGFP fragment amplification (being the ab fragment) is a primer with p9 and p15, a and b fragment are template.

Sig-EGFP fragment amplification (being the cd fragment) is a primer with p9 and p15, and c and d fragment are template.

5×primer?buffer 10μl

2.5mM?dNTPs 4μl

p9(20mM) 0.5μl

p15(20mM) 0.5μl

PrimeSTAR ^TMHS archaeal dna polymerase (2.5U/ μ l) 0.5 μ l

Template x 1 μ l

Template y 1 μ l

ddH ₂O 32.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 98 ℃ of reaction 2min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 98 ℃, 10s (sex change); 68 ℃, 5min (annealing, amplification) carries out 30 circulations, extends 5min in 68 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, the dna fragmentation of visible expection size the results are shown in Figure 10.First swimming lane is sig-tumstatin-linker-EGFP, and second swimming lane is sig-EGFP, and the 3rd swimming lane is Mark.Corresponding product ab, cd fragment are reclaimed in rubber tapping.

7.6 sig-tumstatin-linker-EGFP (being the ab fragment) and sig-EGFP fragment (being the cd fragment) double digestion.

Buffer2 5μl

100×BSA 0.5μl

Nhe?I 1μl

Ab (or cd, pIRESneo3) fragment 10 μ l

ddH ₂O 33.5μl

Amount to 50 μ l

37 ℃ of water-bath 40min add again

BamH?I?Buffer 5μl

BamH?I 1μl

37 ℃ of water-bath 40min, enzyme cut capable 0.8% agarose gel electrophoresis of product, and corresponding product ab, cd fragment and pIRESneo3 are reclaimed in rubber tapping.

7.7 endonuclease bamhi ab, cd and pIRESneo3 ligation.

Ligase enzyme Buffer 1 μ l

Enzyme is cut ab (or cd) fragment 3 μ l

Enzyme is cut pIRESneo3 1 μ l

T ₄Dna ligase 0.5 μ l

ddH ₂O 4.5μl

Amount to 10 μ l

Abundant mixing, 16 ℃ of ligations are spent the night.

7.8 endonuclease bamhi ab, cd are connected the transformed competence colibacillus bacterium E.coli DH5 α of product with pIRESneo3

(2) pipe is put into 42 ℃ circulator bath, heat-shocked 90s;

(3) fast pipe is transferred to 1-2min in the ice bath;

(5) the centrifugal 5min of 4100rpm;

(7) be inverted plate and in 37 ℃ of constant incubators, cultivate 12-16h;

7.9 E.coli DH5 α recombinant plasmid pcr amplification is identified

Bacterium liquid fragmentation pattern: single clone is inoculated in the 4mL LB amicillin resistance selected liq substratum on the picking LB kalamycin resistance selectivity solid medium, and 37 ℃ of 180rpm shaking culture are spent the night; Get bacterium liquid 30 μ l, the centrifugal 1min of 12000rpm abandons supernatant, adds 16.4 μ l ddH ₂O, 99 ℃ of 10min.

10×Taq?buffer+KCl 2.5μl

2mM?dNTPs 2.5μl

p9(20mM) 0.5μl

p15(20mM) 0.5μl

Taq enzyme (5.0U/ μ l) 0.6 μ l

25mM?MgCl ₂ 2μl

Above-mentioned bacterial lysate 16.4 μ l

Amount to 25 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 94 ℃ of reaction 5min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 94 ℃, 30s (sex change); 55 ℃, 30s (annealing); 72 ℃, 1min (amplification); 30 circulations of row are extended 5min in 72 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product.

7.10 E.coli DH5 α recombinant plasmid double digestion is identified (Nhe I+BamH I)

Choose pcr amplification positive bacteria extracting plasmid (method is the same), double digestion (Nhe I+BamH I) (method is the same).Capable 0.8% agarose gel electrophoresis of double digestion product.

The pIRESneo3/sig-tumstatin-linker-EGFP recombinant plasmid is identified figure (Figure 11).First swimming lane is Mark, second swimming lane is recombinant plasmid double digestion (Nhe I+BamH I), the 3rd swimming lane is sig-tumstatin-linker-EGFP pcr amplified fragment (about 1500bp), the 4th swimming lane is the sig-tumstatin-linker pcr amplified fragment, the 5th swimming lane is the EGFPPCR amplified fragments, and the 6th swimming lane is Mark.

The pIRESneo3/sig-EGFP recombinant plasmid is identified figure (Figure 12).First swimming lane is Mark, and second swimming lane is recombinant plasmid double digestion (Nhe I+BamH I), and the 3rd swimming lane is the sig-EGFP pcr amplified fragment, and the 4th swimming lane is the sig pcr amplified fragment, and the 5th swimming lane is the EGFP pcr amplified fragment, and the 6th swimming lane is Mark.And a small amount of extracting plasmid, method is the same.

