CN104388398A - Melittin gene-carrying oncolytic adenovirus with HREAFP as promoter and application of oncolytic adenovirus - Google Patents

Abstract

The invention relates to a Melittin gene-carrying oncolytic adenovirus with HREAFP as a promoter. The oncolytic adenovirus, with the HREAFP as the promoter, carries the major late promoter (MLP)-controlled Melittin gene. The oncolytic adenovirus has the advantages that AFP (Alpha Fetoprotein) positive liver cancer cells can be clearly targeted by the established oncolytic adenovirus which can be duplicated in the AFP positive liver cancer cells at high multiples, the oncolytic adenovirus carries a tumor inhibiting killer gene and the specific expression of the Melittin gene in the AFP positive liver cancer cells along with the duplication of viruses is realized, and therefore, a new method for treating liver cancer by use of the oncolytic adenovirus is developed; the recombinant proliferative adenovirus is capable of specifically inhibiting the proliferation of the AFP positive liver cancer cells; the advantages of virogene treatment are brought into full play, and a new idea is provided for treating solid tumors such as liver cancer.

Description

A kind of take HREAFP as the oncolytic adenovirus carrying Melittin gene and the application thereof of promotor

Technical field

The present invention relates to gene-virotherapy field, specifically, the oncolytic adenovirus carrying Melittin gene and the application thereof of promotor that to be a kind of with HREAFP be.

Background technology

Primary hepatocarcinoma is one of modal malignant tumour clinically, and occupy the 3rd of tumour associated death, current excision is still the prefered method of Hepatoma therapy, but recurrence of PHC rate is high, 5 years recurrence rates about 60% ~ 70%.The methods for the treatment of of other liver cancer also has TACE art, PEI art, radio-frequency ablation procedure, chemicotherapy etc., but result for the treatment of is not satisfactory, current liver cancer treatment there is no the medicine of positive effect, and therefore finding better medicine and treatment means has become the significant problem that liver cancer treatment faces.

Gene-virus treatment is a kind of oncotherapy New Policy in conjunction with viral therapy and gene therapy double dominant.Adenovirus is the conventional carrier of gene therapy, and have that toxicity is low, security is high, host range is wide and the advantage such as inside and outside Absorbable organic halogens existence, but adenovirus infection efficiency is low, targeting is poor, limits it and further applies.The replication competent adenovirus of current restructuring becomes the focus of research with the advantage of its uniqueness, application in gene-virus treatment is more and more extensive, also known as oncolytic adenovirus, there is infection, copy, breed and the characteristic of dissolved cell, it can utilize the difference of structure and pathways metabolism between tumour and normal tissue cell, targeting in tumour cell, copy propagation, final cracking tumour cell, simultaneously can the antineoplastic foreign gene of long-term express, be a kind of oncotherapy New Policy in conjunction with viral therapy and gene therapy double dominant.Therefore, how to improve the targeting of oncolytic virus, and find the focus that antineoplastic foreign gene is research.

The key of oncolytic adenovirus is the propagation of selectivity targeting in tumour cell, these can by artificial deleted virus copy in normal cell institute required, and gene nonessential in tumour cell, as P53, or adopt tumour or tissue-specific promoter to regulate and control gene needed for virus replication, make virus can only copy propagation in the tumor tissues of particular type or cell, as anoxic promotor, telomerase promoter, alpha-fetoprotein (AFP) promotor of liver cancer, carcinomebryonic antigen (CEA) promotor etc. of gastroenteric tumor.

The polypeptide that melittin is made up of 26 amino-acid residues, its encoding gene only has 78bp.Have antibacterial, anti-inflammatory, radioprotective, arthritis effect and the effect such as cardiovascular, its effect in therapeutic field of tumor has been subject to great attention.We have confirmed that the high amount of stopping of melittin can killing hepatoma cell, and have restraining effect to transplanted tumor in nude mice.But have the side effect of haemolysis, safe-dosaging limits is little, and its lethal effect does not have selectivity.

Summary of the invention

The object of the invention is for deficiency of the prior art, providing a kind of take HREAFP as the oncolytic adenovirus carrying Melittin gene of promotor.

Of the present invention again one object be that providing a kind of take HREAFP as the preparation method carrying the oncolytic adenovirus of Melittin gene of promotor.

Another object of the present invention is, providing a kind of take HREAFP as the application of carrying the oncolytic adenovirus of Melittin gene of promotor.

For realizing above-mentioned first object, the technical scheme that the present invention takes is: a kind of take HREAFP as the oncolytic adenovirus carrying Melittin gene of promotor, described oncolytic adenovirus is promotor with HREAFP, takes the oncolytic adenovirus that late promoter MLP regulates and controls peptide in bee venom.

Preferably, described oncolytic adenovirus is for carrier with 11 type adenovirus, build and jointly regulate and control Ela district with hypoxia response elements HRE promotor and afp promoter AFP hybrid promoter, Elb district disappearance 55KDa, carries the oncolytic adenovirus being regulated and controled Melittin gene by late promoter MLP.

Preferably, the preparation method of described oncolytic adenovirus is: the plasmid pENTR-MLP-Melittin and adenoviral backbone plasmid pPE3-F11B that carry Melittin gene are recombinated by LR, build plasmid pPE3-F11B-MLP-Melittin, by itself and the common transfection human embryo kidney 293 cells of plasmid PXC20-△ Elb-55kda-HREAFP containing hybrid promoter HREAFP, obtain regulating and controlling Ela district by HREAFP, the restructuring replication competent adenovirus taking Melittin gene of Elb district disappearance 55kda albumen.

