CN101875920B

CN101875920B - DcR3 and GAD65 double-gene co-expression recombinant adenovirus and preparation method and application thereof

Info

Publication number: CN101875920B
Application number: CN2009102510830A
Authority: CN
Inventors: 杨曌; 吴玉章
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2009-12-29
Filing date: 2009-12-29
Publication date: 2012-04-18
Anticipated expiration: 2029-12-29
Also published as: CN101875920A

Abstract

The invention discloses a DcR3 and GAD65 double-gene co-expression recombinant adenovirus and a preparation method and application thereof, wherein the recombinant adenovirus genome comprises a DcR3 and GAD65 double-gene expression box, and the expression box sequentially comprises a CMV promoter, a DcR3 full-length coding gene, a terminator, an internal ribosome entry site IRES, a GAD65 full-length coding gene and a terminator from upstream to downstream; the preparation method of the recombinant adenovirus is simple; the dendritic cells transfected in vitro are infused back into the body, which can induce the tolerance of specific T cells, effectively inhibit the damage of islet beta cells, and prolong the survival time of transplanted islets, can be used for preparing dendritic cell vaccines for resisting islet transplantation rejection, and has good development and application prospects in the field of type 1 diabetes treatment.

Description

DcR3 and GAD65 double gene coexpression recombinant adenovirus

Technical field

The present invention relates to a kind of recombinant adenovirus, particularly a kind of DcR3 and GAD65 double gene coexpression recombinant adenovirus also relate to the preparation method and the application of this recombinant adenovirus.

Background technology

Along with the raising of living standards of the people and the change of mode of life, the sickness rate of mellitus increases day by day, has become the third-largest disease that after cardiovascular disorder and tumour, influences health of the masses and life.Easy, safe and effective characteristics that pancreatic islets transplantation has, postoperative patient need not rely on exogenous insulin can correct carbohydrate metabolism disturbance in the body, obviously improves the quality of living, and particularly gets most of the attention in the treatment of type 1 diabetes in mellitus.But owing to supply the genetic background difference of acceptor, pancreatic islets transplantation receives host's immunological rejection easily so that post-transplantation complication takes place, thereby influences result of treatment.And inducing specific T cell tolerance is a kind of therapeutic strategy that gets a good chance of to the pancreatic islets transplantation repulsion.

The pancreas islet candidate autoantigen of report has exceeded 20 kinds so far; L-Glutamic decarboxylase 65 (glutamic acid decarboxylase 65 wherein; GAD65) be considered to most important autoantigen; And very likely be the beginning moving-target antigen of type 1 diabetes, there are a large amount of GAD65 specific T-cells to exist among the type 1 diabetes patient in early days.Therefore, can utilize GAD65 inducing specific T cell tolerance to repel to avoid pancreatic islets transplantation.Inveigle acceptor (decoy receptor) to be meant that those cytolemma outskirts are similar with functional receptor extracellular plot structure, the ability binding partner, but the tenuigenin district lacks the acceptor of transduction signal ability.Inveigle acceptor through combining identical part, thereby bring into play regulating and controlling effect with differentiation and immunologic function in that the receptor level pair cell is active with functional receptor is competitive.Inveigle acceptor 3 (decoy receptor; DcR3) be the tumor necrosis factor receptor super family member who finds recently; Have the biological characteristics of regulating apoptosis and immunologic cellular activity and differentiation, in tumour immunity, autoimmunization, transplantation immunity and anti-infectious immunity, bringing into play important effect.The non-obese diabetic rat model of transgenic experiment showed, that DcR3 can stop autoimmunization cell and endoxan to the autoimmunity damage of beta Cell of islet, prevents the generation of mellitus, can also obviously alleviate the pancreas islet Inflammatory response.In addition, the zoografting model experiment proves that DcR3 transgenic islet cell transplantation success ratio is apparently higher than the wild-type islet cells.

