CN101338296A - Construction of attenuation salmonella bacterin with Vp3 and TRAIL co-expression plasmid - Google Patents

Abstract

The invention relates to an attenuated salmonella of a double expression plasmid which carries Vp3 and TRAIL as well as the applications thereof in constructing and preparing a gene treatment medicine for curing or preventing knub. Not only the body of the invention can restrain the knub from growing, but also can be used as a foreign gene excellent expression carrier to submit the characteristic of a pharmaceutical protein in a knub cell to insert the Vp3 and the TRAIL into the double expression plasmid by combining the tumoraffin of an invasion bacterial with the knub characteristics of the Vp3 and the TRAIL for inducing apoptosis, forms a strain of pBud-Vp3-TRAIL/SL7207 by taking the attenuated salmonella as a carrier and researches the effect and mechanism for resisting the knub inside and outside a body by a mode of gastric cancer. Test inside and outside the body shows that the co-expression of the Vp3 and the TRAIL can play the better coordination effect for restraining the growing of the knub.

Description

Take the structure and the application of attenuation salmonella vaccine of the double expression plasmid of Vp3 and TRAIL

Technical field

The invention belongs to biological technical field, be specially a kind of structure and application in the treatment tumour thereof of attenuation salmonella of the pBud-Vp3-TRAIL-EGFP of carrying double expression plasmid.

Background technology

Since nearly half a century, tumour has become the common disease that is seriously threatening human health.Estimate that according to The World Health Organization (WHO) and the scholar of American Society of Clinical Oncology (ASCO) the annual de novo cancer patient in the whole world is 1,000 ten thousand, dead patient in cancer accounts for 12% of death toll between 600-700 ten thousand people.In the past few decades adopt surgical operation, radiotherapy and chemotherapy, carry out the dynamics of anticancer struggle and constantly strengthen, but its result is unsatisfactory so far.Therefore, people constantly explore the novel method of oncotherapy.Along with the develop rapidly of molecular biology research, cell growth, differentiation, mechanism of apoptosis are familiar with gradually, and a kind of new methods of treatment--gene therapy is just becoming the research focus.Gene therapy is that tumour patient has brought dawn, is expected to become the 5th effective way of treatment malignant tumour after operation, radiotherapy, chemotherapy and immunotherapy.

Tumour is not only the disease of cell proliferation and prosoplasia, also is simultaneously to transfer the unusual disease of dying.People recognize that gradually the generation of tumour is the result of cell proliferation and apoptosis dysequilibrium, the cell cycle cell hyperproliferation that causes out of control, but the more important thing is that apoptosis is obstructed, directly caused old and feeble, undermined paracytic life and prolonged.Therefore, eliminating tumour by inducing apoptosis of tumour cell also is a kind of new methods of treatment that should consider.In fact, the radiotherapy of present clinical employing and some chemotherapy regimen play a role by inducing apoptosis of tumour cell.But because their no specificitys, thereby normal cell also had lethality, toxic side effect is obvious; Its inductive apoptosis is the caused by tumor suppressor p 53 mediation mostly simultaneously, because the p53 gene of tumour patient over half has sudden change in various degree, has suppressed apoptosis of tumor cells; Proto-oncogene bcl-2 up-regulated also suppresses apoptosis of tumor cells in the most tumors, has greatly influenced result of treatment, and this also is that tumour cell produces one of chemical sproof reason.In addition, many apoptosis-induced factors can cause non-selective killing and wounding or other side effect and be difficult to enter clinical application, start serious inflammatory reaction, FAS as TNF and cause serious liver injury.Searching has the selective killing tumour cell and absolves one of mark the most all day that Normocellular cancer therapy drug is a tumor research simultaneously.

It has been generally acknowledged that classical apoptosis path has two kinds: death receptor approach (death receptor pathway) and plastosome approach (mitochon-drial pathway).For many years, to consistent apoptosis element (Apoptin) apoptosis induction ligand relevant (tumornecrosis factor-related apoptosis inducing ligand, the tumour-specific of apoptosis effect TRAIL) of having proved of the tumour cell of different sources with tumour necrosis factor with Normocellular research.Research to the tumour-specific Apoptosis Mechanism of Apoptin and TRAIL has in recent years had breakthrough progress, widened its field in cancer therapeutic applications research and tumour generation fundamental research. studies show that, Apoptin is by plastosome approach cell death inducing, relate to the activation of downstream caspase-3, cytochrome C is from Intramitochondrial release, and the forfeiture of mitochondrial membrane potential.And TRAIL is apoptosis-induced by the death receptor approach: death receptor DR5 (or TRAIL-R1) or DR4 (or TRAIL-R2) combine with TRAIL by extracellular region and activation, death domain is assembled mutually in the born of the same parents, further activate the caspase cascade reaction and bring out apoptosis. infer that in view of the above the two may exist intersects or association on the apoptosis induction path.

The apoptosis element is chicken anaemia virus (Chicken anaemia virus, the CAV) albumen of VP3 genes encoding, full length gene 363bp, encode 121 amino acid, the stretching area that includes 2 proline rich and 2 cationic charge districts.For many years discover that (the apoptosis element apoptin) can be induced a kind of p53 of not relying in people's various tumour cells, and to the insensitive apoptosis of Bcl-2, single 1 apoptosis element can trigger apoptosis.In experiment in vitro, yet the apoptosis element can not be induced the programmed death of normal Lymphoid tissue, corium, epidermis, endothelium and smooth muscle cell., after normal cell is converted into tumour cell, just become to the plain inductive apoptotic sensitivity of apoptosis.

Can be in order to inquire into Apoptin as a kind of possibility of tumor cytotoxicity agent, the transient expression effect of Apoptin in dissimilar human blood malignant cells at first studied by Noteborm research group.Transfection experiment shows, cross among the B lymphoma cell DDHH-2 of expression and the lymphoglandula B cell Jobo-0 that Epstein-Barr virus is induced immortalization at acute myeloid leukemia cell KG-1 and K562, BcL-2, after the transfection 5 days, the apoptosis rate that Apoptin expresses positive cell reached 80%-90%.Proof Apoptin can induce the apoptosis of human blood malignant cell.Research subsequently proves that again Apoptin can induce the apoptosis of multiple different people tumour cells such as people's human osteosarcoma cell, liver cancer cell, the shaft-like oncocyte of kidney, lymphoma, mammary cancer, lung cancer, squamous cell carcinoma.And can not induce normal diploid cell such as lymphocyte, epithelial cell, inoblast, endotheliocyte and smooth muscle cell apoptosis, and the continuous passage of the normal fiber archeocyte of transfection VP3 gene, do not find any toxicological effect, do not have the plain activity of conversion of apoptosis yet.Zhang etc. discover that the apoptosis element also can be induced its apoptosis, and the fibroblast from healthy people is not had influence to the cell of the tool tumour genetic predisposition of conversion of part virus and uviolizing.Subsequently, discovery Bcl-2 such as Danen-van Oorschot AA associating protein B AG-1 can suppress the apoptosis of p53 inductive tumour cell Saos-2, and can not suppress apoptin inductive Saos-2 apoptosis.In the experimentation on animals, usefulness such as van der Eb MM are expressed the recombinant adenoviral vector of VP3 gene and are handled the nude mice liver cancer cell knurl of having inoculated transplanting, find that growth of tumor can significantly be suppressed, and nude mice is not had obvious toxic-side effects.The recombinant adenoviral vector transfection people cholangiocarcinoma cell that usefulness such as Pietersen AM are expressed the VP3 gene is CC-LP, and CC-SW, and Mz-ChA-1 find that tumour cell is extensively suppressed, and are not subjected to the influence of p53 sudden change.These have all shown the application prospect that it is tempting.