For realizing above-mentioned second object, the technical scheme that the present invention takes is: described preparation method is: the plasmid pENTR-MLP-Melittin and adenoviral backbone plasmid pPE3-F11B that carry Melittin gene are recombinated by LR, build plasmid pPE3-F11B-MLP-Melittin, by itself and the common transfection human embryo kidney 293 cells of plasmid PXC20-△ Elb-55kda-HREAFP containing hybrid promoter HREAFP, obtain regulating and controlling Ela district by HREAFP, the restructuring replication competent adenovirus taking Melittin gene of Elb district disappearance 55kda albumen.

For realizing above-mentioned 3rd object, the technical scheme that the present invention takes is: the application of described oncolytic adenovirus in the medicine of preparation treatment tumour.

Preferably, described tumour is liver cancer.

Preferably, described liver cancer is the liver cancer of alph-fetoprotein positive.

The invention has the advantages that:

1, the oncolytic adenovirus QG511-HA-Melittin of the present invention's structure, one is the liver cancer cell can specifying the target AFP positive, two is that high multiple copies in the liver cancer cell of the AFP positive, three be carry there is Tumor suppression kill and wound gene, embody the superposition of three aspect factors, achieving Melittin gene specific expressed with copying of virus in the liver cancer cell of the AFP positive, is the new tool of Hepatoma therapy;

2, this restructuring replication competent adenovirus energy specificity is bred in the liver cancer cell of alph-fetoprotein positive, then affects not quite, and can suppress the hepatoma cell proliferation of alph-fetoprotein positive specifically to Examination For The Diagnosis of Afp Negative Hepatoma cell and normal liver cell;

3, given full play to the advantage of gene-virus therapy, the treatment for noumenal tumours such as liver cancer provides a kind of new thinking.

Accompanying drawing explanation

Fig. 1 .pclon7-MLP carrier enzyme cuts qualification figure (ScaI)

M：Lambda DNA/EcoRI+HindIII(NEB)

pclon7-MLP(ScaI):1440bp+2183bp

No. 3, No. 4, No. 5 clones cut out corresponding fragment.

Fig. 2 .Pclon7-MLP carrier enzyme cuts qualification figure (AgeI+EcoRl)

M：Lambda DNA/EcoRI+HindIII(NEB)

Pclon7-MLP(AgeI+EcoRI)：470bp+3153bp

1-5 clone cuts out corresponding fragment

According to the result obtained above, select enzyme to cut correct clone No. 5 and protect bacterium.

Fig. 3 .Pclon7-MLP-Melittin plasmid enzyme restriction qualification figure (PVUII)

M1：Lambda DNA/EcoRI+HindIII(NEB)

M2:100bp ladder(NEB)

Pclon7-mlp-melittin (PVUII): 242bp+430bp+526bp+2513bp, No. 1, No. 2, No. 3 cut out respective segments.

The enzyme of Fig. 4 .pclon7-MLP-Melittin plasmid cuts qualification figure (AgeI+NotI)

M1：Lambda DNA/EcoRI+HindIII(NEB)

M2：l00bp ladder(NEB)

Pclon7-mlp-melittin (AgeI+Notl): 828bp+2883bp, No. l, No. 2, No. 3 cut out respective segments.No. 3 are selected to protect bacterium.

Fig. 5 .pENTR-MLP-Melittin plasmid enzyme restriction qualification figure (PVUII)

M1：l00bp ladder(NEB)

M2：Lambda DNA/EcoRI+HindIII(NEB)

PENTR-MLP-Melittin (PVUII): 242bp+422bp+2483bp, l, 2,4,8, No. 9 cut out respective segments.

Fig. 6 .pENTR-MLP-Melittin plasmid enzyme restriction qualification figure (XhoI)

M1：l00bp ladder(NEB)

M2：Lambda DNA/EcoRI+HindIII(NEB)

PENTR-MLP-Melittin (XhoI): 141bp+441bp+2565bp, 1,2,4,8, No. 9 cuts out respective segments, choose No. 8 protect bacterium, in take out.

Fig. 7 .pPE3-F11B-MLP-Melittin plasmid enzyme restriction qualification figure (HindIII, AgeI, XbaI+PacI, NotI, BamHI)

MI：Lambda DNA/EcoRI+HindIII(NEB)

M2：100bp ladder(NEB)

HINDIII 75bp+1522bp+2081bp+2791bp+3892bp+3983bp+4360bp+4597bp+5322bp+8010bp

AgeI 335bp+593bp+784bp+2755bp+3734bp+4046bp+4379bp+6309bp+13698bp

Xbal+PacI 338bp+604bp+1007bp+12715bP+5697bp+27716bp

NotI 326bp+452b+960bp+1931bp+2589bp+4999bp+12200bp+13176bp

BamHI 265bp+401bp+1741bp+5171bp+8381bp+20674bp

Through identifying that this clone is correct, this clone is selected to take out full-page proof.

