CN102247593B - Preparation and application of recombinant newcastle disease virus-modified autologous tumor vaccine - Google Patents
Preparation and application of recombinant newcastle disease virus-modified autologous tumor vaccine Download PDFInfo
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- CN102247593B CN102247593B CN 201110188380 CN201110188380A CN102247593B CN 102247593 B CN102247593 B CN 102247593B CN 201110188380 CN201110188380 CN 201110188380 CN 201110188380 A CN201110188380 A CN 201110188380A CN 102247593 B CN102247593 B CN 102247593B
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Abstract
The invention discloses a preparation method of a recombinant newcastle disease virus-modified autologous tumor vaccine. The preparation method comprises the following steps of: (1) constructing an IL-7 gene-carrying recombinant newcastle disease virus; and (2) amplifying the IL-7 gene-carrying recombinant newcastle disease virus obtained by the step (1), and then infecting tumor cells to prepare the IL-7 gene-carrying recombinant newcastle disease virus-modified autologous tumor vaccine. In the invention, the IL-7 gene-carrying recombinant newcastle disease virus-modified autologous tumor vaccine is utilized for the first time; and immunoprophylaxis and treatment effects of the vaccine on tumor are proved through a mouse tumor model, so that the vaccine can be applied to the field of preparing an immunoprophylaxis and treatment medicament for the tumor.
Description
Technical field
The present invention relates to a kind of preparation of mouse tumor vaccine, particularly preparation and the application thereof of the autologous tumor vaccine modified of a kind of recombinant Newcastle disease virus that carries mice IL-7.
Background technology
ATV-NDV handles autologous tumor cell through gamma-rays, (Newcastle disease virus NDV), and directly uses as tumor vaccine with this tumor cell that has infected NDV to infect Avian pneumo-encephalitis virus again.NDV is a kind of birds paramyxovirus, has the immunostimulating characteristics of many tissue affinities, and it can optionally copy in tumor cell matter and not rely on the propagation of cell, belongs to the virus of non-pathogenic, effectively and safely infected tumor's cell.ATV-NDV be with NDV infect gone out can but remain the tumor vaccine that patient's autologous tumor cell alive is made, tumor patient is carried out postoperative active specific immunotherapy treatment (ASI).Behind the NDV infected tumor cell, neuraminidase molecule (HN) and the warm albumen (F) of virus self have been introduced at cell surface, and then the generation of the common stimulating activity of inducing T cell and cytokine and local chemotactic factor, strengthen the expression of tumor specific antigen, thereby produced the specificity antineoplastic effect.In ATV-NDV, NDV is the modification carrier that carries HN and F gene, plays the effect that abduction delivering strengthens the non-characteristic component of immunogenicity of tumor, and the autologous tumor living cells of the energy that goes out plays the special component of expressing tumor related antigen (TAAs).NDV has the cell binding characteristic, in minutes, can be combined with the component that tumor cell surface extensively exists by the neuraminidase molecule of self.Then, outer virionic membrane and the host cell membrane of the fusion protein F that NDV comprises and HN mediation merge, thus infected tumor's cell.ATV-NDV is in inoculation locally starting immunologic process.
Because the introducing of NDV, in tumor cell, introduce danger signal and raised the MHC molecule of cell surface, improved the immunogenicity of tumor vaccine.ATV-NDV a plurality of countries obtained application clinically, show from the II phase clinical effectiveness in Europe, accept the ATV-NDV treatment after intestinal cancer, breast carcinoma, the head and neck squamous cell carcinoma patient operation, can be improved 25%, 36% and 23% 5 year life cycle respectively; And the treatment group finds no tangible autoimmunity symptom.China had begun the clinical use of ATV-NDV, and had obtained effect preferably through the approval of the Committee of Experts of Ministry of Public Health biotechnology leading group in 2002.At present ATV-NDV can only be as the complementary therapy of tumor operation treatment clinically, for some grade malignancies higher or some non-solid tumors, its curative effect is also undesirable.