The mode of gene combination therapy mainly contains two kinds: the one, carry heterogeneic recombinant expression vector transfection simultaneously target cell respectively with multiple; This mode can each recombinant expression vector of free adjustment ratio; But need to make up respectively to carry heterogeneic recombinant expression vector; Structure work is loaded down with trivial details, time-consuming, and influence factor is many; In addition, because transfection process has certain randomness, there is the inhomogenous problem of gene copy in the cell after the transfection, both has been unfavorable for the expression and the successive treatment of goal gene, causes experimental result to be difficult to analysis and assessment again; Above-mentioned drawbacks limit the further application of this kind mode.The 2nd, two or more genes are carried expression by a carrier; This mode can improve gene through control measures such as multiple promoter carrier, polycistron carrier or fusion genes and change seven efficient; Increase expression intensity; And keep relative expression ratio, thereby a kind of deficiency of mode before having overcome receives investigator's attention in recent years day by day.

BMDC is not only the most powerful antigen presenting cell of function in the body, can effectively activate T cells and start immune response, and BMDC itself has the effect of regulating immunoreation, inducing immune tolerance.External structure is carried the recombinant virus and the transfection BMDC of immunosuppression molecule gene and is processed dendritic cell vaccine, it is fed back in the body again, has been widely used in the treatment of aspects such as inducing immune tolerance.

Summary of the invention

In view of this, one of the object of the invention is to provide a kind of DcR3 and GAD65 double gene coexpression recombinant adenovirus, can inducing specific T cell tolerance, and the destruction of effectively suppressing beta Cell of islet, and prolong lifetime of islet transplantation; Two of purpose is to provide the preparation method of said DcR3 and GAD65 double gene coexpression recombinant adenovirus; Three of purpose is to provide the application of said DcR3 and GAD65 double gene coexpression recombinant adenovirus.

For achieving the above object, the present invention adopts following technical scheme:

1, DcR3 and GAD65 double gene coexpression recombinant adenovirus; Comprise a DcR3 and GAD65 double gene expression box in the said recombinant adenovirus genome, said DcR3 and GAD65 double gene expression box comprise CMV promotor, DcR3 total length encoding sox, terminator, internal ribosome entry site IRES, GAD65 total length encoding sox and terminator successively by the upper reaches to downstream.

2, the preparation method of said DcR3 and GAD65 double gene coexpression recombinant adenovirus may further comprise the steps:

A, RT-PCR amplification DcR3 full-length cDNA;

B, RT-PCR amplification GAD65 full-length cDNA;

C, with the IRES upper reaches that step a gained DcR3 full-length cDNA inserts the pStar carrier, step b gained GAD65 full-length cDNA inserts the IRES downstream of pStar carrier, recombinant vectors pStar-DcR3-IRES-GAD65;

D, be template with step c gained recombinant vectors pStar-DcR3-IRES-GAD65, pcr amplification DcR3-IRES-GAD65 fragment is also changed the restriction enzyme site at these fragment two ends;

E, the CMV promotor downstream that steps d gained DcR3-IRES-GAD65 fragment is inserted adenovirus shuttle vector pShuttle-CMV, recombinant adenovirus shuttle vectors pShuttle-CMV-DcR3-IRES-GAD65;

F, step e gained recombinant adenovirus shuttle vectors pShuttle-CMV-DcR3-IRES-GAD65 and adenovirus skeleton carrier pAdEasy-1 are carried out homologous recombination in the born of the same parents in intestinal bacteria BJ5183, recombinant adenoviral vector pAd-DcR3/GAD65;

G, recombinant adenoviral vector pAd-DcR3/GAD65 is packaged into recombinant adenovirus Ad-DcR3/GAD65 in 293 cells.