Fig. 8 .J47-Δ E1b-55kda plasmid enzyme restriction qualification figure (AgeI+Bg1II)

MI：100bp ladder(NEB)

M2：Lambda DNA/EcoRI+HindIII(NEB)

J47-Δ E1b-55kda (AgeI+Bg1II): 1832bp+2940bp, 1,2,3, No. 4 cuts out respective segments.

Fig. 9 .J47-Δ E1b-55kda plasmid enzyme restriction qualification figure (XbaI+ScaI)

M1：Lambda DNA/EcoRI+HindIII(NEB)

M2：100bp ladder(NEB)

J47-ΔE1b-55kda(XbaI+ScaI)：1058bp+1795bp+919bp

L, 2,3,4,5,6,7, No. 8 cut out respective segments

Through qualification I, 2,3, No. 4 correct, select No. 2 J47-Δ E1b-55kda to send handsome company check order, order-checking is correctly.

Figure 10 .pXC20-Δ E1b-55kda plasmid enzyme restriction qualification figure (HindIII+XbaI)

M1：100bp ladder(NEB)

M2：Lambda DNA/EcoRl+HindIII(NEB)

PXC20-Δ E1b-55kda-HREAFP (HindIII+XbaI): 1712bP+7235bp, No. 6 cut out respective segments, select No. 6 continuation PVUII to identify.

Figure 11 .pXC20-Δ E1b-55kda-HREAFP plasmid enzyme restriction qualification figure (PVUII)

M1：100bp ladder(NEB)

M2：Lambda DNA/EcoRI+HindIII(NEB)

PXC20-Δ E1b-55kda-HREAFP (PVUII): 27bp+76bp+610bp+908bp+2226bp+2388bp+3080bp, l, 2, No. 3 cut out respective segments.

Figure 12 .pXC20-Δ E1b-55kda-HREAFP plasmid enzyme restriction qualification figure (PstI)

MI：100bp ladder(NEB)

M2：Lambda DNA/EcoRI+HindIII(NEB)

PXC20-Δ E1b-55kda-HREAFP (PstI): 27bp+430bp+733bp+996bp+3422bp+3772bp, 1,2, No. 3 cuts out respective segments.

Figure 13 .pXC20-Δ E1b-55kda-HREAFP plasmid enzyme restriction qualification figure (SacI+SpeI)

M1：Lambda DNA/EcoRI+HindIII(NEB)

M2：100bp ladder(NEB)

pXC20-ΔE1b-55kda-HREAFP(SacI+SpeI)：122bp+916bp+1287bp+2003bp+5052bp

1,2, No. 3 cut out respective segments

Through identify 1,2, No. 3 all correct.

Figure 14 .QG511-HA-Melittin viruses indentification figure (MLP-Melittin): PCR identifies whether QG511-HA-Melittin carries MLP-Melittin gene in virus

M1：Lambda DNA/EcoRI+HindIII(NEB)

M2：100bp ladder(NEB)

N: negative control; P: positive control (pPE3-F11b-MLP-Melittin plasmid);

1-4:QG511-HA-Melittin virus.

Whether containing 11 type Fiber in Figure 15 .QG511-HA-Melittin viruses indentification figure (Fiber 11): PCR qualification QG511-HA-Melittin virus, if containing promising 728bp

M：l00bp DNA Ladder；

N: negative control; P: positive control (pPE3-F11b-MLP-Melittin plasmid);

1-4:QG511-HA-Melittin virus.

Figure 16 .QG511-HA-Melittin viruses indentification figure (HRE): PCR identifies whether QG511-HA-Melittin carries HRE gene in virus, if containing promising 166bp

M1：100bp DNA Ladder；

N: negative control; P: positive control (J47-HREAFP plasmid);

1-4:QG511-HA-Melittin virus.

Whether Figure 17 .QG511-HA-Melittin viruses indentification figure (AFP): PCR identifies containing AFP in QG511-HA-Melittin virus, if containing promising 269bp

M1：100bp DNA Ladder；

N: negative control; P: positive control (J47-HREAFP plasmid);

1-4:QG511-HA-Melittin virus.

Whether containing △ E1B55Kda in Figure 18 .QG511-HA-Melittin viruses indentification figure (△ E1B55Kda): PCR qualification QG511-HA-Melittin virus, if containing promising 1832bp

M1：Lambda DNA/EcoRI+HindIII(NEB)M2：100bp ladder(NEB)

N: negative control; P: positive control (pXCl plasmid);

1-4:QG511-HA-Melittin virus.

Figure 19. take peptide in bee venom restructuring replication competent adenovirus QG511-HA-Melittin schematic diagram.

Figure 20 .QG511-HA-Melittin infects proliferative conditions after different cell.

Figure 21. the QG511-HA-Melittin of different MOI is to the suppression situation of hepatoma cell proliferation.

Figure 22. the knurl weight of each group Hep3B tumor bearing nude mice.

Figure 23. each group nude mouse tumor pathological section.

Embodiment

Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.

Object: the present invention with 11 type adenovirus for carrier, build and jointly regulate and control Ela district with hypoxia response elements (HRE) promotor and afp promoter (AFP) hybrid promoter, Elb district disappearance 55KDa, carry the oncolytic adenovirus being regulated and controled Melittin gene by late promoter MLP, observe the suppression of its propagation in the liver cancer cell of the AFP positive and the tumor growth to lotus people knurl nude mice thereof.