As adjuvant, be the important method that improves the tumor vaccine effect with cytokine.Yet part or whole body direct injection cytokine often may cause some bio-safety problems: as autoimmune disease etc.; And the cytokine of injection in the concentration of injection site along with the very fast reduction of metabolism, be not enough to give full play to its effect.Therefore utilizing the Avian pneumo-encephalitis virus of expressing recombinant cytokine to make up ATV-NDV, continue at immune position to discharge these cytokines, is the effective way that improves the ATV-NDV effect, avoids occurring autoimmune disease.
Body is mainly removed tumor by cellular immunization, and the T cell is the main cell of carrying out cellular immunization.IL-7 the generation of differentiation, growth, propagation and the memory t cell of T cell with keep in play a crucial role.On the one hand, IL-7 has improved function and the quantity of T effector cell by the expression of downward modulation Socs3; Can also promote simultaneously the propagation of the T cell in thymus source.On the other hand, IL-7 can reduce the expression of ubiquitin ligase Cbl-b in the T cell, causes the T cell insensitive to the down regulation of the inhibition factor such as TGF-β, Treg and cell, and then improves the therapeutic effect of tumor vaccine.
At present, do not see that the reorganization NDV that has utilization to carry IL-7 makes up the report that ATV-NDV carries out tumour immunity prevention and treatment.
Summary of the invention
Goal of the invention of the present invention provides autologous tumor vaccine of a kind of recombinant Newcastle disease virus modification and preparation method thereof.
To achieve the above object of the invention, the technical solution used in the present invention is: the preparation method of the autologous tumor vaccine that a kind of recombinant Newcastle disease virus is modified may further comprise the steps:
(1) at first makes up the recombinant Newcastle disease virus that carries the IL-7 gene;
(2) the described recombinant Newcastle disease virus that carries the IL-7 gene of amplification step (1), infected tumor's cell prepares the autologous tumor vaccine that a kind of recombinant Newcastle disease virus that carries mice IL-7 is modified then.
In the technique scheme, in the step (1), but structure carries the method reference literature of the recombinant Newcastle disease virus of IL-7 gene: Hu S L, Sun Q, Liu X F, et al. Generation of a recombinant Newcastle disease virus Expressing green fluorescence protein gene. Virologica Sinica, 2007,22 (1): 34-40.
In the technique scheme, in the step (2), the recombinant Newcastle disease virus step of the described IL-7 of the carrying gene of described amplification step (1) is specially: adopt the normal saline dilution to carry the recombinant Newcastle disease virus LX/IL-7 of IL-7 gene, be inoculated in 9～10 age in days SPF Embryo Gallus domesticus then, discard the dead embryo in the 24h, collect the allantoic fluid of the interior dead embryo of 24～96h and 96h survival embryo; Described allantoic fluid is got the centrifugal and collecting precipitation of supernatant through the centrifugal precipitation that discards, and is every milliliter of 50～100 HU(blood clotting valencys with the resuspended virus of phosphate buffer (PBS) to the concentration of pH7.0～7.4); Wherein, the centrifugation step of centrifugal allantoic fluid is specially: under 4 ℃ of conditions, the centrifugal 40-45 of 5,000 rpm minute; The centrifugation step of centrifuged supernatant is specially: 4 ℃, 35,000 rpm ultracentrifugation 2-2.5h.
In the technique scheme, in the step (2), described infected tumor cell step is specially: adopt trypsin digestion to collect the tumor cell of exponential phase, after cell is suspended with phosphate buffer (PBS), handle through radiation irradiation; With the cell after handling with 10
7The ratio of individual cell infection 100HU virus is hatched behind the mixing, and the centrifugal collecting cell precipitation namely gets the autologous tumor vaccine that the described recombinant Newcastle disease virus that carries mice IL-7 is modified then; Wherein, the condition of described radiation irradiation processing is: the radiation gamma of 50-100 Gry is handled; The condition of described incubation step is specially: 37 ℃, contain in the cell culture incubator of 5% carbon dioxide and hatched 1 hour.