8.1 G418 preparation: among the 300mg G418, add 3ml deionization H ₂O solution, concentration are after 100mg/ml dissolves fully, and 0.22um filters, and-20 degree are preserved.

8.2 the best susceptibility concentration of the G418 of CHO

To 1000cell/mL, every hole 500uL adds 24 orifice plates that substratum is arranged with cell dilution, with the G418 concentration dilution to 0 in every hole, and 300,400,500,600,700,800,8 ranks such as 900ug/mL.Liquid is changed every 2d in each concentration 3 multiple hole, cultivated 10-14 days, and be benchmark with the whole dead concentration of minimum cell, than the high again rank of this concentration, keep half that use screening concentration during screening.The best susceptibility concentration of the G418 of CHO concentration is 600mg/L.

8.3CHO the transfection of cell:

8.3.1 transfection the day before yesterday, 24 hole culture dish, every hole 500ul substratum 0.5～2 * 10 ⁵Cell (counting 1～4 * 10 ⁵/ mL), add in the short period of time before liposome/DNA mixture on transfection same day, DMEM washes once, change 500ul fresh serum or serum free medium arranged.

8.3.2 transfection composite

1. .0.8 μ g DNA+50ul serum-free DMEM dilution, mixing gently.

2. lipofectamine ^TM2000 (with preceding mixing gently), 2ul+50ul serum-free DMEM dilution, mixing gently, room temperature 5min.

The lipofectamine of the DNA 50ul+ dilution of 3. diluting ^TM2000 (50ul), mixing gently, room temperature 20min.

8.3.3 do not remove substratum, dropwise add 100 μ l liposome/DNA mixtures (from culture hole on one side to the other side), limit edged jog culture plate.

8.3.4 37 ℃ of CO ₂Hatch 6hr.Change substratum behind the 6hr, add the 500ul fresh growth medium.

8.3.5 the after-applied screening pressure of transfection 24hr diluting cells (1: 10) is used the culture medium culturing that contains G418 instead.

8.3.6 G418 screening: after continuing to cultivate 14 days under the G418 screening concentration, choose mono-clonal, enlarged culturing.

Recombinant plasmid pIRESneo3/sig-tumstatin-linker-EGFP, pIRESneo3/sig-tumstatin, pIRESneo3/sig-TNF, pIRESneo3/sig-tumstatin-linker-TNF carry out the transfection of Chinese hamster ovary celI by above method.The Chinese hamster ovary celI of recombinant plasmid pIRESneo3/sig-tumstatin-linker-EGFP transfection shows green fluorescence, and the secreting, expressing success is described.All recombinant plasmids are chosen mono-clonal, enlarged culturing.

9.1 the Chinese hamster ovary celI RT-PCR of transfection sig-tumstatin-linker-TNF gene identifies

After the Chinese hamster ovary celI of transfection sig-tumstatin-linker-TNF gene was cultured to logarithmic phase, counting, centrifugal collecting cell extracted total RNA (press RNA extraction agent box specification sheets operates).With Oligo (dT) is synthetic cDNA first chain of primer, and getting 2 μ l reverse transcription products is template,, carry out conventional pcr amplification sig-tumstatin-linker-TNF gene with primer p4 and primer p8.

5×PrimeSTAR?buffer+MgCl ₂ 10μl

2.5mM?dNTPs 4μl

p4(20mM) 0.5μl

p8(20mM) 0.5μl

cDNA 2μl

ddH ₂O 32.5μl

Amount to 50 μ l

The preparation of reaction tubes is all carried out in ice bath, in the process for preparation, and Xian Jiashui, template DNA and other reactive component, 98 ℃ of reaction 2min carry out the PCR reaction then, and the loop parameter of pcr amplification is: 98 ℃, 10s (sex change); 68 ℃, 4min (annealing, amplification) carries out 30 circulations, extends 5min in 64 ℃ again.Capable 0.8% agarose gel electrophoresis of pcr amplification product, first swimming lane as seen expect the size dna fragmentation (about 1300bp) Figure 13.