Method: common molecular clone and viral Packaging Method build HREAFP hybrid promoter and jointly regulate and control Ela district, Elb district disappearance 55KDa, carries by 11 type oncolytic adenovirus QG511-HA-Melittin of late promoter MLP regulation and control Melittin gene and the contrast virus QG511-HA not carrying Melittin gene.After gene sequencing, round pcr qualification correctly, amplicon virus, cesium chloride density gradient centrifugation purifying, and measure virus titer by TCID50 method.This virus of virus multiplication experimental observation is at alph-fetoprotein positive liver cancer cell, Examination For The Diagnosis of Afp Negative Hepatoma cell and normal liver cell infection, proliferative conditions.MTT experiment observes the proliferation inhibiting effect of this Adenovirus on Human alph-fetoprotein positive liver cancer cell, Examination For The Diagnosis of Afp Negative Hepatoma cell and Normal Human Liver cell.For lotus Hep3B cells Nude Mouse Model after security authentication in Hartley cavy body, with the viral QG511-HA group of zero load for contrast, observe its suppression situation to tumour.

Result: successfully building with HREAFP is promotor, take oncolytic adenovirus QG511-HA-Melittin and the viral QG511-HA of contrast that late promoter MLP regulates and controls peptide in bee venom, in alph-fetoprotein positive Hep3B liver cancer cell, Examination For The Diagnosis of Afp Negative Hepatoma cell SSMC-7721, human normal liver cell L 02, the proliferation times of 48h is respectively 12800 times, 130 times and 19.8 times.MTT experiment shows that it has obvious lethal effect to Hep3B, as compared to SMMC-7721 with L02 significant difference (p<0.05).After 9 days are intervened to Hep3B cells tumor-bearing mice, tumour inhibiting rate is 62%, extend nude mice lifetime, difference has statistical significance (P<0.05) compared with physiological saline group, compared with QG511-HA group, difference not obvious (P>0.05), obviously alleviates the pathological change of tumor tissues.

Conclusion: take HREAFP as the propagation that oncolytic adenovirus that promotor carries MLP promotor peptide in bee venom in late period effectively can suppress the liver cancer cell vivo and vitro of the AFP positive.

Embodiment

One, method

1 cell cultures: HEK293 cell strain, the hepatoma cell strains such as Huh7 are from cell institute of the Shanghai City Chinese Academy of Sciences.Each cell routine is cultivated.

The construction process of 2plasmid and plasmid thereof

Wild adenoviral backbone plasmid pXCl is from Canada Company MICROBIX, and adenoviral backbone plasmid pPE3-F11B, plasmid pXC20, plasmid pclon7-MLP-p53, plasmid pclon7, plasmid pENTR11-linker, plasmid J47-1inker, PUC 19-AFP270,5 type adenovirus shuttle plasmid pSG502, the plasmid PMD-Melittin that carries Melittin gene provide by TakaRa company.

Pclon7-MLP-p53 obtains pclon7-MLP after AgeI+EcoRI enzyme is cut, then with obtain melttin through HindIII+XhoI double digestion and link, obtain plasmid pclon7-MLP-Melittin.Pclon7-MLP-Melittin, pENTR11-linker are all obtained plasmid pENTR-MLP-Melittin with link after AgeI+NotI double digestion, itself and pPE3-F11B-RC (+) are carried out the restructuring of LR technology, obtain plasmid pPE3-F11B-MLP-Melittin. and pclon7-MLP and pPE3-F11B-RC (+) is carried out the restructuring of LR technology, obtain plasmid pPE3-F11B-MLP.

PXC1 is that primer amplification goes out segment Δ E1b-55kda according to GT139 and GT141, to be connected to obtain J47-Δ E1b-55kda with J47-linker through the digestion products of AgeI+BelII.J47-Δ E1b-55kda and PXC20 is carried out connection after AgeI+BelII enzyme is cut and obtains PXC20-Δ E1b-55kda.Take PUCl9-AFP270 as template, amplify AFP segment with primer GT113 and GT114, after HindIII+BstBI enzyme is cut, insert the J47-1inker carrier that same enzyme cuts obtain J47-AFP; Application primer GTlll and GTll2, is template with SG502, amplifies HRE fragment, inserts the J47-linker that same enzyme cuts and obtain J47-HRE after HindIII+AgeI enzyme is cut.J47-AFP with J47-HRE is connected after HindIII+AgeI enzyme is cut and obtains J47-HREAFP.J47-HREAFP is cut through AgeI+BstBI enzyme, reclaim 442bp object band insert same enzyme cut after pXC20-Δ E1b-55kda carrier in obtain PXC20-Δ E1b-55kda-HREAFP.Plasmid construction flow process is as follows, and each primer sequence sees the following form 1.

(1) structure of pPE3-F11B-MLP-Melittin carrier

1. the enzyme of pclon7-MLP carrier cuts qualification

Plasmid pclon7-MLP-p53 AgeI+EcoRI enzyme is cut, obtains MLP (AgeI+EcoRI), then plasmid pclon7 AgeI+EcoRI enzyme is cut, the two is connected, obtains pclon7-MLP, then carry out enzyme and cut qualification.Scal enzyme is cut and is identified that object band is 1440bp+2183bp (Fig. 1).AgeI and EcoRI double digestion is identified, band is 470bp+3153bp (Fig. 2).