The recombinant Newcastle disease virus that carries mice IL-7 that adopts technique scheme to prepare makes up the autologous tumor vaccine, through the bright effect with tumour immunity prevention and treatment of internal and external test probatio inspectionem pecuoarem, therefore, the simultaneously claimed above-mentioned application of autologous tumor vaccine in preparation tumor prevention and medicine of carrying the recombinant Newcastle disease virus modification of IL-7 of the present invention; Preferably, described tumor is melanoma, lymphoma, glioma, hepatocarcinoma, pulmonary carcinoma, intestinal cancer, cancer of pancreas or carcinoma of prostate.
Because technique scheme is used, the present invention compared with prior art has following advantage:
1. the present invention utilizes the autologous tumor vaccine of the recombinant Newcastle disease virus modification of carrying mice IL-7 first, by mouse tumor model, has verified immunoprophylaxis and the therapeutic effect of this vaccine to tumor; Thereby it can be applied in the immunoprophylaxis and medicine field of preparation tumor; The autologous tumor vaccine that the recombinant Newcastle disease virus that carries mice IL-7 of the present invention is modified can be for the preparation of medicine, particularly melanoma, lymphoma, glioma, hepatocarcinoma, pulmonary carcinoma, intestinal cancer, cancer of pancreas, the carcinoma of prostate of prevention and treatment kinds of tumors.
Description of drawings
Fig. 1. carry the sketch map of the LX strain cDNA of IL-7 among the embodiment one;
Fig. 2. HN protein immunization fluorescence evaluation behind the reorganization NDV infection cell among the embodiment one; Wherein, the cell that A.LX/IL-7 infects, HN immunofluorescence result; B. matched group, the cell of uninfecting virus;
Fig. 3. IL-7 expresses dynamic (dynamical) testing result figure in the embodiment one autologous tumor vaccine;
Fig. 4. the tumor prevention effect of autologous tumor vaccine among the embodiment two;
Fig. 5. the oncotherapy effect of autologous tumor vaccine among the embodiment three;
Fig. 6. carry the recombinant Newcastle disease virus of mice IL-7 among the embodiment four to the infection conditions of the tumor of other type.
The specific embodiment
Below in conjunction with drawings and Examples the present invention is further described:
Embodiment one: the preparation of carrying the autologous tumor vaccine that the recombinant Newcastle disease virus of mice IL-7 modifies
(1) the method Hu S L of reference literature 1 at first, Sun Q, Liu X F, et al. Generation of a recombinant Newcastle disease virus Expressing green fluorescence protein gene. Virologica Sinica, 2007,22 (1): 34-40. makes up the recombinant Newcastle disease virus that carries the IL-7 gene:
1.1 carry the structure of the LX strain cDNA of IL-7: get the C57BL/6 mouse spleen, become cDNA by Trizol-chloroform method extracted total RNA, reverse transcription, between the P of reference literature 1 reported method clone IL-7 to LX and the M gene (Fig. 1), construct recombiant plasmid pLX/IL-7.
1.2 cotransfection obtains virus: reference literature 1 report, the method that adopts liposome transfection with plasmid pLX/IL-7 and 3 helper plasmid P, N deriving from ZJ-1 and L cotransfection to BSR T7/5 cell.Behind the 48-72h 9 ~ 11 age in days SPF Embryo Gallus domesticus are inoculated in 2-3 back of cells transfected multigelation.Take out the Embryo Gallus domesticus of surviving behind the 24h, collect allantoic fluid, obtain virus.With the virus inoculation chick embryo fibroblast of obtaining, utilize the method identifying virus (Fig. 2) of immunofluorescence, the recombinant Newcastle disease virus that carries the IL-7 gene as shown in Figure 2 successfully constructs.