9.2 the Western blot of tumstatin-linker-TNF recombinant protein detects

Cultivate through pcr amplification male Chinese hamster ovary celI mono-clonal, 4 ℃ centrifugal, and 1000rpm gets supernatant, adopts Laemmli method, resolving gel concentration 12%, gel-colored employing Coomassie brilliant blue R-250 method behind the electrophoresis through SDS-PAGE.After immunoblotting reaction: SDS-PAGE finishes, press the Bio-Rad description of product, gel is near negative electrode one side, nitrocellulose membrane (NC film) is near anode one side, transfering buffering liquid (25mmol/L Tris, 192mmol/L Glycine, 20% methyl alcohol), 100V constant voltage 1h, with albumen from the gel electrotransfer to the NC film, after electrotransfer finishes, take out the NC film, (contain 0.02mol/L with washings, pH 7.4TBS, 0.4%Tween 20) room temperature washes 3 times, immerses 37 ℃ of 1h in the confining liquid (TBST that contains 2%BSA), and washings (TBST) room temperature is washed 3 times, add the anti-TNF alpha monoclonal antibody and be one anti-, hatch 1h for 37 ℃, the TBST room temperature is washed 3 times, and the anti-IgG of horseradish peroxidase mark is two anti-, hatch 1h for 37 ℃, the TBST room temperature is washed 3 times, washes 3 times with TBS again, and the NC film immerses in the colour developing liquid, room temperature lucifuge colour developing 5min, the distilled water flushing termination reaction.

The result shows that the expressing cho cell albumen of transfection sig-tumstatin-linker-TNF gene locates to present strong specific reaction at about 45ku (tumstatin-linker-TNF).Be about 17ku according to the sophisticated people TNF of our design molecular weight, people tumstatin molecular weight is about 28ku, the molecular weight of fusion rotein then should be about 45ku, the result (45ku) that Western detects is consistent with expection, show that we have obtained people tumstatin-linker-TNF fusion rotein, 2 among Figure 14,4 bands.

The Western blot of 10 sig-TNF recombinant proteins detects, and method is the same.The result that Western detects presents strong specific reaction at about 17ku place, 1 among Figure 14,3 bands.

11 recombinant protein assays: adopt the Lowry method.

12.1 cell inhibitory effect test

The every hole of 96 well culture plates adds 100 μ l and contains 5 * 10 ³The nutrient solution of ECV304 cell (DMEM+0.5%FCS) is put 37 ℃, 5%CO ₂Behind the 24h, spend the night at incubator, discard nutrient solution, every hole adds 100 μ l and contains different concns tumstatin-linker-TNF or tumstatin or TNF α+DMEM+20% FCS sample liquid, simultaneously, as positive control, PBS is a blank with the endostatin of 50 μ g/ml, and three parallel repeating holes are established in each processing, after cultivating 48h, in culture plate, add 20 μ lMTT solution, continue to cultivate 4-6h, abandon supernatant, every hole adds 100 μ l DMSO to detecting with microplate reader after the dissolving fully, and wavelength is 570nm.Calculate inhibiting rate (%)=(A _Blank-A _{Experimental group})/A _Blank* 100% or cells survival rate (%)=A _{Experimental group}/ A _Blank* 100%.。The result shows the remarkable inhibition of endothelial cell proliferation of tumstatin-linker-TNF fusion rotein and is dose-dependently, ED ₅₀Be about 5 μ g/ml.Tumstatin also suppresses ECV304 cell proliferation, its ED ₅₀Be about 12 μ g/ml, higher 2.4 times than tumstatin-linker-TNF fusion rotein, TNF does not have the obvious suppression effect to ECV304 propagation.

12.2 cell in vitro fragmentation test

With l cell L929 or 0.25% trysinization of ECV304 cell, adjusting cell concn with the DMEM that contains 10% foetal calf serum (FBS) is 5 * 10 ⁵/ ml spreads in the flat plastics microtest plate in 96 holes, every hole 100 μ l, 37 ℃, 5% CO ₂Overnight incubation adds dactinomycin (50 μ l/ holes, final concentration 2 μ g/ml) and TNF α or tumstatin-linker-TNF fusion rotein solution (50 μ l/ holes, final concentration 0.003-3ng/ml), standard substance TNF α (5 * 10 then ⁷μ g/mg) presses with 10 ⁵IU plays do dilution in 1: 10 and makes parallel control, and 37 ℃, 5%CO ₂Cultivate 20h, adding 20 μ l MTT solution are quantitative in culture plate.Inverse with the maximum dilution multiple that can make 50% necrocytosis is its activity unit.Tumstatin-linker-TNF fusion rotein and contrast TNF α are hatched 20h with target cell in the presence of dactinomycin, both are similar to the killing activity of ECV304 cell, are respectively 3.4 * 10 ⁶IU/mg and 4.4 * 10 ⁶IU/mg, lower 10 times to the killing activity tumstatin-linker-TNF fusion rotein of L929 cell than TNF α, be respectively 4.6 * 10 ⁶IU/mg and 4.4 * 10 ⁷IU/mg.