2. the enzyme of pclon7-MLP-Melittin cuts qualification

The plasmid PMD-Melittin synthesized in TakaRa company is carried out HindIII+Xhol double digestion, obtain melttin (HindIII+XhoI) fragment, be connected with pclon7-MLP (SalI+HindIII) digestion products, obtain pclon7-MLP-Melittin.PVUII single endonuclease digestion pclon7-MLP-Melittin (Fig. 3).AgeI and NotI double digestion is identified, obtains pclon7-MLP-Melittin (Fig. 4).

3. the qualification of pENTR-MLP-Melittin carrier

The correct pclon7-MLP-Melittin of qualification is carried out (AgcI+NofI) double digestion, obtains MLP-Melittin (AgeI+NotI) fragment and be connected with pENTR11-linker (AgeI+NotI) digestion products.PVUII enzyme cuts qualification pENTR-MLP-Melittin (Fig. 5).XhoI enzyme cuts qualification plasmid pENTR-MLP-Melittin (Fig. 6).

④pPE3-F11B-MLP-Melittin

Gateway (Invitrogen company) the technology restructuring of pENTR-MLP-Melittin and pPE3-F11b-Rc (+).PPE3-F11B-MLP-Melittin plasmid enzyme restriction qualification figure (Fig. 7).

(2) qualification of pXC20-Δ EIb-55kda-HREAFP270 carrier

1. the qualification of J47-Δ E1b-55kda carrier

Application primer GT139 and GT141, is template with PXC1, amplifies the object fragment of 1832bp, after enzyme is cut, insert J47-1inker.AgeI+Bg1II enzyme cuts qualification J47-Δ E1b-55kda (Fig. 8).XbaI+SeaI enzyme cuts qualification J47-Δ E1b-55kda (Fig. 9).

2. the qualification of pXC20-Δ Elb-55kda carrier

The correct J47-Δ E1b-55kda of qualification is carried out (AgeI+Bg1II) double digestion, obtain Δ Elb-55kda (AgeI+Bg1II) fragment, in pXC20 (AgeI+Bg1II) carrier that this fragment insertion same enzyme is cut, is obtained pXC20-△ E1b-55kda.HindIII+XbaI enzyme cuts qualification plasmid pXC20-△ E1b-55kda (Figure 10).PVUII enzyme cuts qualification plasmid pXC20-△ EIb-55kda-HREAFP (Figure 11).PstI enzyme cuts qualification plasmid pXC20-△ E1b-85kda-HREAFP (Figure 12).Fig. 3-5SacI+SpeI enzyme cuts qualification plasmid pXC20-△ E1b-55kda-HREAFP (Figure 13).

(3) restructuring of replication competent viral QG511-HA-Melittin and qualification

QG511-HA-Melittin adenovirus is recombinated successfully, extracting viral DNA, carries out PCR qualification to virus recombinant.

1. PCR identifies whether QG511-HA-Melittin carries MLP-Melittin gene (Figure 14) in virus.

2. whether PCR identifies containing 11 type Fiber in QG511-HA-Melittin virus, if containing promising 728bp (Figure 15), as Fig. 1-No. 4 adenovirus all can amplify 728bp band, consistent with positive control (P).Illustrate that 1-4 adenovirus type XI Fiber sequence exists.

3. PCR identifies whether QG511-HA-Melittin carries HRE gene in virus, if containing promising 166bp (Figure 16).As Fig. 1-No. 4 adenovirus all can amplify 166bp band, consistent with positive control (P).1-4 adenovirus is described all containing HRE gene.

4. whether PCR identifies containing AFP in QG511-HA-Melittin virus, if containing promising 269bp (Figure 17).As shown in the figure, 1-4 adenovirus all can amplify 269bp band, consistent with positive control (P).Illustrate that 1-4 adenovirus AFP sequence exists.

5. whether PCR identifies containing △ E1b-55kda in QG511-HA-Melittin virus, if containing promising 1832bp (Figure 18).As shown in the figure, 1-4 adenovirus all can amplify 1832bp band, consistent with positive control (P).Illustrate that 1-4 adenovirus △ E1b-55kda sequence exists.

The primer sequence that table 1 the present invention adopts

Primer numbers	Primer sequence
			GT111	CCC ACC GGT AGG CCT ACC AAG AGG ACC	SEQ ID NO.1

GT112	CCC AAG CTT GAG ATT AGT AGT CCA CAG TGC	SEQ ID NO.2
			GT113	CCC AAG CTT TAC GTA TGT TAT TGG CAG TGG	SEQ ID NO.3
GT114	CTG CAG AAT TCG AAT CGA GAT TTG TTA TAT TTG C	SEQ ID NO.4
			GT139	GAT CTC ACA GAC GCC CAG GA	SEQ ID NO.5
GT141	GGA AGA TCT TTA CCT AGCCTC CTC TGT AGC CTC	SEQ ID NO.6
			GT227	TCC TCC TCG TAT TAG AAA C	SEQ ID NO.7
GT228	CTA CTG TTG TCT CTT CCT	SEQ ID NO.8
			VT669	CAA CCA CAG GCG GAT CTC TAC	SEQ ID NO.9
VT670	GTT CCA GGA CCA AGT TAT ACG	SEQ ID NO.10

The structure of 3 oncolytic virus QG511-HA-Melittin and QG511-HA and qualification

Utilize Lipofectamine2000 test kit, by plasmid PXC20-Δ E1b-55kda-HREAFP and viral backbone carrier pPE3-F11B-MLP-Melittin or plasmid pPE3-F11B-MLP cotransfection to 293 cell not carrying Melittin.After transfection there is virus plaque in 9-14 days cells, and through 3 virus plaque purifying, application QIAamp DNABlood Mini Kit test kit to specifications step extracts adenovirus DNA.