(2) carry amplification and the purification of the recombinant Newcastle disease virus (LX/IL-7) of mice IL-7 gene: the LX/IL-7 kind poison of getting-70 ℃ of preservations doubly dilutes with the normal saline 1:10000 that sterilizes, press 0.2mL/ embryonic breeding kind 9～10 age in days SPF Embryo Gallus domesticus, discard the dead embryo in the 24h, collect the allantoic fluid of the interior dead embryo of 24-96h and 96h survival embryo.The allantoic fluid of results is 4 " under the C condition, 5,000 rpm discarded precipitation in centrifugal 40 minutes.Supernatant is in 4 ℃ of ultrahigh speed refrigerated centrifugers, 35,000 rpm ultracentrifugation 2h, and collecting precipitation is every milliliter of 100HU with an amount of resuspended virus of PBS buffer (pH7.4) to concentration.
(3) preparation of the autologous tumor vaccine of LX/IL-7 modification: adopt trypsin digestion to collect the tumor cell of exponential phase, after cell is suspended with PBS, through 50Gry's
60The Go treatment with irradiation.With the cell after handling with 10
7The ratio of the virus of individual cell infection 100HU, mixing in the centrifuge tube of 10mL, the cell culture incubator that put into 37 ℃, contains 5% carbon dioxide was hatched 1 hour.The centrifugal collecting cell precipitation is the autologous tumor vaccine.
(4) IL-7 expresses dynamic (dynamical) detection in the autologous tumor vaccine of LX/IL-7 modification:
Collect well-grown B16 cell, be prepared into cell suspension, adjust cell concentration to 1 * 10 with the DMEM culture medium of serum-free
7Individual/mL.Get 1 mL cell suspension, with 50Gry's
60The Go treatment with irradiation adds the viral liquid that 100uL contains the LX/IL-7 of 100HU then, 37 ℃, 5%CO
2Incubator was cultivated 1 hour.Under 4 ℃ of conditions, centrifugal 10 minutes of 2,000 rpm, the cell of collecting precipitation places 37 ℃, 5%CO with the suspend cell of precipitation of the DMEM of 1mL serum-free
2Cultivate in the incubator.Drew cell conditioned medium in 24,48 and 72 hours respectively at cultivating the back, carry out doubling dilution, each is organized dilution factor and all establishes 3 multiple holes.According to R﹠amp; The amount of IL-7 is measured in the explanation of the Mouse IL-7 Immunoassay test kit of D company.The result is as shown in Figure 3: cultivated back 24 hours, the IL-7 secretory volume can reach 200ng/mL in the tumor vaccine supernatant, reaches the highest after 48 hours.
Embodiment two: the tumor prevention Research on effect of carrying the autologous tumor vaccine that the recombinant Newcastle disease virus of mice IL-7 modifies
(1) preparation of murine melanoma vaccine:
The preparation of B16 tumor vaccine: collect well-grown B16 cell, be prepared into cell suspension, adjust cell concentration to 1 * 10 with the DMEM culture medium of serum-free
7Individual/mL.Get 1 mL cell suspension, with 50Gry's
60The Go treatment with irradiation adds 100uL then and contains PBS, 37 ℃, 5%CO
2Incubator was cultivated 1 hour.Under 4 ℃ of conditions, centrifugal 10 minutes of 2,000 rpm, the cell of collecting precipitation with the aseptic PBS suspension cell of 1mL, places standby on ice.
Carry crt gene (red fluorescent protein, the preparation of the murine melanoma vaccine (B16-LX/RFP) that Avian pneumo-encephalitis virus RFP) (LX/RFP) is modified: collect well-grown B16 cell, be prepared into cell suspension, adjust cell concentration to 1 * 10 with the DMEM culture medium of serum-free
7Individual/mL.Get 1 mL cell suspension, with 50Gry's
60The Go treatment with irradiation adds the viral liquid that 100uL contains the LX/RFP of 100HU then, 37 ℃, 5%CO
2Incubator was cultivated 1 hour.Under 4 ℃ of conditions, centrifugal 10 minutes of 2,000 rpm, the cell of collecting precipitation with the aseptic PBS suspension cell of 1mL, places standby on ice.