Can the apoptosis element why the specificity inducing tumor cell and the apoptosis of tool tumour genetic predisposition and normal cell is not had influence? research thinks that the plain activity of apoptosis may be relevant with its nucleus location.Current research shows that its 82-88 amino acid (NLS1) and its C end basic region (11 amino acid) is nuclear localization signal (NLS2), and it is necessary to be that the apoptosis element is appraised and decided the position.This specific character make its very easily with the cyto-chromatin structure in histone and (or) the nonhistones combination, and its high proline content may destroy the superhelix tissue, thereby cause the cracking of chromatin superhelix, finally causing the fracture of cell DNA and concentrate. the expressed apoptosis element of the cell of normal health individuality is confined in the tenuigenin, the apoptosis element of tumor susceptibility cell is then moved in the nucleus, then concentrated on nucleus by apoptosis element in the apoptosis-induced cell, be positioned to examine on the interior chromatin.Current research is thought and is had a kind of kinases in tumour cell and the transformant, the plain 108 Threonine phosphorylations of catalysis apoptosis, rely on two nuclear localization signals, the apoptosis element of phosphorylation enters nucleus and exists with the polymer activity form, activate downstream capases cascade reaction, the plastosome membrane potential destroys, and cytochrome c discharges from plastosome, finally causes the apoptosis of cell.In view of above characteristics, apoptosis have prestige and brings into play enormous function at anti-tumor aspect.But tumour is multifactor a, too many levels, multistage complex disease, and the intervention of single factors often can not reach ideal and press down the knurl effect.So seeking new treatment point of penetration and existing treatment approach is effectively united is a kind of strategy of therapy of tumor and biotherapy research.

(tumor necrosis factor-related apoptosisinducing ligand is the TNF superfamily newcomer of discovered in recent years TRAIL) to the relevant apoptosis induction ligand of tumour necrosis factor, also claims Apo-2L.In the experiment in vitro, TRAIL can induce multiple tissue-derived apoptosis of tumor cells rapidly in vivo, and its apoptosis-induced effect does not rely on Fas/Apo-1L and TNF acceptor, but by TRAIL receptor pathway cell death inducing.Known its acceptor mainly contains death receptor DR4, DR5 and decoy receptor DcR1, DcR2 at present.TRAIL is with after DR4, DR5 combine, can activate a series of integrated enzyme reactions and cause the apoptosis of target cell, and DcR1 and DcR2 since its intracellular portion lack as or block, with the apoptosis that can not induce target cell after TRAIL combines.Because most of tumor tissues high expression level DR4 and DR5, and lowly express or do not express DcR1 and DcR2, thus TRAIL with DR4 or/and DR5 combines, can cause the apoptosis of tumour cell, play antitumor action.And most healthy tissues high expression level DcR1 and/or DcR2 can compete in conjunction with TRAIL, and therefore, general healthy tissues can not suffer damage.

This shows, compare with multiple medicine, apoptin and TRAIL may be alternative inducing apoptosis of tumour cell truly, and the apoptotic proteins of injuring normal cell not, be expected to solve in the oncotherapy high efficiency and treat that the opposite sex is difficult to unify and is easy to produce a difficult problem such as resistance, become a kind of anti-tumor agent, have higher development and utilize prospect with direct bullet lethal effect.

Whether useful a kind of therapy of tumor method is except a kind of effective therapeutic gene will be arranged, and also depends on: must have the carrier of a certain amount of expression alien gene to reach in the tumour; Carrier expression alien gene effectively in the microenvironment of tumour; By certain mechanism the sequential and the expression amount of vector expression to be regulated and control. currently used virus vector (retrovirus, adenovirus, adeno-associated virus etc.) and non-virus carrier (as liposome etc.) be astatism all, and expression level was not ideal enough after gene was led in the human body, and this becomes the bottleneck that gene therapy can not have breakthrough.In several years of past, some scholars begin one's study microorganism in the effect aspect the oncotherapy: facultative anaerobe can preferentially assemble in tumor tissues, this phenomenon is because the microenvironment of tumour itself determines, the hypoxemia necrotic area can be bred these bacteriums in the tumour, bacterium is spread in tumor tissues easily, can also remove by microbiotic in the case of necessary, this makes them occupy certain advantage in the Antioncogene treatment.Attenuation salmonella just can find the institute of settling down of a safety in tumor tissues, optionally accumulate in the solid tumor, and Salmonellas levels of replication in tumor tissues is higher 1000 to 10000 times than healthy tissues.Salmonella typhi not only itself can suppress tumor growth, and can present pharmaceutical protein to tumour cell in vivo as good exogenous gene expression carrier.Someone utilizes the Salmonella mattress as the IL-2 gene of having presented external source pharmaceutical protein vector expression in vivo.

As in the therapy of tumor study on the carrier, carrier has the mechanism of high target like this also to be not very clear now to tumour with salmonella typhi.Pawelek etc. think that content in normal cell such as the auxotrophic strain desired substance that adopts such as die aromatischen Aminosaeuren is few, be not enough to satisfy the nutritional need of bacterial growth, and very rich at the continuous splitted tumour of cell " necrotic area " content, cause the concentrated relatively of bacterium; Also might be that some adhesion molecule existence causes bacterium that tumour cell is had target.More interesting is why bacterium itself just has so strong restraining effect to tumour.May there be many-sided reason in this.The contained lipopolysaccharides (LPS) of bacterium can stimulate lymphopoiesis and differentiation, brings out production of cytokines such as TNF, thereby strengthens and enlarge the ability of immunne response. and also may be to carry out nutrient competition between bacterium and the tumour cell in addition; Bacterium secretory protein lytic enzyme killing tumor cell.