12.3 ECV304 cell adhesion test

With the tumstatin-linker-TNF of different concns and TNF α bag by 96 orifice plates, every hole 50 μ l (150mmol/L NaCl, 50mmol/L Na ₃PO ₄, pH7.3), 4 ℃ are spent the night, and with after the 0.9%NaCl washing, add the 2%BSA+DEME fluid-tight and close, and place 45min for 37 ℃, again with 0.9%NaCl wash 3 times standby.Collect logarithmic phase ECV304 cell, physiological saline washing three times, DEME liquid suspension ECV304 cell, every hole 100 μ l ECV304 cells (3 * 10 ⁴Individual cell) be added on 96 orifice plates of above-mentioned tumstatin-linker-TNF and TNF bag quilt, 37 ℃, 5%CO ₂Hatch 1 ~ 1.5h, with DEME flush away free cell, use then 3% formaldehyde (pH7.3,2% sucrose, PBS) fixing, 0.5% violet staining, microplate reader 540nm wavelength reads the absorbance A value.The result shows, in the plate hole of 15 μ g/ml tumstatin-linker-TNF bag quilt endotheliocyte arranged, in the plate hole of TNF α bag quilt then seldom or do not have an endotheliocyte.These results point out the tumstatin part of fusion rotein tumstatin-linker-TNF correct folding and in conjunction with the adhesion receptor of endothelial cell surface strongly.

12.4 Annexin V-FITC is in conjunction with test

5 * 10 ⁶The nutrient solution of ECV304 cell (DMEM+10%FBS) adds 6 well culture plates, puts 37 ℃, 5%CO ₂Overnight incubation, change fresh DMEM+10%FBS and add 5 μ g/ml tumstatin or 100ng/mltumstatin-linker-TNF or 80ng/ml TNF α next day, and contrast adds the PBS of same amount, behind the 18h, collecting cell, 3,000 * g is centrifugal, after cell washs with PBS, press test kit specification sheets mark Annexin V-FITC and PI, the cell of mark is counted with flow cytometer, 15000 cells of each sample counting, the data Cell Quest software analysis of standard.Simple Annexin V-FITC positive person be early, mid-term apoptotic cell, the equal positive person of Annexin V-FITC and PI is an apoptosis or dead cell in late period.The result shows the energy force rate tumstatin (5 μ g/ml) strong 50 times (Figure 15) of tumstatin-linker-TNF (100ng/ml) inducing endothelial cell early apoptosis.

12.5 tubular structure forms test

The ECMatrix of 50 μ l dilution ^TM(CHEMICON) add 96 well culture plates, hatch 1h for 37 ℃ and treat ECMatrix ^TMSolidify, 150 μ l are not contained antibiotic ECV304 cell inoculation arrive above-mentioned bag by ECMatrix ^TMCulture plate in, it is 5 * 10 that every hole contains cell quantity ⁵～1 * 10 ⁴Individual, add tumstatin-linker-TNF or the tumstatin or the TNF of different concns then.Contrast adds the BSA of same amount.The test triplicate.Cell is at 37 ℃, 5% CO ₂Continue to cultivate under the condition, 1,2,4,8, the 12h time point difference of observing endotheliocyte tubular arrangement situation, unit surface inner tubular structure quantity and integrated degree between the different treatment group.Detailed process and judging criterion are according to In Vitro Angiogenesis Assay Kit specification sheets.Compare with control group, the tumstatin-linker-TNF fusion rotein significantly suppresses the formation of endotheliocyte tubular structure and is the dose-dependently effect.It is 23.0 ± 3.1% that 5 μ g/ml BSA group tubular structure forms percentage, tumstatin-linker-TNF fusion rotein group be 3.2 ± 1.6% (P＜0.01) (Figure 16).Tumstatin is not so good as tumstatin-linker-TNF to the inhibition ability that the endotheliocyte tubular structure forms, and TNF α treatment group forms not obviously influence to the endotheliocyte tubular structure

12.6 inhibition test in the body

Aseptic technique is subcutaneous with 2 * 10 at the BALB/c mouse back ⁶Inoculation HepG2 cell grows to 60mm etc. tumour ³Be divided into 5 groups (5 every group) at random.Abdominal injection tumstatin (5mg/kg/day), tumstatin-linker-TNF (1mg/kg/day or 0.5mg/kg/day) or TNF α (0.3mg/kg/day), PBS compares, and injects 12d continuously, puts to death mouse after the off-test, peel off the knurl body, tumour is weighed.The mouse volume.Gross tumor volume calculates according to following formula: gross tumor volume (mm ³)=(is long * and wide ²)/2.Body weight change rate=(treatment back body weight-treatment precursor weight-average value)/contrast precursor weight-average value * %.From Figure 17 as seen, tumstatin treatment group (5mg/kg/day) is similar with the gross tumor volume of high dosage tumstatin-linker-TNF fusion rotein treatment group (1mg/kg/day), is respectively 230 ± 11.6mm ³With 208 ± 15.1mm ³Yet low dosage tumstatin-linker-TNF fusion rotein treatment group (0.5mg/kg/day) shows best tumor killing effect, and its mean tumour volume is 1/3 (118 ± 16.6mm of control group ³Vs 375 ± 24.5mm ³).