Carry the preparation from body melanoma vaccines (B16-LX/IL-7) of the recombinant Newcastle disease virus modification of mice IL-7: collect well-grown B16 cell, be prepared into cell suspension, adjust cell concentration to 1 * 10 with the DMEM culture medium of serum-free
7Individual/mL.Get 1 mL cell suspension, with 50Gry's
60The Go treatment with irradiation adds the viral liquid that 100uL contains the LX/IL-7 of 100HU then, 37 ℃, 5%CO
2Incubator was cultivated 1 hour.Under 4 ℃ of conditions, centrifugal 10 minutes of 2,000 rpm, the cell of collecting precipitation with the aseptic PBS suspension cell of 1mL, places standby on ice.
(2) the immunoprophylaxis test of the B16 melanoma vaccines of LX/IL-7 modification:
The experiment grouping: test divides 4 groups, is respectively immune PBS, irradiated B16, LX/RFP and LX/IL-7.The C57BL/6 mice is subcutaneous uses 1 * 10 respectively
6The PBS immunity of individual vaccine bebcell or 100uL 1 time, 3 week back offside subcutaneous vaccinations 1 * 10
5Individual B16-F10 cell, the tumor growth situation.The B16 cell (B16-LX/RFP) of inoculation LX/RFP modified, the more immune B16 group of tumor growth rate is slack-off.And immune B16-LX/IL-7 group, tumor growth rate all has remarkable decline, significant difference (Fig. 4) than other group.This result shows, carry that the recombinant Newcastle disease virus of mice IL-7 modifies from the body melanoma vaccines, have good immunoprophylaxis effect.
Embodiment three: the immunization therapy test of the B16 melanoma vaccines that LX/IL-7 modifies:
The foundation of murine melanoma model: C57BL/6 mouse hypodermic inoculation 1 * 10
5Individual B16 cell is that black point-like lump appears in visible inoculation position after 3 days.
Therapeutic test: test divides 3 groups, is respectively inoculation irradiated B16, LX/RFP and LX/IL-7.Tumor-bearing mice tumor offside is subcutaneous inoculates 1 * 10 respectively
6Individual vaccine bebcell is inoculated vaccine weekly 2 times, inoculated for 3 weeks altogether after, the tumor growth situation.The result is as shown in Figure 5: with respect to matched group (inoculating irradiated B16 cell), B16-LX/RFP group tumor growth rate obviously reduces the two significant difference.And B16-LX/IL-7 group tumor growth rate also has obvious reduction than B16-LX/RFP, and the two significant difference has two tumor to disappear fully in 10 mices.This result shows, carry that the recombinant Newcastle disease virus of mice IL-7 modifies from the body melanoma vaccines, have good immunization therapy effect.
Embodiment four: carry the recombinant Newcastle disease virus of mice IL-7 to the infection conditions of the tumor of other type
Collect well-grown MCF-7(MCF-7 respectively), the HepG2(Bel7402), Hep1/6(rat liver cancer cell line) and Raw 264.7(mouse monokaryon macrophage leukaemia system) cell, be prepared into cell suspension, adjust cell concentration to 1 * 10 with the DMEM culture medium of serum-free
7Individual/mL.Get 1 mL cell suspension, with 50Gry's
60The Go treatment with irradiation adds the viral liquid that 100uL contains the LX/IL-7 of 100HU then, 37 ℃, 5%CO
2Incubator was cultivated 1 hour.Under 4 ℃ of conditions, centrifugal 10 minutes of 2,000 rpm, the cell of collecting precipitation places 37 ℃, 5%CO with the suspend cell of precipitation of the DMEM of 1mL serum-free
2Cultivate in the incubator.Drew cell conditioned medium in 24,48 and 72 hours respectively at cultivating the back, carry out doubling dilution, each is organized dilution factor and all establishes 3 multiple holes.According to R﹠amp; The amount of IL-7 is measured in the explanation of the Mouse IL-7 Immunoassay test kit of D company.The result is as shown in Figure 6: cultivated back 24 hours, each organizes the expression that all can detect IL-7 in the tumor cell supernatant.Show that the recombinant Newcastle disease virus that carries mice IL-7 can be used for the structure of the autologous tumor vaccine of polytype tumor.