Salmonellas can the target kinds of tumors as the remote injection of the advantage of gene delivery vector; Can under the condition of aerobic and anaerobic, grow, optionally in tumor microenvironment, assemble; Can transmit the expression effector; Have high invasiveness and low pathogenicity; Can the by oral route administration; The bacterium that accumulates in single tumour cell can be used as the storage vault of bacterium, finally makes bacteriological action in tumour at a distance; Penetrance with motility and homogeneous; Can express multiple human cytokines; Tissue there is single-minded taxis; Bacteria effect to organize the normally natural target spot of these bacteriums; And expense is low, and the Non-Invasive administration is fit to long-term treatment.

The engineered method of humans such as clairmont has been eliminated Salmonella typhimurium msbB gene, but still kept auxotrophic characteristics such as the high invasiveness of this bacterial strain and die aromatischen Aminosaeuren be synthetic. no longer be subjected to the restriction of temperature, still keep it at tumour cell and (2000: 1) at high proportion in normal cell and inhibition tumor promotion, and no longer including a large amount of TNF-a produces. simultaneously, and msbB ^-Mutant strain is all responsive to multiple microbiotic, and this has just been avoided bacterium to breed in a large number in vivo and has caused septicopyemia and microbemia, makes Salmonellas more can be suitable for human body and carries out gene therapy.

Summary of the invention

The object of the present invention is to provide the attenuation salmonella vaccine of the double expression plasmid of a kind of Vp3 of taking and TRAIL, in its building process, adopt the TRAIL/VP3 gene eucaryon double expression plasmid of energy specificity inducing apoptosis of tumour cell; Attenuation salmonella is as carrier, by it is bacterium in the aggressive born of the same parents, not only itself can suppress tumor growth, and can present pharmaceutical protein to tumour cell in vivo as good exogenous gene expression carrier, directionally shifts VP3 and trail dna and carries out genetic treatment of tumor.Therefore, another object of the present invention relates to the purposes of attenuation salmonella vaccine in preparation gene therapy tumor disease medicine of the double expression plasmid of described Vp3 of taking and TRAIL.

The technical scheme that adopts is so for achieving the above object: the attenuation salmonella vaccine of the double expression plasmid of promptly a kind of Vp3 of taking and TRAIL is characterized in that adopting following steps to make up:

1.pBud-EGFP structure

XbaI enzyme cutting pEGFP and pBudCE4.1 reclaim T behind the EGFP fragment of 757bp and the big fragment pBudCE4.1 of linearized vector respectively ₄Dna ligase connects, and transformed competence colibacillus cell Jm109 screens positive recombinant plasmid, through enzyme cut, pcr amplification and order-checking obtain pBud-EGFP after identifying;

2.pBud-TRAIL-EGFP structure

(1) design primer P1 and P2:

P1：5’-GCCCTCGAG-ATGCTAGTGAGAGAAAGAGGTC-3’(XhoI)

P2：5’-GCCAGATCT-TTTCCAGGTCAGTTAGCCA-3’(BglII)

(2) extract human peripheral lymphocytes RNA and change into cDNA, for touching plate, use the amino acid whose gene order 525bp of coding extracellular region 112-287 in primer P1 and the P2 amplification people trail dna with cDNA;

(3) cut pBud-EGFP and TRAIL with XhoI and BglII enzyme, glue reclaims the big fragment of linearized vector pBud-EGFP of TRAIL and gained, T ₄Dna ligase connects, and transformed competence colibacillus cell Jm109 screens positive recombinant plasmid, through enzyme cut, pcr amplification and order-checking obtain pBud-TRAIL-EGFP after identifying;

3.pBud-Vp3-TRAIL-EGFP structure

(1) design primer P3 and P4

P3：5’-GCCGTCGAC-AATGAACGCTCTCCAAGAA-3’(SalI)

P4：5’-GCCAGTACT-CAGTCTTATACGCCTTCT-3’(ScaI)

(2) with pMD18-T/VPX be template, with primer P3 and P4 amplification apoptin full length sequence;

(3) cut pBud-TRAIL-EGFP and Vp3 with SalI and ScaI enzyme, glue reclaims the big fragment of linearized vector pBud-TRAIL-EGFP of Vp3 and gained, T ₄Dna ligase connects, and transformed competence colibacillus cell Jm109 screens positive recombinant plasmid, through enzyme cut, pcr amplification and order-checking obtain pBud-vp3-TRAIL-EGFP after identifying;

4. pBud-vp3-TRAIL-EGFP recombinant plasmid electricity is transformed into attenuation salmonella SL7207

(1) the electric transformed competence colibacillus SL7207 of preparation

(2) after electricity transforms, the positive reorganization of screening bacterium, through enzyme cut, pcr amplification identifies and obtains the attenuation salmonella vaccine that pBud-Vp3-TRAIL-EGFP/SL7207 is the present invention's double expression plasmid of taking Vp3 and TRAIL.

Advantage of the present invention and positively effect are:

(1) apoptosis element and TRAIL can specially lure imported apoptosis of tumor cells, and exciting is without any side effects to the diploid cell of normal people and mouse.Be subjected to extensive concern at present, but because lacked efficient and directed gene transfer vector system.Can't be widely used in clinical.It is not ideal enough that all astatisms, and gene of currently used virus vector (retrovirus, adenovirus, adeno-associated virus etc.) and non-virus carrier (as liposome etc.) import in the body back expression level.This becomes the bottleneck that gene therapy can not have breakthrough.The present invention attempts close tumprigenicity (target) of aggressive bacterium and the plain apoptosis-induced tumour-specific of apoptosis are combined, further strengthened the specificity of therapy of tumor, make it at therapy of tumor, especially wide application prospect is being arranged aspect the gene therapy of cancer of the stomach.

(2) the present invention unites Vp3 and TRAIL and inserts the eucaryon double expression plasmid, is death receptor approach (death receptor pathway) and plastosome approach (mitochon-drialpathway) inducing apoptosis of tumour cell from apoptotic two paths of classics.The generation that meets tumour and development are the theories of multifactor, multistage, polygenic complicated change procedure.

(3) the present invention has adopted double-promoter carrier for expression of eukaryon pBudCE4.1, this dual-expression vector is by two two foreign genes of promotor control independently, is transcribed into two mRNA molecules and translation and produces separately antigen protein. vp3 is placed expression cytomegalovirus (CMV) strong promoter under; Trail dna is inserted Ef-1a promotor downstream, and structure can be expressed the eukaryon expression plasmid pBud-vp3-TRAI1 of two kinds of genes simultaneously.

(4) with attenuation Salmonella SL7207 bacterial strain as recombinant vectors, have that toxic side effect is little, safety, production technique is simple, have more direct easy advantage than the lead-in mode of liposome transfection and particle gun injection, and be more suitable for experiment in the body.Simultaneously itself be again the immunological adjuvant of very strong work, the immunotherapy and the therapy of tumor of tumour combined the therapy of tumor scheduling theory field of enriching that the further confirmation of this theory will be bigger.