Nucleotide and aminoacid sequence table:

Primer (artificial sequence)

p1：5’-gccggatccc?caggtttgaa?aggaaaacgt-3’

p2：5’-cggaagcttt?cagtgtcttt?tcttcat-3’

p3：5’-gctgccgctc?ctgctggtgc?tcctggcggc?ggcgcccgca?gccagcccag?gtttgaaagg?aaaac-3’

p4：5’-attgctagca?tgagcgcccg?gaccgccccc?aggccgcagg?tgctcctgct?gccgctcctgctggtg-3’

p5：5’-gatggatcca?ccagatccac?ctgagccacc?accgtgtctt?ttcttcatgc?ac-3’

p6：5’-agatccacct?gagccaccac?cgtgtctttt?cttcatgcac-3’

p7：5’-ggtggctcag?gtggatctgg?tgtcagatca?tcttctcg-3’

p8：5’-gatggatcct?cacagggcaa?tgatcccaaa?gtag-3’

p9：5’-attgctagca?tgagcgcccg?gaccgccccc-3’

P10：5’-tcgagaagat?gatctgacgc?tggctgcggg?cgccgc-3’

p11：5’-gcggcgcccg?cagccagcgt?cagatcatct?tctcg-3’

p12：5’-gcaggatcct?cagtgtcttt?tcttcatgca?c-3’

p13：5’-ctcgcccttg?ctcaccatac?cagatccacc?tgagcc-3’

p14：5’-ggctcaggtg?gatctggtat?ggtgagcaag?ggcgag-3′

p15：5’-aatggatcct?cacttgtaca?gctcgtccat?gcc-3’

p16：5’ctcgcccttg?ctcaccatgc?tggctgcggg?cgccgc-3’

p17：5’-gcggcgcccg?cagccagcat?ggtgagcaag?ggcgag-3’