Claims (8)
1. the preparation method of a recombinant Newcastle disease virus that the carries mice IL-7 B16 melanoma vaccines of modifying is characterized in that, may further comprise the steps:
(1) at first makes up the recombinant Newcastle disease virus that carries the IL-7 gene;
(2) the described recombinant Newcastle disease virus that carries the IL-7 gene of amplification step (1) infects the B16 melanoma cell then, prepares the B16 melanoma vaccines that a kind of recombinant Newcastle disease virus that carries mice IL-7 is modified;
In the step (2), described infection B16 melanoma cell step is specially: adopt trypsin digestion to collect the B16 melanoma cell of exponential phase, after cell is suspended with phosphate buffer, handle through radiation irradiation; With the cell after handling with 10
7The ratio of the virus of individual cell infection 100 HU is hatched behind the mixing, and the centrifugal collecting cell precipitation namely gets the B16 melanoma vaccines that the described recombinant Newcastle disease virus that carries mice IL-7 is modified then.
2. the preparation method of the B16 melanoma vaccines of modifying according to the described recombinant Newcastle disease virus that carries mice IL-7 of claim 1, it is characterized in that, in the step (2), the step of the recombinant Newcastle disease virus of the described IL-7 of the carrying gene of amplification step (1) is specially: adopt the normal saline dilution to carry the recombinant Newcastle disease virus of IL-7 gene, be inoculated in 9～10 age in days SPF Embryo Gallus domesticus then, discard the dead embryo in the 24h, collect the allantoic fluid of the interior dead embryo of 24～96h and 96h survival embryo; Described allantoic fluid is got the centrifugal and collecting precipitation of supernatant through the centrifugal precipitation that discards, and is every milliliter of 50～100HU with the resuspended virus of the phosphate buffer of pH7.0～7.4 to concentration.
3. the preparation method of the B16 melanoma vaccines of modifying according to the described recombinant Newcastle disease virus that carries mice IL-7 of claim 2, it is characterized in that, in the step (2), the centrifugation step of centrifugal allantoic fluid is specially: under 4 ℃ of conditions, centrifugal 40～45 minutes of 5,000 rpm.
4. the preparation method of the B16 melanoma vaccines of modifying according to the described recombinant Newcastle disease virus that carries mice IL-7 of claim 2, it is characterized in that, in the step (2), the centrifugation step of centrifuged supernatant is specially: 4 ℃, 35,000 rpm ultracentrifugations, 2～2.5h.
5. the preparation method of the B16 melanoma vaccines of modifying according to the described recombinant Newcastle disease virus that carries mice IL-7 of claim 1, it is characterized in that the condition that described radiation irradiation is handled is: the radiation gamma of 50～100Gry is handled.
6. the preparation method of the B16 melanoma vaccines of modifying according to the described recombinant Newcastle disease virus that carries mice IL-7 of claim 1 is characterized in that the condition of described incubation step is specially: 37 ℃, contain in the cell culture incubator of 5% carbon dioxide and hatched 1 hour.
7. the B16 melanoma vaccines that the recombinant Newcastle disease virus that carries mice IL-7 that adopts the preparation method of the B16 melanoma vaccines that the described recombinant Newcastle disease virus that carries mice IL-7 of claim 1 modifies to prepare is modified.
8. the described application of B16 melanoma vaccines in preparation B16 melanoma prevention and medicine of carrying the recombinant Newcastle disease virus modification of mice IL-7 of claim 7.
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| US20090175826A1 (en) * | 2006-06-05 | 2009-07-09 | Elankumaran Subbiah | Genetically-engineered newcastle disease virus as an oncolytic agent, and methods of using same |
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Address after: Suzhou City, Jiangsu province 215137 Xiangcheng District Ji Road No. 8 Patentee after: Soochow University Patentee after: Liu Haiyan Address before: 215123 Suzhou City, Suzhou Province Industrial Park, No. love road, No. 199 Patentee before: Soochow University Patentee before: Liu Haiyan |
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