(5) in genetic treatment of tumor, the controllability of exogenous gene expression is a very crucial problem.After if goal gene imports human body, expression is in runaway condition, will cause serious consequence.Therefore, effectively regulating and control expression of exogenous gene is the breach that gene therapy makes progress.In the antitumor research after therapeutic gene imports tumour cell, attenuation salmonella is a selectable instrument of ideal, can stop its expression by the method for microbiotic intervention.

Description of drawings

Figure 1A is the synoptic diagram of the structure of pBud-Vp3-TRAIL-EGFP plasmid, and Figure 1B is the synoptic diagram of the structure of pBud-Vp3-TRAIL-EGFP/SL7207 and the effect in cancer of the stomach;

Fig. 2 cuts with PCR for the enzyme of recombinant plasmid pBud-TRAIL-EGFP provided by the invention and pBud-Vp3-TRAIL-EGFP and identifies.Wherein the enzyme of A.pBud-TRAIL-EGFP is cut evaluation: 1.DL2000 Marker; 2. λ-EcoT14I digest; 3.pBud through the XbaI single endonuclease digestion; 4.pBud-EGFP through the SalI single endonuclease digestion; 5 pBud-EGFP cut evaluation through XbaI single endonuclease digestion .6.pBud-TRAIL-EGFP through the enzyme of XhoI and BglII double digestion .B.pBud-Vp3-TRAIL-EGFP: the 1.SalI single endonuclease digestion; 2.XhoI and BglII double digestion; 3.SalI and ScaI double digestion; 4.XbaI single endonuclease digestion; 5.VP3 PCR identify; 6.DL2000Marker; 7.VP3 PCR identify; 8.Vp3 positive control; 9.TRAIL PCR identify; 10.TRAIL positive control; 11. negative control;

Fig. 3 detects collection of illustrative plates, wherein A:expressionof sTRAIL:M.DL2000Maker for the RT-PCR of sTRAIL mRNA of the present invention and Vp3 mRNA; 1.pBud-Vp3-TRAIL-EGFP/SGC7901; 2.pBud-TRAIL-EGFP/SGC7901; 3.SGC7901; B:expression of Vp3 M:DL2000Maker; 1.pBud-Vp3-TRAIL-EGFP/SGC7901; 2.pBud-Vp3-EGFP/SGC7901; 3.SGC7901;

Fig. 4 analyzes collection of illustrative plates, wherein 1.pBud-TRAIL-EGFP for the Western Blot of sTRAIL albumen of the present invention and VP3 protein expression; 2.pBud-EGFP; 3.control; 4.pBud-TRAIL-Vp3-EGFP; 5.pBud-Vp3-EGFP; 6.control;

The fluorogram that Fig. 5 expresses in the SGC7901 cell for gene eucaryon expression plasmid provided by the invention, wherein

A.pBud-Vp3-TRAIL-EGFP/SGC7901；B.pBud-Vp3-EGFP/SGC7901；C.pBud-TRAIL-EGFP/SGC7901；D.pEGFP/SGC7901；

Fig. 6 is fluorescence location and cytotoxicity figure behind the pBud-Vp3-TRAIL-EGFP/SL7207 transfection SGC7901 cell of the present invention, among the figure: A-C fluorescence location (* 8000); D is the cell contrast, and the SGC7901 cell of untransfected does not fluoresce, and (* 4000) are indicated in the lower right corner; The SGC7901 cell of E transfection pEGFP control plasmid; The apoptosis (* 4000) that causes behind the F-G pBud-Vp3-TRAIL-EGFP/SL7207 transfection SGC7901 cell; H shows AO-EB dyeing, the red non-viable apoptotic cell (* 200) of representing;

Fig. 7 is sTRAIL and the Vp3 proliferation inhibiting effect figure to the SGC7901 cell;

Fig. 8 is the apoptosis (* 200) of TUNEL method detection SGC-7901 cell, A.pBud-Vp3-TRAIL-EGFP/SGC7901 among the figure; B.pBud-Vp3-EGFP/SGC7901; C.pBud-TRAIL-EGFP/SGC7901; D.SGC7901;

Fig. 9 is anti-knurl effect and the security HE colored graph of pBud-Vp3-TRAIL-EGFP/SL7207 of the present invention, among the figure: A. tumor-bearing mice (6w), B. the mouse (6W) of handling through SL7207, C. the mouse (6W) of handling through pBud-Vp3-TRAIL-EGFP/SL7207, HE dyes (D-I * 200): among the figure: the D tumor tissues: cell has atypia, karyon increases, nuclear hyperchromatism, and the caryoplasm ratio increases; The E stomach: the coat of the stomach four-layer structure is complete, and the body of gland marshalling does not see that mucosal epithelium comes off, ulcer, cell infiltration, hyperplasia and hemorrhagic pathological change.The F kidney: structure is as usual, do not see ooze out, pathological change such as hyperplasia.The G heart: histological structure is clear, and the cardiac muscle fibre marshalling is not seen cell infiltration.The H lungs: alveolar structure is clear, and segmental bronchuses at different levels, bronchiole mucous membrane are complete, do not see inflammatory cell infiltration.The I liver: liver lobule clear-cut, liver cell are not seen sex change and necrosis, and central vein, liver sinusoid are not seen hyperemia and thrombosis.

Embodiment

Embodiment 1

Present embodiment has been described the building process of attenuation salmonella SL7207 provided by the invention strain (pBud-Vp3-TRAIL-EGFP/SL7207) and the expression in mammalian cell, and may further comprise the steps:

1.pBud-EGFP structure

(1) XbaI enzyme cutting pEGFP, pBudCE4.1, the EGFP fragment that reclaims 757bp respectively is connected construction recombination plasmid pBud-EGFP with the laggard row of the big fragment of linearized vector;

Carrier and EGFP all use XbaI enzyme cutting, and reaction system is as follows:

vector EGFP

DNA 10uL 10uL

KpnI 1uL 1uL

10×buffer 2uL 2uL

ddH ₂O 7uL 7uL

total?volume 20uL 20uL

Mixing is put in 37 ℃ of water baths enzyme and is cut 6h.

(2) enzyme is cut the recovery and the purifying (Omiga DNA glue reclaims test kit) of product

Operation is undertaken by explanation, reclaims to obtain 757bp EGFP fragment and the big fragment of linearized vector;

(3) ligation

By the UV spectrophotometer measuring nucleic acid content be: vector (100ng/ μ l), EGFP (150ng/ μ l).According to the external source fragment: the carrier mol ratio is 3～10 principle, and design ligation system is as follows:

EGFP 7μl

The big fragment 1 μ l of linearized vector

10×buffer 1μl

T ₄Dna ligase 1 μ l

total?volume 10μl

Set up negative control simultaneously, 16 ℃ of connections of spending the night.