Further; The concrete grammar of step a is: extract the total RNA of human liver tissue; Rt obtains total cDNA, is template with this total cDNA again, is the upstream and downstream primer with sequence shown in SEQ ID No.1 and the SEQ ID No.2; The pcr amplification two ends have the DcR3 full-length cDNA of Pst I and EcoR I restriction enzyme site respectively, and PCR cycling condition parameter is: 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes; The purified rear clone of PCR product is gone into the pT-Easy carrier, gets recombinant vectors pT-DcR3;

Further; The concrete grammar of step b is: extract the total RNA of Human Pancreas; Rt obtains total cDNA, is template with this total cDNA again, is the upstream and downstream primer with sequence shown in SEQ ID No.4 and the SEQ ID No.5; The pcr amplification two ends have the GAD65 full-length cDNA of BamH I and Sal I restriction enzyme site respectively, and PCR cycling condition parameter is: 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes; The purified rear clone of PCR product is gone into the pT-Easy carrier, gets recombinant vectors pT-GAD65;

Further, the concrete grammar of step c is: go out the DcR3 full-length cDNA with Pst I and EcoR I double digestion among the carrier pT-DcR3 that recombinates certainly, purified back be connected with the pStar carrier of Pst I equally with EcoR I double digestion, must recombinant vectors pStar-DcR3; Go out GAD65 full-length cDNA with BamH I and Sal I double digestion from recombinating among the carrier pT-GAD65 again, purified afterwards be connected with the recombinant vectors pStar-DcR3 of BamH I equally with Sal I double digestion, recombinant vectors pStar-DcR3-IRES-GAD65;

Further; The concrete grammar of steps d is: with recombinant vectors pStar-DcR3-IRES-GAD65 is template; With sequence shown in SEQ ID No.7 and the SEQ ID No.8 is that the upstream and downstream primer carries out PCR; The restriction enzyme site at DcR3-IRES-GAD65 fragment two ends is replaced by Sal I restriction enzyme site and Not I restriction enzyme site, and PCR cycling condition parameter is: 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes; The purified rear clone of PCR product is gone into the pT-Easy carrier, gets recombinant vectors pT-DcR3-IRES-GAD65;

Further; The concrete grammar of step e is: go out the DcR3-IRES-GAD65 fragment with Sal I and Not I double digestion among the carrier pT-DcR3-IRES-GAD65 that recombinates certainly; Purified back be connected with the adenovirus shuttle vector pShuttle-CMV of Sal I equally with Not I double digestion, must recombinant adenovirus shuttle vectors pShuttle-CMV-DcR3-IRES-GAD65;

Further; The concrete grammar of step f is: with recombinant adenovirus shuttle vectors pShuttle-CMV-DcR3-IRES-GAD65 with Pme I linearization for enzyme restriction; Again with adenovirus skeleton carrier pAdEasy-1 electric transformed into escherichia coli BJ5183 competent cell of while; Carry out homologous recombination in the born of the same parents, get recombinant adenoviral vector pAd-DcR3/GAD65;

Further; The concrete grammar of step g is: the human embryonic kidney 293 cell is seeded in the culturing bottle with 40%～60% cell density, treats that cell grows to 30%～50% when merging, and adopts Lipofectamine 2000 reagent with recombinant adenoviral vector pAd-DcR3/GAD65 rotaring redyeing 293 cell; Centrifugal collecting cell when treating that obvious pathology appears in cell; With phosphate buffered saline buffer resuspended after, multigelation makes lysis, centrifugal removal cell debris; Collection contains the supernatant of virus, promptly gets recombinant adenovirus Ad-DcR3/GAD65.

3, said DcR3 and the GAD65 double gene coexpression recombinant adenovirus application in the dendritic cell vaccine that the anti-pancreatic islets transplantation of preparation is repelled.