Be used for detecting the existence of MLP-Melittin gene according to primer GT227, GT228, using pPE3-F11b-MLP-Melittin as positive control; Primer GT111, GT112 are used for detecting the existence of HRE promotor, with J47-AFP as positive control; Primer GT113, G114 are used for detecting the existence of AFP promotor, positive control J47-HREAFP; Primer (GT139, GT141) is used for detecting the existence of Δ E1b-55kda, positive control pXCl; Primer (VT669, VT670) be used for identifying Fiber, positive control pPE3-F11b-MLP-Melittin, namely to carry Melittin gene amplification 11 type adenovirus QG511-HA-Melittin consistent with framework result for called after, do not carry Melittin genetic contrast virus QG511-HA.Further, through gene sequencing qualification important feature (Melittin, Δ E1b-55kda, AFP, HER), namely qualification correctly carries out repeated amplification in 293 cells, and application cesium chloride density gradient centrifugation carries out purifying.Primer sequence is in table 1.

4 virus titers detect: get and be in logarithmic phase cell HepG2, Hep3B, SMMC-7721, L02, and digestion, spreads six orifice plates, 1 × 10 ⁵/ hole, 37 DEG C, 5%CO ₂incubator cultivates 24h, and use serum-free medium instead, add QG511-HA-Melittin respectively by MOI=5, cross method shakes up; Change containing 5% serum free culture system liquid after 2h; Collect cell strain and the supernatant liquor of virus infection Oh, 48h and 96h ,-80 DEG C frozen; TCID50 method detects virus titer.

5 cell proliferation detection methods: the cell collecting logarithmic phase, with 10% serum free culture system liquid, is mixed with individual cells suspension, spreads 96 orifice plates, 5 × 10 ³cells/well, 37 DEG C 5%, CO ₂cultivate 24 hours; Serum-free medium virus dilution, by MOI=0,0.1,0.5, l, 2,5,10,20,50,100, add viral 100ul/ hole, often organize 4 holes, 37 DEG C 5%, CO ₂cultivate 7 days; The impact of viral on cell proliferation is detected afterwards by tetrazolium salts colorimetric test (MTT).

6 laboratory animal and intervene grouping: BALB/C-nu/nu nude mice, 4-6 week age, body weight 18-20g, male, purchased from Shanghai Slac Experimental Animal Co., Ltd., animal conformity certification number: SCHG (Shanghai) 2007-0005.Sub-cage rearing under The 2nd Army Medical College animal experimental center is without special pathogenic bacteria (Specificpathogens free, SPF) condition, freely looks for food, drinks water.Hep3B cell is washed, counting adjustment cell count to 2 × 10 with serum-free MEM ⁷/ ml, gets 0.2ml cell suspension in nude mice left shoulder back of the body single subcutaneous injection, continues raising 2 weeks, set up model of nude mice bearing tumor.The one-tenth knurl nude mice of the most major diameter 5 ~ 8mm of tumour, is divided into three groups, QG511-HA-Melittin group, QG511-HA group and physiological saline group at random by it, often organize 8, inject QG511-HA-Melittin2 × 10 respectively ⁸pfu/0.2ml/ time, QG511-HA2 × 10 ⁸pfu/0.2ml/ time, physiological saline 0.2ml/ time, the next day, injects, multi-point injection totally 5 times in knurl.Treated rear 48h, cervical dislocation puts to death tumor bearing nude mice, cuts tumour surrounding skin, complete stripping tumour, removes fatty tissue and claims knurl quality, scales/electronic balance weighing knurl block weight.Get tumor specimen routine section HE dyeing, observe pathological change situation.

7HE dyes: nude mouse tumor tissue is fixed 12h through 10% formalin solution room temperature, and ensure that liquid will be organized and cover completely, paraffin embedding, HE dyes.

Two, result

The restructuring of 1 oncolytic adenovirus QG511-HA-Melittin and qualification

The order of the structure of the plasmid introduced in MATERIALS METHODS, builds pclon7-MLP carrier, pclon7-MLP-Melittin, pENTR-MLP-Melittin and pXC20-Δ E1b-55kda-HREAFP270 respectively.Build and name plasmid pPE3-F11B-MLP-Melittin.The order of the structure of the plasmid introduced in MATERIALS METHODS, builds J47-Δ E1b-55kda, PXC20-Δ E1b-55kda, J47-HREAFP and PXC20-Δ E1b-55kda-HREAFP respectively.The qualification result of each step is shown in Fig. 1-13, and through gene sequencing, proves the exactness of plasmid construction further.Build and name plasmid pXC20-Δ E1b-55kda-HREAFP270.Packaging and the amplification of virus is carried out, through qualification, containing corresponding gene in viral genome according to the method in MATERIALS METHODS.The results are shown in Figure 14-18.Called after QG511-HA-Melittin, the viral nomenclature that corresponding same structure lacks Melittin gene is QG511-HA.Figure 19 is shown in by Organization of viral genome schematic diagram.