(4) transform

1). get the competent cell 150 μ l of prepared fresh under the sterile state, place the aseptic EP pipe of 1.5ml.

2). every pipe adds and connects product 10 μ l, ice bath 30min behind the mixing.

3) .42 ℃ of heat-shocked 2min.

4). every pipe adds the LB substratum of 800 μ l antibiotic-frees, 37 ℃ of gentle vibration 45min.

5). the centrifugal 2min of room temperature 5000g, stay 200 μ l supernatants and bacterial precipitation mixing after, evenly coat on the LB training fat flat board that contains Zeocin with aseptic elbow glass shop bacterium device.

(5) bacterium colony is identified

1). random choose transform bacterium colony several, be inoculated in the LB substratum that contains Zeocin concussion and cultivate.

2). prepare plasmid DNA in a small amount.

3). carry out with Xba I restriction enzyme that enzyme is cut and the agarose gel electrophoresis analysis.

2.pBud-TRAIL-EGFP structure

(1) design of primers with reference to the pBudCE4.1 collection of illustrative plates, according to the design of primers principle, design primer P1 and P2, and the downstream is introduced xhoI and Bgl II restriction enzyme site respectively thereon according to Genebank BC020220 number coding people trail dna sequence:

P1：5’-GCCCTCGAG-ATGCTAGTGAGAGAAAGAGGTC-3’(xhoI)

P2：5’-GCCAGATCT-TTTCCAGGTCAGTTAGCCA-3’(Bgl?II)

(2) the amino acid whose gene order 525bp of coding extracellular region 112-287 in the amplification people trail dna

Extract human peripheral lymphocytes RNA, getting 2 μ l RNA is primer with oligo dT, adopts the transcript reagent box of Takara to change into cDNA

Primer: upstream 5 '-GGTACCATGC-3 ', downstream 5 '-GATATCGGTACC-3 ',

The reverse transcription reaction system is as follows:

Rnase?Free?H ₂O 10μl

5×RT?Buffer 4μl

dNTP?Mixture 2μl

Rnase?Inhibitor 1μl

Oligo(dT) 1μl

RNA 1μl

ReverTra?Ace 1μl

total?volume 20μl

RT reaction conditions: 30 ℃ of 10min, 42 ℃ of 40min, 99 ℃ of 5min

Get 2 μ l RT products and carry out PCR

The PCR reaction system is as follows:

P1 1μl

P2 1μl

Mg ²⁺ 2μl

Taq 0.3μl

cDNA 1.2μl

dNTP 2μl

10×Buffer 2.5μl

ddH2O 15μl

total?volume 25μl

PCR reaction conditions: 94 ℃ of 5min, 94 ℃ of 40s, 56 ℃ of 40s, 72 ℃ of 1min, 35 circulations, 72 ℃ of 10min.

Adopt the PCR purification kit purified pcr product of Omiga

(3) ligation

Cut pBud-EGFP and TRAIL with XhoI and BglII enzyme, Omiga DNA glue reclaims the big fragment T of linearized vector that test kit reclaims TRAIL and gained ₄Dna ligase connects.

TRAIL 7μl

The big fragment 1 μ l of linearized vector

10×buffer 1μl

T ₄Dna ligase 1 μ l

total?volume 10μl

Set up negative control simultaneously, 16 ℃ of connections of spending the night.

(4) transform

1). get the competent cell 150 μ l of prepared fresh under the sterile state, place the aseptic EP pipe of 1.5ml.

2). every pipe adds and connects product 10 μ l, ice bath 30min behind the mixing.

3) .42 ℃ of heat-shocked 2min.

4). every pipe adds the LB substratum of 800 μ l antibiotic-frees, 37 ℃ of gentle vibration 45min.

5). the centrifugal 2min of room temperature 5000g, stay 200 μ l supernatants and bacterial precipitation mixing after, evenly coat on the LB training fat flat board that contains Zeocin with aseptic elbow glass shop bacterium device.

(5) bacterium colony is identified

1). random choose transform bacterium colony several, be inoculated in the LB substratum that contains Zeocin concussion and cultivate.

2). prepare plasmid DNA in a small amount.

3). carry out with XhoI and BglII restriction enzyme that enzyme is cut and the agarose gel electrophoresis analysis.

4). positive colony carries out the cDNA sequencing through key lab of the infectious diseases molecular biology the Ministry of Education of Medical University Of Chongqing automatic sequencer.

3.pBud-Vp3-TRAIL-EGFP structure

(1) design of primers is according to Genebank AY171617 number coding apoptin full length gene sequence, with reference to the pBudCE4.1 collection of illustrative plates, according to the design of primers principle, design primer P3 and P4, and the downstream is introduced Sal I and Sca I restriction enzyme site respectively thereon, removes termination codon TAA.

By the software design primer:

P3：5’-GCCGTCGAC-AATGAACGCTCTCCAAGAA-3’(SalI)

P4：5’-GCCAGTACT-CAGTCTTATACGCCTTCT-3’(Sca?I)

(2) amplification chicken anaemia virus apoptin full length sequence

With pMD18-T/VPX (the Harbin veterinary institute is buied) is that template is carried out pcr amplification apoptin full length sequence, adds the 12bp restriction enzyme site, altogether 375bp:

The reaction cycle condition is: 94 ℃ of pre-sex change 5min; 94 ℃ of 30Sec, 56 ℃ of 45Sec, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 10min.1.5% agarose gel electrophoresis, the PCR purification kit purified pcr product of employing Omiga.

(3) ligation

Cut pBud-TRAIL-EGFP and Vp3 with SalI and ScaI enzyme, Omiga DNA glue reclaims the big fragment T of linearized vector that test kit reclaims Vp3 and gained ₄Dna ligase connects.

Vp3 6μl

The big fragment 2 μ l of linearized vector

10×buffer 1μl

T ₄Dna ligase 1 μ l

total?volume 10μl

Set up negative control simultaneously, 16 ℃ of connections of spending the night.

(4) transform

1). get the competent cell 150 μ l of prepared fresh under the sterile state, place the aseptic EP pipe of 1.5ml.

2). every pipe adds and connects product 10 μ l, ice bath 30min behind the mixing.

3) .42 ℃ of heat-shocked 2min.

4). every pipe adds the LB substratum of 800 μ l antibiotic-frees, 37 ℃ of gentle vibration 45min.

5). the centrifugal 2min of room temperature 5000g, stay 200 μ l supernatants and bacterial precipitation mixing after, evenly coat on the LB training fat flat board that contains Zeocin with aseptic elbow glass shop bacterium device.

(5) bacterium colony is identified

1). random choose transform bacterium colony several, be inoculated in the LB substratum that contains Zeocin concussion and cultivate.

2). prepare plasmid DNA in a small amount.