Beneficial effect of the present invention is: the present invention unites expression with pancreas islet specific antigens GAD65 and novel immunosuppression molecule DcR3; Can induce the specific lymphocyte immunity tolerance of GAD65; Make islet cells can locality escape immune identification and attack, and other healthy tissuess and organ can not be affected.DcR3 of the present invention and GAD65 double gene coexpression recombinant adenovirus in-vitro transfection BMDC are fed back mellitus susceptibility NOD mouse again and carry out pancreatic islets transplantation simultaneously; The result finds; Recombinant adenovirus of the present invention can reduce the multiplication capacity and the cytokine secretion ability of mellitus susceptibility NOD mouse lymphocyte; Can postpone the disease time of NOD mouse mellitus and reduce sickness rate, and can prolong the lifetime of islet transplantation, show effectively inducing specific T cell tolerance of recombinant adenovirus of the present invention; The destruction of effectively suppressing beta Cell of islet; And the lifetime of prolongation islet transplantation, can be used to prepare the dendritic cell vaccine that anti-pancreatic islets transplantation is repelled, have the excellent development application prospect in type 1 diabetes treatment field.Simultaneously, the preparation method of recombinant adenovirus of the present invention is simple, and cost is low.

Description of drawings

In order to make the object of the invention, technical scheme and advantage clearer, will combine accompanying drawing that the present invention is made further detailed description below, wherein:

Fig. 1 has shown the agarose gel electrophoresis result (1 swimming lane is a dna molecular amount standard, and 2 swimming lanes are the PCR product) of DcR3 full-length cDNA PCR product;

Fig. 2 has shown the agarose gel electrophoresis result (1 swimming lane is a dna molecular amount standard, and 2 swimming lanes are the PCR product) of GAD65 full-length cDNA PCR product;

Fig. 3 has shown immunoblotting (Western Blot) result (1 swimming lane is the total protein of Ad-DcR3/GAD65 virus transfection BMDC, and 2 swimming lanes are the total protein of empty virus transfection BMDC) of DcR3 and GAD65 double gene coexpression;

Fig. 4 has shown the detected result of lymphopoiesis ability;

Fig. 5 has shown the detected result of lymphocytic emiocytosis cytokine ability;

Fig. 6 has shown the average attack rate of NOD mouse mellitus;

Fig. 7 has shown pancreas islet mean survival time (MST) detected result.

Embodiment

Below will carry out detailed description to the preferred embodiments of the present invention with reference to accompanying drawing.The experimental technique of unreceipted actual conditions in the preferred embodiment, usually according to normal condition, the molecular cloning experiment guide (third edition for example; J. work such as Sa nurse Brooker, Huang Peitang etc. translate, Science Press; 2002) described in condition, or the condition of advising according to manufacturer.

One, the preparation of recombinant adenovirus Ad-DcR3/GAD65

1, DcR3 Full Length cDNA Cloning

Press Trizol test kit specification sheets and extract the total RNA of human liver tissue, rt obtains total cDNA, is template with this total cDNA again, with F1:5 '-aat CtgcagTcgcgagcggccgcacaacttt-3 ' (SEQ ID No.1, underscore partly are Pst I restriction enzyme site) and R1:5 '-tac GaattcGtgcacagggaggaagcgctcacg-3 ' (SEQID No.2, underscore partly are EcoR I restriction enzyme site) is the upstream and downstream primer, pcr amplification DcR3 full-length cDNA, and the PCR system is 50 μ l, the cycling condition parameter is: 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes.The PCR product is after agarose gel electrophoresis evaluation, gel recovery test kit are cut glue recovery purifying; Be connected with the pT-Easy carrier; Connect product transformed into escherichia coli DH5 α competent cell,, extract plasmid with the LB plate screening positive colony that contains penbritin; Order-checking is identified, with positive colony plasmid called after pT-DcR3.

The agarose gel electrophoresis result shows that the PCR product is single specificity band (Fig. 1) at about 900bp place, conforms to expected results; The insertion gene order (SEQ ID No.3) that sequencing result shows the positive colony plasmid and GenBank accession number are that the DcR3 full length cDNA sequence of NM_003823 is consistent.