The Sig-tumstatin-linker-TNF nucleotide sequence

atgagcgccc?ggaccgcccc?caggccgcag?gtgctcctgc?tgccgctcct?gctggtgctc

ctggcggcgg?cgcccgcagc?cagcccaggt?ttgaaaggaa?aacgtggaga?cagtggatca

cctgcaacct?ggacaacgag?aggctttgtc?ttcacccgac?acagtcaaac?cacagcaatt

ccttcatgtc?cagaggggac?agtgccactc?tacagtgggt?tttcttttct?ttttgtacaa

ggaaatcaac?gagcccacgg?acaagacctt?ggaactcttg?gcagctgcct?gcagcgattt

accacaatgc?cattcttatt?ctgcaatgtc?aatgatgtat?gtaattttgc?atctcgaaat

gattattcat?actggctgtc?aacaccagct?ctgatgccaa?tgaacatggc?tcccattact

ggcagagccc?ttgagcctta?tataagcaga?tgcactgttt?gtgaaggtcc?tgcgatcgcc

atagccgttc?acagccaaac?cactgacatt?cctccatgtc?ctcacggctg?gatttctctc

tggaaaggat?tttcattcat?catgttcaca?agtgcaggtt?ctgagggcac?cgggcaagca

ctggcctccc?ctggctcctg?cctggaagaa?ttccgagcca?gcccatttct?agaatgtcat

ggaagaggaa?cgtgcaacta?ctattcaaat?tcctacagtt?tctggctggc?ttcattaaac

ccagaaagaa?tgttcagaaa?gcctattcca?tcaactgtga?aagctgggga?attagaaaaa

ataataagtc?gctgtcaggt?gtgcatgaag?aaaagacact?gaggtggtgg?ctcaggtgga

tctggtgtca?gatcatcttc?tcgaaccccg?agtgacaagc?ctgtagccca?tgttgtagca

aaccctcaag?ctgaggggca?gctccagtgg?ctgaaccgcc?gggccaatgc?cctcctggcc

aatggcgtgg?agctgagaga?taaccagctg?gtggtgccat?cagagggcct?gtacctcatc

tactcccagg?tcctcttcaa?gggccaaggc?tgcccctcca?cccatgtgct?cctcacccac

accatcagcc?gcatcgccgt?ctcctaccag?accaaggtca?acctcctctc?tgccatcaag

agcccctgcc?agagggagac?cccagagggg?gctgaggcca?agccctggta?tgagcccatc

tatctgggag?gggtcttcca?gctggagaag?ggtgaccgac?tcagcgctga?gatcaatcgg

cccgactatc?tcgactttgc?cgagtctggg?caggtctact?ttgggatcat?tgccctgtga

Tumstatin-linker-TNF fusion rotein aminoacid sequence

Pro?Gly?Leu?Lys?Gly?Lys?Arg?Gly?Asp?Ser?Gly?Ser?Pro?Ala?Thr?Trp

Thr?Thr?Arg?Gly?Phe?Val?Phe?Thr?Arg?His?Ser?Gln?Thr?Thr?AlaIle

Pro?Ser?Cys?Pro?Glu?Gly?Thr?Val?Pro?Leu?Tyr?Ser?Gly?Phe?Ser?Phe

Leu?Phe?Val?Gln?Gly?Asn?Gln?Arg?Ala?His?Gly?Gln?Asp?Leu?Gly?Thr

Leu?Gly?Ser?Cys?Leu?Gln?Arg?Phe?Thr?Thr?Met?Pro?Phe?Leu?Phe?Cys

Asn?Val?Asn?Asp?Val?Cys?Asn?Phe?Ala?Ser?Arg?Asn?Asp?Tyr?Ser?Tyr

Trp?Leu?Ser?Thr?Pro?Ala?Leu?Met?Pro?Met?Asn?Met?Ala?Pro?Ile?Thr

Gly?Arg?Ala?Leu?Glu?Pro?Tyr?Ile?Ser?Arg?Cys?Thr?Val?Cys?Glu?Gly

Pro?Ala?Ile?AlaI?le?Ala?Val?His?Ser?Gln?Thr?Thr?Asp?Ile?Pro?Pro

Cys?Pro?His?Gly?Trp?Ile?Ser?Leu?Trp?Lys?Gly?Phe?Ser?Phe?Ile?Met

Phe?Thr?Ser?Ala?Gly?Ser?Glu?Gly?Thr?Gly?Gln?Ala?Leu?Ala?Ser?Pro

Gly?Ser?Cys?Leu?Glu?Glu?Phe?Arg?Ala?Ser?Pro?Phe?Leu?Glu?Cys?His

Gly?Arg?Gly?Thr?Cys?Asn?Tyr?Tyr?Ser?Asn?Ser?Tyr?Ser?Phe?Trp?Leu

Ala?Ser?Leu?Asn?Pro?Glu?Arg?Met?Phe?Arg?Lys?Pro?Ile?Pro?Ser?Thr

Val?Lys?Ala?Gly?Glu?Leu?Glu?Lys?Ile?Ile?Ser?Arg?Cys?Gln?Val?Cys

Met?Lys?Lys?Arg?His?Gly?Gly?Gly?Ser?Gly?Gly?Ser?Gly?Val?Arg?Ser

Ser?Ser?Arg?Thr?Pro?Ser?Asp?Lys?Pro?Val?Ala?His?Val?Val?Ala?Asn

Pro?Gln?Ala?Glu?Gly?Gln?Leu?Gln?Trp?Leu?Asn?Arg?Arg?Ala?Asn?Ala

Leu?Leu?Ala?Asn?Gly?Val?Glu?Leu?Arg?Asp?Asn?Gln?Leu?Val?Val?Pro

Ser?Glu?Gly?Leu?Tyr?Leu?Ile?Tyr?Ser?Gln?Val?Leu?Phe?Lys?Gly?Gln

Gly?Cys?Pro?Ser?Thr?His?Val?Leu?Leu?Thr?His?Thr?Ile?Ser?Arg?Ile

Ala?Val?Ser?Tyr?Gln?Thr?Lys?Val?Asn?Leu?Leu?Ser?Ala?Ile?Lys?Ser

Pro?Cys?Gln?Arg?Glu?Thr?Pro?Glu?Gly?Ala?Glu?Ala?Lys?Pro?Trp?Tyr

Glu?Pro?Ile?Tyr?Leu?Gly?Gly?Val?Phe?Gln?Leu?Glu?Lys?Gly?Asp?Arg

Leu?Ser?Ala?Glu?Ile?Asn?Arg?Pro?Asp?Tyr?Leu?Asp?Phe?Ala?Glu?Ser

Gly?Gln?Val?Tyr?Phe?Gly?Ile?Ile?Ala?Leu

Linker connecting arm nucleotide sequence

ggtggtggc?tcaggtggat?ctggt

Linker connecting arm aminoacid sequence

Gly?Gly?Gly?Ser?Gly?Gly?Ser?Gly

Claims

1, a kind of recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon, be secretor type reorganization pIRESneo3/sig-tumstatin-linker-TNF carrier for expression of eukaryon, it is characterized in that: sig-tumstatin-linker-TNF fusion gene sequence:

atgagcgccc?ggaccgcccc?caggccgcag?gtgctcctgc?tgccgctcct?gctggtgctc

ctggcggcgg?cgcccgcagc?cagcccaggt?ttgaaaggaa?aacgtggaga?cagtggatca

cctgcaacct?ggacaacgag?aggctttgtc?ttcacccgac?acagtcaaac?cacagcaatt

ccttcatgtc?cagaggggac?agtgccactc?tacagtgggt?tttcttttct?ttttgtacaa

ggaaatcaac?gagcccacgg?acaagacctt?ggaactcttg?gcagctgcct?gcagcgattt

accacaatgc?cattcttatt?ctgcaatgtc?aatgatgtat?gtaattttgc?atctcgaaat

gattattcat?actggctgtc?aacaccagct?ctgatgccaa?tgaacatggc?tcccattact

ggcagagccc?ttgagcctta?tataagcaga?tgcactgttt?gtgaaggtcc?tgcgatcgcc

atagccgttc?acagccaaac?cactgacatt?cctccatgtc?ctcacggctg?gatttctctc

tggaaaggat?tttcattcat?catgttcaca?agtgcaggtt?ctgagggcac?cgggcaagca

ctggcctccc?ctggctcctg?cctggaagaa?ttccgagcca?gcccatttct?agaatgtcat

ggaagaggaa?cgtgcaacta?ctattcaaat?tcctacagtt?tctggctggc?ttcattaaac

ccagaaagaa?tgttcagaaa?gcctattcca?tcaactgtga?aagctgggga?attagaaaaa

ataataagtc?gctgtcaggt?gtgcatgaag?aaaagacact?gaggtggtgg?ctcaggtgga

tctggtgtca?gatcatcttc?tcgaaccccg?agtgacaagc?ctgtagccca?tgttgtagca

aaccctcaag?ctgaggggca?gctccagtgg?ctgaaccgcc?gggccaatgc?cctcctggcc

aatggcgtgg?agctgagaga?taaccagctg?gtggtgccat?cagagggcct?gtacctcatc

tactcccagg?tcctcttcaa?gggccaaggc?tgcccctcca?cccatgtgct?cctcacccac

accatcagcc?gcatcgccgt?ctcctaccag?accaaggtca?acctcctctc?tgccatcaag

agcccctgcc?agagggagac?cccagagggg?gctgaggcca?agccctggta?tgagcccatc

tatctgggag?gggtcttcca?gctggagaag?ggtgaccgac?tcagcgctga?gatcaatcgg

cccgactatc?tcgactttgc?cgagtctggg?caggtctact?ttgggatcat?tgccctgtga。

2, a kind of preparation method of recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon, with the signal peptide sequence gene clone of VI Collagen Type VI α 3 chain N end on tumor chalone and tumour necrosis factor gene, between tumor chalone and tumour necrosis factor, add the connecting arm sequence by gene clone, constitute signal peptide-tumor chalone-connecting arm-tumour necrosis factor fusion gene sequence, it is characterized in that: concrete steps are as follows:

(1) human cloning tumstatin

1. RT-PCR human cloning tumstatin total length encoding gene, human embryonic kidney cell line 293 cell cultures are to logarithmic phase, counting, centrifugal collecting cell extract total RNA, Oligo (dT) is synthetic cDNA first chain of primer, getting 2 μ l reverse transcription products is template, upstream primer P1, downstream primer P2 carries out conventional pcr amplification tumstatin gene;

2. pcr amplification tumstatin cDNA product is connected to the pGEM-T carrier with the T4DNA ligase enzyme, pGEM-T/tumstatin transformed competence colibacillus bacterium E.coli DH5 α, extracting plasmid in a small amount;

(2) recombination fusion protein sig-tumstatin-linker-TNF PCR clone

1. pcr amplification sig-tumstatin-linker gene fragment, upstream primer P3, downstream primer P5, with pGEM-T/tumstatin is that template is carried out pcr amplification, and capable 0.8% agarose gel electrophoresis of pcr amplification product, purified recovery test kit reclaim as template, again with upstream primer P4, downstream primer P6, row second is taken turns pcr amplification, and amplified production is the sig-tumstatin-linker that carries Nhe I+BamH I restriction enzyme site;

2. pcr amplification linker-TNF fragment, upstream primer P7, downstream primer P8 is the linker-TNF fragment of template amplification belt lacing with PBV220-TNF;

3. overlap amplification splicing method amplification sig-tumstatin-linker-TNF, with the PrimeSTARTMHS archaeal dna polymerase upstream primer P4 and the downstream primer P8 of high-fidelity, be the amplification of template overlap amplification splicing method with sig-tumstatin-linker fragment and linker-TNF fragment;

(3) sig-TNF gene clone

Use upstream primer P9, downstream primer P10, pGEM-T/sig-tumstatin-linker are the sig+ fragment of template amplification and TNF band overlap;

2. use upstream primer P11, downstream primer P8 is the TNF+ fragment of template amplification and sig band overlap with PBV220-TNF;