3). carry out with Sal I and ScaI restriction enzyme that enzyme is cut and the agarose gel electrophoresis analysis.

4). positive colony carries out the cDNA sequencing through key lab of the infectious diseases molecular biology the Ministry of Education of Medical University Of Chongqing automatic sequencer.

4. above-mentioned positive recombinant plasmid electricity is transformed into attenuation salmonella SL7207

(1) preparation of electric transformed competence colibacillus

With sterilization toothpick picking mono-clonal attenuation salmonella bacterium colony, put in the centrifuge tube that fills the 3mlLB liquid nutrient medium.37 ℃, 300rpm cultivated 14-16 hour.Second day, 1ml bacterium liquid is poured in the 100ml LB liquid nutrient medium into 37 ℃ with 1: 100 ratio, the 300rpm jolting, when the OD value reaches 0.5, with bacterium liquid precooling on ice 50 minutes, subsequently bacterium liquid branch is installed in the centrifuge tube of 50ml precooling, 4 ℃, the centrifugal 10min of 4000rpm.Abandon supernatant, the sterile glycerol washing of 10% ice precooling, 4 ℃, the centrifugal 10min of 4000rpm.Repeat for several times.Abandon supernatant, add after a small amount of 10% precooling sterile glycerol suspends precipitation-80 ℃ of preservations.

(2) electricity transforms

Get recombinant plasmid behind the 5 μ g purifying in the centrifuge tube of 1.5ml, the pole cup of itself and 0.1CM is placed precooling on ice together.The competent cell that 100ul is thawed shifts in the centrifuge tube of 1.5ml so far, and careful mixing is placed 10min on ice.Adopt the BioRAD electroporation, regulating voltage is 2.5kV, 25 μ F, 200 Ω.Mixture is transferred to electric shock back in the pole cup of precooling and adds the LB liquid nutrient medium of 1mL rapidly, transfer to behind the re-suspended cell in the centrifuge tube of 20ml.37 ℃, 200rpm recovery 1h.Get 200ul LZ coated plate, 37 ℃ of incubated overnight are checked conversion results next day.

(3). carry the evaluation of the attenuation Salmonella SL7207 bacterial strain of eukaryon expression plasmid pBud-Vp3-TRAIL-EGFP

1) extracts plasmid DNA and carry out restricted enzyme cutting spectrum analysis, determine whether contain VP3 and trail dna in the plasmid.

2) be template with the pBud-VP3-TRAIL-EGFP plasmid, add that vp3 upstream, downstream primer carry out pcr amplification, its product if the DNA band of 375bp occurs, proves and contains the VP3 gene in the plasmid when agarose gel electrophoresis; With the pBud-VP3-TRAIL-EGFP plasmid is template, adds that TRAIL upstream, downstream primer carry out pcr amplification, and its product if the DNA band of 525bp occurs, proves and contains trail dna in the plasmid when agarose gel electrophoresis.

Embodiment 2

Present embodiment has been described the restraining effect of attenuation salmonella SL7207 provided by the invention strain (pBud-Vp3-TRAIL-EGFP/SL7207) to growth of tumour cell, and may further comprise the steps:

Observation index:

1.SL7207/pBud-Vp3-TRAIL the experimental study of bacterial strain inducing apoptosis in gastric cancer

1) cell growth state detects and adopts mtt assay to detect stomach cancer cell after the effect of the pBud-Vp3-TRAIL-EGFP/SL7207 of different concns different time bacterial strain expression product, the variation of cell growth state.

The cell of taking the logarithm vegetative period with 0.25 trypsinase and 0.02%EDTA digestion, is made single cell suspension, with 5.0 * 10 ⁴/ ml concentration is inoculated in 96 orifice plates, every hole 200 μ l, and experiment divides 3 groups, and promptly control group, empty bacterium are organized (SL7207), experimental group (pBud-vp3-TRAIL-EGFP/SL7207), empty bacterium group and experimental group each minute 5 dosage group (Salmonellas number/cell count=200/1; 100/1; 50/1; 25/1; 5/1), establishes 4 the blank zeroing of multiple Kong Bingshe holes (except that not adding the extracellular, surplus processing is respectively organized identical with other) for every group.Cell is placed 37 ℃, 5%CO ₂, 95% humidity incubator in cultivate 24h, treat that cell enters logarithmic phase, discarding former substratum washes twice with PBS liquid, add the processing factor respectively by above-mentioned grouping then, put in the incubator and cultivate, take out culture plate respectively in 24h, 48h and 72h, add MTT20 μ l to every hole, stop cultivating after continuing to cultivate 4h, the careful suction removed supernatant liquor in the hole, every hole adds DMSO 150 μ l, at micro-oscillator concussion 15min, crystallisate is fully dissolved read each hole optical density value (OD value) afterwards at enzyme-linked immunosorbent assay instrument 490nm place.

Oncocyte inhibiting rate=blank group average optical density value one experimental group average optical density value/blank group average optical density value.

Behind attenuation salmonella invasion and attack stomach cancer cell 24h, the attenuation salmonella invasion and attack back cell growth of finding different ratios has different influences, and along with the increasing progressively of invasion and attack ratio, the cell growth-inhibiting is obvious more, ratio is 50/1,25/1 and 5/1 not have significant difference.Behind the 24h between empty bacterium group and experimental group (200/1 and 100/1) and stomach cancer cell control group the OD value marked difference is arranged, can significantly suppress stomach cancer cell and grow, but respectively between 200/1 and 100/1 each ratio group the stomach cancer cell growth-inhibiting not had difference.Experimental group and empty bacterium are organized between each ratio group OD value behind 48h and the 72h marked difference.The above results explanation attenuation salmonella is influential to the stomach cancer cell growth, TRAIL and VP3 genetic expression albumen also do not present apoptosis effect when 24h, begin to bring into play apoptosis-induced effect behind the 48h, TRAIL and the effect of VP3 combined induction apoptosis are better than the apoptotic effect of both independent pair cells.

Mtt assay detects cytotoxicity, can be used as the dosage foundation of follow-up apoptosis experiment.Experimental result shows that the activity of various dose pair cell all has restraining effect, with control group relatively, difference all has significance (P＜0.05) (Fig. 7), therefore, chooses 50/1 concentration and carry out apoptosis research in subsequent experimental.

2) the dna fragmentation detection adopts TUNEL apoptosis detection kit (Roche company) to measure the fragmentation of DNA in the stomach cancer cell of the above-mentioned processing of process.