2, GAD65 Full Length cDNA Cloning

Press Trizol test kit specification sheets and extract the total RNA of Human Pancreas, reverse transcription obtains total cDNA, is template with this total cDNA again, with F2:5 '-gatc GgatccTaccgtagaggccc-3 ' (SEQ ID No.4, underscore partly are BamH I restriction enzyme site) and R2:5 '-acgc GtcgacAatatttagaacaggttcc-3 ' (SEQ ID No.5, underscore partly are Sal I restriction enzyme site) is the upstream and downstream primer, pcr amplification GAD65 full-length cDNA, and the PCR system is 50 μ l, the cycling condition parameter is: 94 ℃ of preparatory sex change 5 minutes; 94 ℃ of sex change are 50 seconds then, 58 ℃ of annealing 50 seconds, and 72 ℃ were extended totally 30 circulations 2 minutes; Last 72 ℃ were extended 10 minutes.The PCR product is after agarose gel electrophoresis evaluation, gel recovery test kit are cut glue recovery purifying; Be connected with the pT-Easy carrier; Connect product transformed into escherichia coli DH5 α competent cell,, extract plasmid with the LB plate screening positive colony that contains penbritin; Order-checking is identified, with positive colony plasmid called after pT-GAD65.

The agarose gel electrophoresis result shows that the PCR product is single specificity band (Fig. 2) at about 1800bp place, conforms to expected results; The insertion gene order (SEQ ID No.6) that sequencing result shows the positive colony plasmid and GenBank accession number are that the GAD65 full length cDNA sequence of NM_000818 is consistent.

3, the structure of recombinant vectors pStar-DcR3-IRES-GAD65

According to the restriction enzyme site that DcR3 and GAD65 full-length cDNA two ends are designed, the IRES upper reaches of at first the DcR3 full-length cDNA being inserted the pStar carrier, the IRES downstream of again the GAD65 full-length cDNA being inserted the pStar carrier.Concrete grammar is: earlier with the pT-DcR3 carrier with Pst I and EcoR I double digestion; The double digestion product and is connected connection product transformed into escherichia coli DH5 α competent cell equally through the pStar carrier of Pst I with EcoR I double digestion after gel reclaims test kit and cuts glue and reclaim purifying; With the LB plate screening positive colony that contains penbritin; Extract plasmid, Pst I and EcoR I double digestion are identified, with positive colony plasmid called after pStar-DcR3; Again with the pT-GAD65 carrier with BamH I and Sal I double digestion; The double digestion product and is connected connection product transformed into escherichia coli DH5 α competent cell equally through the pStar-DcR3 carrier of BamH I with Sal I double digestion after gel reclaims test kit and cuts glue and reclaim purifying; With the LB plate screening positive colony that contains penbritin; Extract plasmid, BamH I and Sal I double digestion are identified, with positive colony plasmid called after pStar-DcR3-IRES-GAD65.

4, the structure of recombinant vectors pShuttle-CMV-DcR3-IRES-GAD65

With the pStar-DcR3-IRES-GAD65 carrier is template, with F3:5 '-acgc GtcgacTcgcgagcggccg-cacaacttt-3 ' (SEQ ID No.7, underscore partly are Sal I restriction enzyme site) and R3:5 '-agaatc Gcggccgc-aatatttagaacaggttcc-3 ' (SEQ ID No.8; Underscore partly is a Not I restriction enzyme site) be the upstream and downstream primer; Pcr amplification DcR3-IRES-GAD65 fragment also is replaced by Sal I restriction enzyme site and Not I restriction enzyme site with the restriction enzyme site at these fragment two ends, and PCR system and cycling condition parameter are the same.The PCR product is after agarose gel electrophoresis evaluation, gel recovery test kit are cut glue recovery purifying; Be connected with the pT-Easy carrier; Connect product transformed into escherichia coli DH5 α competent cell,, extract plasmid with the LB plate screening positive colony that contains penbritin; Order-checking is identified, with positive colony plasmid called after pT-DcR3-IRES-GAD65.