2 oncolytic adenovirus QG511-HA-Melittin proliferated specifically in the hepatoma cell strain of the AFP positive

Through qualification, after virus formulation success, we are first at the AFP positive and its multiplication capacity of detection in negative hepatoma cell strain and normal liver cell thereof.The proliferation times of QG511-HA-Melittin 48h in the hepatoma cell strain Hep3B of alph-fetoprotein positive is up to 12800 times, in alph-fetoprotein positive hepatoma H22 cells, the proliferation times of 48 hours is 5000 times, and the proliferation times of 48h is 130 times in the Liver cancer cell SMMC-7721 of α-fetoprotein-negative, and in normal cell strain L02, the proliferation times of 48h is only 196 times.Within by this virus infection 48h, virus titer obviously rises, and between 48h to 96h, virus multiplication slows down.See Figure 20.

3 oncolytic adenovirus QG511-HA-Melittin specificity can suppress the propagation of the hepatoma cell strain of the AFP positive

Have detected QG511-HA-Melittin, QG511-HA can proliferated specifically in the hepatoma cell strain of alph-fetoprotein positive, we have detected its restraining effect to this several cell proliferation simultaneously, find the Hep3B cell that can kill and wound half at MOI=5, and SMMC-7721 and L02 cell half kills and wounds MOI value is all greater than 100, difference has significance (p<0.05).See Figure 21.

4 oncolytic adenovirus QG511-HA-Melittin can suppress the growth of the liver cancer Hep3B nude mouse tumor of the lotus people AFP positive

QG511-HA-Melittin, QG511-HA all can suppress the growth of Hep3B tumor bearing nude mice tumour, and visual inspection transplanted tumor is rounded, oval or lobulated growth.Rapidly, tumour quality is softer for the growth of physiological saline group, and vascular surface distribution is closeer, and part coating is imperfect, has local infiltration to be inclined to; QG511-HA-melittin group, QG511-HA group growth of xenografted are suppressed, and growth is comparatively limited to, and quality is slightly hard, and coating is more complete.Compared with physiological saline group, the knurl method of double differences is different statistical significance (P < 0.05).But under identical sky said conditions, compare between QG511-HA-melittin group with QG511-HA group, no significant difference (P > 0.05) (see Figure 22).Microscopic observation physiological saline group transplanted tumor cell shape is irregular, size heterogeneity, the large dense dye of core, and core heteromorphism is obvious, and caryoplasm ratio obviously increases, visible pathological mitotic figure.Cancer cells dense arrangement, does not have polarity, in bulk, strip, and visible blood capillary proliferation in cancerous tissue, based on interstitial and borderline tumor, the visible a little tissue necrosis of tumor center.QG511-HA-melittin group, QG511-HA group oncocyte present sex change in various degree, and Partial tumors cell is pyknosis, mesenchyma stroma of tumors vascular components less (see Figure 23).

Three, discuss

Adenovirus, as the conventional carrier of gene therapy, has that toxicity is low, security is high, host range is wide and the advantage such as inside and outside Absorbable organic halogens existence.Adenovirus is used to the treatment of tumour gradually in recent years, but adenovirus infection efficiency is low, and targeting is poor, and the adenovirus require repeated infection of replication defective limits it further applies.The recombinant adenovirus that E1B district 55KD lacks optionally can be bred in P53 sudden change or the tumour cell of disappearance, when discharging the virion made new advances after tumor cell lysis and again infect the tumour cell of surrounding, to go round and begin again the copying of repetition, propagation, the process of cracking, form water fall effect until tumour cell is all killed, therefore such virus is again oncolytic type adenovirus, as the U.S. ONYX pharmaceutical companies development E1B district 55KD lack ONYX-015 oneself demonstrate good clinical efficacy, Effective multiplication in the normally functioning tumour cell of some p53, and do not breed in the tumour cell of some p53 functional defect.Oncolytic type adenovirus becomes the focus of research in recent years, how can improve the targeting of oncolytic type adenovirus, and it is crucial for reducing it to Normocellular toxicity.A lot of people realizes the targeting propagation of virus by the structure transforming viral framework.Some investigator's fixed points add the promotor of some tumoral character, strengthen targeting.As anoxic promotor, telomerase promoter or the promotor for specific tumor feature, as alpha-fetoprotein (AFP) promotor, prostate specific antigen (PSA) promotor etc. of liver cancer.

HRE is intracellular hypoxia response elements, and its particular combination site in conjunction with HIF-1 (hypoxiz-inducible factor-1, HIF-1), thus can start the transcriptional expression of target gene.Inside tumor cells, by a series of signal transducting system, causes the expression of HIF-1 transcription factor, finally causes activation and the expression of HRE gene.The activation of HRE promotor is the specific change of noumenal tumour, there is scholar abroad once by the strategies in gene therapy treatment tumour of hypoxia response promotor, find the active compared with normal liver of HRE promotor in the tumour cell of weary oxygen or the high l000 of activity in spleen doubly left and right, foreign gene can be made mainly to express in tumour, and express in normal cell and obviously weaken.Qian Qijun seminar successfully constructs the replicative adenovirus CNHK500 of human telomerase reverse transcriptase's (hTERT) promotor and the two regulation and control of hypoxia response (HRE) promotor, proves that CNHK500 has good specificity antineoplastic effect in body with experiment in vitro.