Reference reagent box specification sheets carries out:

(1) with the fixing 1h of 4% Paraformaldehyde 96, with PBS damping fluid rinsing 3 times; (2) add 3%H ₂O ₂20 ℃ of effects of blocking-up liquid 10min, PBS rinsing 3 times; (3) add penetrating fluid (0.1%TritonX-100 sodium citrate buffer solution) in 4 ℃ of effect 20min, PBS rinsing 3 times; (4) add 50 μ L TUNEL reaction mixtures, add mulch film, 37 ℃ of effect 1h in the wet box, PBS rinsing 3 times; (5) with 37 ℃ of sealings of 3% bSA (BSA) 20min, PBS rinsing 3 times; (6) add 50 μ L conversion POD solution, add mulch film, be placed on 37 ℃ of effect 30min in the wet box, PBS rinsing 3 times; (7) add 50 μ L DAB substrates, lucifuge colour developing 10min in 15 ℃～25 ℃ wet boxes, PBS rinsing; (8) dimethylbenzene is transparent, mounting, and microscopically is observed.

See the obvious pyknosis of apoptotic cell under the TUNEL dyeing light microscopic, nuclear is fine and close, and engrain is that pale brown look, multi-blade or crescent particle often are collected at the nuclear periphery.It is brown xanchromatic apoptotic cell by engrain that control group has a little nucleus.The results are shown in (Fig. 8), experimental group is compared with cellular control unit, and difference is (P＜0.05) extremely significantly.

3) adopt the stomach cancer cell of laser co-focusing fluorescent microscope detection after above-mentioned processing to have or not the morphological change of apoptosis.

Laser scanning co-focusing microscope is observed the plain localization and expression of visible apoptosis in nucleus, and the parts of fine karyon breaks, be rendered as differ in size, the irregular punctate fluorescence of form or plum blossom shape karyomorphism.And can see apoptotic body and cellular swelling, volume increases, ill-defined non-viable non-apoptotic cell.(seeing Fig. 6 F-G)

4) adopt AO-EB dyeing to detect the apoptosis rate of the stomach cancer cell after above-mentioned processing.

The cell in vegetative period of taking the logarithm is used 0.25 tryptic digestion.The RPMI1604 nutrient solution that contains 10FCS is made into the individual cells suspension, and (final concentration is 5 * 10 in 6 well culture plates in application of sample 1ml/ every hole ⁴/ ml) cell is put into the incubator cultivation.Add behind the 24h and mix fluorescent dye liquid at 1: 1: 100 μ g/ml bifurcation heavy stone used as an anchor oranges (AO) and 100 μ g/ml smell second pyridine (EB) mixing, press cover glass.Observing apoptosis cellular form under the fluorescent microscope.The dead cell (NVN) of difference living cell counting (VN), viable apoptotic cell (VA), non-viable apoptotic cell (NVA) and non-apoptosis, repeat count is got its mean value.Calculate apoptosis rate:

Apoptosis rate (%)=(VA+NVA)/(VA+NVA+VN+NVN) * 100%

Cell is through AO, EB double staining, and fluorescent microscope is observed down, and negative control group is based on normal cell, and it is complete to show as cellularstructure, and size is normal.Cell is the green of uniformity by AO dyeing, and can see a small amount of viable apoptotic cell, shows as cell dyeing and strengthens, and fluorescence is hyperfunction, and cell is colored and is yellow-green colour one yellow fluorescence.Visible viable apoptotic cell increases in the experimental group, showing as nucleus is that AO dyeing is yellow-green fluorescence, densely gather into crescent or particulate state, be positioned at a side of cell, and visible a large amount of non-viable apoptotic cell, showing as cell and be orange by EB dyeing, cell volume dwindles, the parts of fine karyon breaks, be rendered as differ in size, the irregular fluorescent dye fragment of form or plum blossom shape karyomorphism.And can see apoptotic body and cellular swelling, and volume increases, and edge blurry is dyed the non-viable non-apoptotic cell of orange, and cell volume increases, and is uneven fluorescent red-orange, and profile is unclear, has disintegrated or near disintegration (seeing Fig. 6 H).The apoptosis rate of undressed BGC-823 is 2%, and apoptosis rate is respectively 26.63% (P＜0.05) and 49.69% (P＜0.05) after SL7207 and pBud-Vp3-TRAIL-EGFP/SL7207 processing.

2. bearing mouse model is set up

In Bechtop, aseptic technique is 1 * 10 with the SGC7901 dilution ⁷Individual/the ml cell suspension, in the aseptic subcutaneous vaccination of back, BALB/C nude mice right side row, inoculum size 0.2ml cell suspension, total cellular score is 2 * 10 ⁶/.Observe its inoculation position behind the mouse inoculation, inoculation back 14d mouse right hind thigh portion forms obvious lump, is divided into three groups at random, indifference between group: A group: 4 pBud-Vp3-TRAIL-EGFP/SL7207 groups; B group: 4 empty bacterium groups of SL7207; The C group: 4, the PBS group.Inject above-mentioned treatment soln 0.2ml in the knurl piece respectively.

Observe the growth conditions of every mouse tumor piece, per 3 days with tumour size of slide calliper rule records, gross tumor volume by formula: V=L * W * W/2 (L is the major diameter of tumour, w for and L the wideest vertical footpath) calculate.

Tumor control rate=(the average knurl volume of the average knurl volume-experimental group of the control group)/average knurl volume of control group * 100%

3.SL7207/pBud-Vp3-TRAIL-EGFP bacterial strain is to the restraining effect research of tumor-bearing mice knurl bulk-growth

Carry out following experiment respectively, to observe the restraining effect of SL7207/pBud-Vp3-TRAIL-EGFP bacterial strain to tumour.

1) 4 weeks back execution mouse peels off the knurl body, detects the size and the weight of each experimental mice knurl body;

2) expression of Vp3 and TRAIL in the RT-PCR detection cancerous tissue

3) protein expression of Weatern Blot and immunohistochemical analysis Vp3 and TRAIL

4) pathological examination: get liver,kidney,spleen, lung, stomach 10% formaldehyde fixed of mouse, the routine paraffin wax embedding, HE dyeing, opticmicroscope is done the pathology inspection.The concrete grammar of above-mentioned anti-tumor in vivo effect research sees bureau of drug administration of the Ministry of Health for details compiles " new drug preclinical study governing principle ".