3. be that the amplification of template overlap amplification splicing method obtains sig-TNF with upstream primer P9 and downstream primer P8 with sig+ fragment and TNF+ fragment;

(4) sig-tumstatin fragment PCR amplification, upstream primer P9 and downstream primer P12, pGEM-T/sig-tumstatin-linker are template amplification;

(5) sig-tumstatin-linker-EGFP and sig-EGFP fragment PCR amplification

1. the sig-tumstatin-linker fragment amplification of overlap is arranged with EGFP, and upstream primer P9 and downstream primer P13, pGEM-T/sig-tumstatin-linker are template, product called after a;

2. the EGFP fragment amplification of overlap is arranged with sig-tumstatin-linker, use upstream primer P14, downstream primer P15, pEGFP-C2 are template, product called after b;

3. the sig fragment amplification of overlap is arranged with EGFP, use upstream primer P9, downstream primer P16, pGEM-T/sig-tumstatin-linker are template, product called after c;

The EGFP fragment amplification that 4. overlap is arranged with sig, with upstream primer P17 and downstream primer P15, pEGFP-C2 is a template, product called after d;

5. sig-tumstatin-linker-EGFP fragment amplification (being the ab fragment) is a primer with P9 and P 15, and a and b fragment are template,

Sig-EGFP fragment amplification (being the cd fragment) is a primer with P9 and P15, and c and d fragment are template;

98 ℃, 10s (sex change); 68 ℃, 5min (annealing, amplification) carries out 30 circulations, extends 5min, capable 0.8% agarose gel electrophoresis of pcr amplification product, the corresponding product ab of rubber tapping recovery, cd fragment in 68 ℃ again.

3, according to claim 2 with the signal peptide sequence gene clone of VI type glue source α 3 chain N end on tumor chalone and the tumour necrosis factor gene the preparation method, it is characterized in that: the PCR reaction tubes of recombination tumor chalone-tumor putrescence factor fusion rotein consists of:

5×PrimeSTAR?buffer+MgCl2 10μl

2.5mM?dNTPs 4μl

P4(20mM) 0.5μl

P8(20mM) 0.5μl

Sig-tumstatin-linker fragment 1 μ l

Linker-TNF fragment 1 μ l

The PrimeSTARTMHS archaeal dna polymerase of high-fidelity (2.5U/ μ l) 0.5 μ l

ddH2O 32.5μl

Amount to 50 μ l.

4, recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon according to claim 1 and its production and use is characterized in that: the structure of recombinant plasmid pIRESneo3/sig-EGFP, pIRESneo3/sig-tumstatin-linker-EGFP, pIRESneo3/sig-tumstatin, pIRESneo3/sig-TNF, pIRESneo3/sig-tumstatin-linker-TNF;

Cloned genes fragment sig-EGFP, sig-tumstatin-linker-EGFP, sig-tumstatin, sig-TNF, sig-tumstatin-linker-TNF are through Nhe I+BamH I double digestion, same enzyme is cut the pIRESneo3 plasmid, the gene fragment of utilizing the T4DNA ligase enzyme that enzyme is cut connects product Transformed E .coli DH5 α with the pIRESneo3 plasmid vector that enzyme is cut, and prepares recombinant plasmid in a small amount.

5, recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon according to claim 1 and its production and use, it is characterized in that: recombinant plasmid pIRESneo3/sig-tumstatin-linker-EGFP, pIRESneo3/sig-tumstatin, pIRESneo3/sig-TNF, the eukaryotic cell of pIRESneo3/sig-tumstatin-linker-TNF transfection, recombinant plasmid lipofectamineTM2000 transfection CHO-K1 cell, through the best susceptibility concentration of G418 concentration is the screening of 600mg/L, transfection pIRESneo3/sig-EGFP, the mono-clonal Chinese hamster ovary celI of IRESneo3/sig-tumstatin-linker-EGFP shows green fluorescence, the secreting, expressing success is described, picking mono-clonal pure culture respectively; The Chinese hamster ovary celI RT-PCR of transfection pIRESneo3/sig-TNF, pIRESneo3/sig-tumstatin-linker-TNF gene and culture supernatant detect through Western blot and identify the pure culture of positive cell picking mono-clonal.

6, recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon according to claim 1 and its production and use, it is characterized in that: the linker connecting arm, it has the dna sequence dna of ggtggtggc tcaggtggat ctggt, its expression has the aminoacid sequence of Gly Gly Gly Ser Gly Gly Ser Gly, corresponding fusion gene have tumstatin-linker, sig-tumstatin-linker ,-inker-TNF.

7, a kind of purposes of recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon is characterized in that: the application of recombination tumor chalone-tumor putrescence factor secretion type carrier for expression of eukaryon in the preparation antitumor drug.