AFP promotor is the promotor of a typical tumour-specific.The expression of the AFP promotor control E1A of tumour-specific such as Hallenbeck, the recombinant adenovirus of structure can be bred in the hepatocellular carcinoma cell lines of the AFP positive, and breeds hardly in the HCC cell strain of AFP negative.Takikawa etc. build the carrier containing AFP promotor and activating transcription factor and the carrier containing AFP promotor and Cre sequence, are used for the treatment of hepatocellular carcinoma.Hou J etc. carries suicide gene therapy using AFP as transcription activator and acts on liver cancer cell, significantly improves curative effect.

Melittin is one of main component of apis mellifera bee venom, accounts for 50% of bee venom dry weight.The polypeptide that melittin is made up of 26 amino-acid residues, melittin tool is antibacterial, anti-inflammatory, radioprotective, arthritis effect and the effect such as cardiovascular, its effect in therapeutic field of tumor has been subject to great attention.Melittin high dosage has the effect of killing tumor cell BEL-7402, and low dosage can induce its apoptosis, and melittin obviously can suppress the growth of human hepatocellular carcinoma BEL-7402 cell's transplanted tumor in nude mice.Melittin has the side effect of haemolysis, and safe-dosaging limits is little, and its lethal effect does not have selectivity, limits clinical application.Because its encoding gene only has 78bp, be easy to transform at gene level, we once built and took melittin replication-defective adenoviral vector, in vitro after transfection HepG2, can suppress the propagation of AFP masculine liver cancer cell, obviously reduced the growing amount of its AFP.But not only there is no clear and definite targeting, and not reproducible, peptide in bee venom is expressed seldom.

The present invention adopts E1B district to lack 55kda albumen, the Suppressor p53 of deactivation normal sudden change in tumour cell, adopt the Ela district that anoxic promotor HRE and afp promoter AFP promotor regulate and control jointly first, further with late promoter (MLP) for promotor, by the gene clone of Melittin to wherein, construct with HREAFP be promotor carry Melittin gene oncolytic adenovirus QG511-HA-Melittin, this virus is lower in the Liver cancer cell SMMC-7721 propagation of normal cell strain L02 and α-fetoprotein-negative, by contrast, after infecting 48h, in hepatoma cell strain Hep3B and HepG2 of alph-fetoprotein positive, proliferation times significantly raises.In cavy body after injection, through the pathological observation of Liver function grade and important organ, do not find any untoward reaction.After being applied to the nude mice of lotus people Hep3B cell, obviously inhibit the growth of tumour, and alleviate the pathological change of tumour.

The oncolytic adenovirus QG511-HA-Melittin that the present invention builds, be intended to the feature of embodiment three aspect, one is the liver cancer cell can specifying the target AFP positive, two is that high multiple copies in the liver cancer cell of the AFP positive, three be carry there is Tumor suppression kill and wound gene, embody the superposition of three aspect factors.Achieving Melittin gene specific expressed with copying of virus in the liver cancer cell of the AFP positive, is the new tool of Hepatoma therapy.

The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing from the inventive method; can also make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.

Claims (7)

1. be the oncolytic adenovirus carrying Melittin gene of promotor with HREAFP, it is characterized in that, described oncolytic adenovirus is promotor with HREAFP, takes the oncolytic adenovirus that late promoter MLP regulates and controls peptide in bee venom.

2. oncolytic adenovirus according to claim 1, it is characterized in that, described oncolytic adenovirus is for carrier with 11 type adenovirus, build and jointly regulate and control Ela district with hypoxia response elements HRE promotor and afp promoter AFP hybrid promoter, Elb district disappearance 55KDa, carries the oncolytic adenovirus being regulated and controled Melittin gene by late promoter MLP.

3. oncolytic adenovirus according to claim 1, it is characterized in that, the preparation method of described oncolytic adenovirus is: the plasmid pENTR-MLP-Melittin and adenoviral backbone plasmid pPE3-F11B that carry Melittin gene are recombinated by LR, build plasmid pPE3-F11B-MLP-Melittin, by itself and the common transfection human embryo kidney 293 cells of plasmid PXC20-△ Elb-55kda-HREAFP containing hybrid promoter HREAFP, obtain regulating and controlling Ela district by HREAFP, the restructuring replication competent adenovirus taking Melittin gene of Elb district disappearance 55kda albumen.

4. the preparation method of oncolytic adenovirus according to claim 1, it is characterized in that, described preparation method is: the plasmid pENTR-MLP-Melittin and adenoviral backbone plasmid pPE3-F11B that carry Melittin gene are recombinated by LR, build plasmid pPE3-F11B-MLP-Melittin, by itself and the common transfection human embryo kidney 293 cells of plasmid PXC20-△ Elb-55kda-HREAFP containing hybrid promoter HREAFP, obtain regulating and controlling Ela district by HREAFP, the restructuring replication competent adenovirus taking Melittin gene of Elb district disappearance 55kda albumen.

5. according to the application of the arbitrary described oncolytic adenovirus of claim 1-3 in the medicine of preparation treatment tumour.

6. application according to claim 5, is characterized in that, described tumour is liver cancer.

7. application according to claim 6, is characterized in that, described liver cancer is the liver cancer of alph-fetoprotein positive.