Observed result:

Suppressing growth of tumor speed or tumour knurl body is dwindled, is the assessment antitumor drug to an oncotherapy effective important indicator whether.Test shows that there are obvious restraining effect in SL7207 and SL7207/pBud-Vp3-TRAIL-EGFP bacterial strain to the growth of mouse transplanting tumor.Mouse general signs, behavior, activity, color and luster show no obvious abnormalities; Diet, ight soil show no obvious abnormalities; The morphological structure of all internal organs there is no tangible pathological change.(see figure 9)

The nucleotides sequence tabulation that the present invention is relevant

1.apoptin(Vp3)

atgaacgctc?tccaagaaga?tactccaccc?ggaccatcaa?cggtgttcag?gccaccaaca 60

agttcacggc?cgttggaaac?ccctcactgc?agagagatcc?ggattggtat?cgctggaatt?120

acaatcactc?tatcgctgtg?tggctgcgcg?aatgctcgcg?ctcccacgct?aagatctgca?180

actgcggaca?attcagaaag?cactggtttc?aagaatgtgc?cggacttgag?gaccgatcaa?240

cccaagcctc?cctcgaagaa?gcgatcctgc?gacccctccg?agtacagggt?aagcgagcta?300

aaagaaagct?tgattaccac?tactcccagc?cgaccccgaa?ccgcaagaag?gcgtataaga?360

ctg 363

2.tumor?necrosis?factor-related?apoptosis?inducing?ligand(TRAIL)

atgctagtga?gagaaagagg?tcctcagaga?gtagcagctc?acataactgg?gaccagagga 60

agaagcaaca?cattgtcttct?ccaaactcc?aagaatgaaa?aggctctggg?ccgcaaaata?120

aactcctggg?aatcatcaag?gagtgggcat?tcattcctga?gcaacttgca?cttgaggaat?180

ggtgaactgg?tcatccatga?aaaagggttt?tactacatctat?tcccaaaca?tactttcga?240

tttcaggagg?aaataaaaga?aaacacaaag?aacgacaaac?aaatggtcca?atatatttac?300

aaatacacaa?gttatcctga?ccctatattg?ttgatgaaaa?gtgctagaaa?tagttgttgg?360

tctaaagatg?cagaatatgg?actctattcc?atctatcaag?ggggaatatt?tgagcttaag?420

gaaaatgaca?gaatttttgt?ttctgtaaca?aatgagcact?tgatagacat?ggaccatgaa?480

gccagttttt?tcggggcctt?tttagttggc?taa 513

3.EGFP

ggatccccgg?gtaccggtcg?ccaccatggt?gagcaagggc?gaggagctgt?tcaccggggt 60

ggtgcccatc?ctggtcgagc?tggacggcga?cgtaaacggc?cacaagttca?gcgtgtccgg?120

cgagggcgag?ggcgatgcca?cctacggcaa?gctgaccctg?aagttcatct?gcaccaccgg?180

caagctgccc?gtgccctggc?ccaccctcgt?gaccaccctg?acctacggcg?tgcagtgctt?240

cagccgctac?cccgaccaca?tgaagcagca?cgacttcttc?aagtccgcca?tgcccgaagg?300

ctacgtccag?gagcgcacca?tcttcttcaa?ggacgacggc?aactacaaga?cccgcgccga?360

ggtgaagttc?gagggcgaca?ccctggtgaa?ccgcatcgag?ctgaagggca?tcgacttcaa?420

ggaggacggc?aacatcctgg?ggcacaagct?ggagtacaac?tacaacagcc?acaacgtcta?480

tatcatggcc?gacaagcaga?agaacggcat?caaggtgaac?ttcaagatcc?gccacaacat?540

cgaggacggc?agcgtgcagc?tcgccgacca?ctaccagcag?aacaccccca?tcggcgacgg?600

ccccgtgctg?ctgcccgaca?accactacct?gagcacccag?tccgccctga?gcaaagaccc?660

caacgagaag?cgcgatcaca?tggtcctgct?ggagttcgtg?accgccgccg?ggatcactct?720

cggcatggac?gagctgtaca?agtaaagcgg?ccgcgac 757

The amplification trail dna dna upstream primer sequence (P1)

gccctcgaga?tgctagtgag?agaaagaggt?c 31

The amplification trail dna DNA downstream primer sequence (P2)

gccagatct?tttccagg?tcag?tagcca 28

6. the dna upstream primer sequence (P3) of amplification VP3 gene

gccgtcgaca?atgaacgctc?tccaagaa 28

7. the DNA downstream primer sequence (P4) of amplification VP3 gene

gccagtactc?agtcttatac?gccttct 27

Claims (2)

1, a kind of attenuation salmonella vaccine of taking the double expression plasmid of Vp3 and TRAIL is characterized in that adopting following steps to make up:

(1) structure of .pBud-EGFP

XbaI enzyme cutting pEGFP and pBudCE4.1 reclaim T behind the EGFP fragment of 757bp and the big fragment pBudCE4.1 of linearized vector respectively ₄Dna ligase connects, and transformed competence colibacillus cell Jm109 screens positive recombinant plasmid, through enzyme cut, pcr amplification and order-checking obtain pBud-EGFP after identifying;

(2) structure of .pBud-TRAIL-EGFP

A, design primer P1 and P2:

P1：5’-GCCCTCGAG-ATGCTAGTGAGAGAAAGAGGTC-3’(XhoI)

P?2：5’-GCCAGATCT-TTTCCAGGTCAGTTAGCCA-3’(BglII)

B, extraction human peripheral lymphocytes RNA change into cDNA, for touching plate, use the amino acid whose gene order 525bp of coding extracellular region 112-287 in primer P1 and the P2 amplification people trail dna with cDNA;

C, cut pBud-EGFP and TRAIL with XhoI and BglII enzyme, glue reclaims the big fragment of linearized vector pBud-EGFP of TRAIL and gained, T ₄Dna ligase connects, and transformed competence colibacillus cell Jm109 screens positive recombinant plasmid, through enzyme cut, pcr amplification and order-checking obtain pBud-TRAIL-EGFP after identifying;

(3) structure of .pBud-Vp3-TRAIL-EGFP

A, design primer P3 and P4

P3：5’-GCCGTCGAC-AATGAACGCTCTCCAAGAA-3’(SalI)

P4：5’-GCCAGTACT-CAGTCTTATACGCCTTCT-3’(ScaI)

B, be template with pMD18-T/VPX, with primer P3 and P4 amplification apoptin full length sequence;

C, cut pBud-TRAIL-EGFP and Vp3 with SalI and ScaI enzyme, glue reclaims the big fragment of linearized vector pBud-TRAIL-EGFP of Vp3 and gained, T ₄Dna ligase connects, and transformed competence colibacillus cell Jm109 screens positive recombinant plasmid, through enzyme cut, pcr amplification and order-checking obtain pBud-vp3-TRAIL-EGFP after identifying;

(4). pBud-vp3-TRAIL-EGFP recombinant plasmid electricity is transformed into attenuation salmonella SL7207

A, the electric transformed competence colibacillus SL7207 of preparation

After B, electricity transform, the positive reorganization of screening bacterium, through enzyme cut, pcr amplification identifies and obtains the attenuation salmonella vaccine that pBud-Vp3-TRAIL-EGFP/SL7207 is the present invention's double expression plasmid of taking Vp3 and TRAIL.

2, the described application of attenuation salmonella vaccine in the gene therapy medicament of preparation treatment or prophylaxis of tumours of taking the double expression plasmid of Vp3 and TRAIL of claim 1.

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