With the pT-DcR3-IRES-GAD65 carrier with Sal I and Not I double digestion; The double digestion product and is connected connection product transformed into escherichia coli DH5 α competent cell equally through the pShuttle-CMV carrier of Sal I with Not I double digestion after gel reclaims test kit and cuts glue and reclaim purifying; With the LB plate screening positive colony that contains penbritin; Extract plasmid, Sal I and Not I double digestion are identified, with positive colony plasmid called after pShuttle-CMV-DcR3-IRES-GAD65.

5, the structure of recombinant adenoviral vector pAd-DcR3/GAD65

The pShuttle-CMV-DcR3-IRES-GAD65 carrier with Pme I linearization for enzyme restriction, with electric transformed into escherichia coli BJ5183 competent cell of pAdEasy-1 carrier while, is carried out homologous recombination in the born of the same parents again; Cultivate with the LB that contains kantlex is dull and stereotyped; Picking with the LB liquid nutrient medium overnight cultures that contains kantlex, extracts plasmid than small colonies; Pac I enzyme is cut evaluation, with positive plasmid called after pAd-DcR3/GAD65.

6, the packing of recombinant adenoviral vector pAd-DcR3/GAD65

The human embryonic kidney 293 cell is seeded in the T-25 culturing bottle with 40%～60% cell density; Treat that cell grows to 30%～50% and discards nutrient solution when merging, with Lipofectamine 2000 reagent with pAd-DcR3/GAD65 carrier rotaring redyeing 293 cell, through the microscope observing cell pathological change; Centrifugal collecting cell when waiting to occur obvious cytopathic effect; Cell precipitation with PBS resuspended after, multigelation makes lysis 4 times, centrifugal removal cell debris; Collection contains the supernatant of virus, promptly gets the Ad-DcR3/GAD65 virus stock solution used; Virus stock solution used is infected 293 cells again in 1: 10 ratio, collects the supernatant that contains virus, repeat aforesaid operations 3 times, the 4th generation recombinant adenovirus Ad-DcR3/GAD65.

Two, the preparation of recombinant adenovirus Ad-DcR3/GAD65 transfection dendritic cell vaccine

1, the sorting of BMDC

Get 4 age in week the Balb/c mouse, take off neck and put to death, using volume(tric)fraction is 75% alcohol solution dipping 5 minutes; Aseptic condition cuts down skin and subcutis, separates mouse femur and shin bone, uses concentration to be the aseptic PBS cleaning of 0.01mol/L 3～5 times; Cut off femur and shin bone two ends, use concentration to wash medullary space repeatedly, process single cell suspension as the aseptic PBS of 0.01mol/L; 1000r/min removed lipid layer in centrifugal 5 minutes, and cell uses concentration resuspended as the aseptic PBS of 0.01mol/L, slowly added equal-volume Percoll cellular segregation liquid again; Centrifugal 30 minutes of 3000r/min, collecting monocytic cell uses concentration to be the aseptic PBS washing of 0.01mol/L 2 times; Resuspended with the DMEM substratum that contains serum again; The gained single cell suspension is inoculated in the T-25 culturing bottle, and additional granulocyte-macrophage colony stimutaing factor (GM-CSF) and interleukin 4 (IL-4), and putting temperature is 37 ℃, CO ₂The gas volume mark be 5% with the incubator of saturated humidity in cultivate, the next day half amount change liquid and replenish cytokine, the 7th day harvested cell promptly gets BMDC.

2, recombinant adenovirus Ad-DcR3/GAD65 changes seven BMDC

Is 1 * 10 with BMDC with cell concn ⁶Individual/ml is inoculated in 6 orifice plates, is 100 addings the 4th generation recombinant adenovirus Ad-DcR3/GAD65 by infection multiplicity (MOI), infects back 48 hours collecting cells, with PBS washing and resuspended, promptly gets Ad-DcR3/GAD65 virus transfection BMDC.

Western Blot detects: difference ultrasonic degradation Ad-DcR3/GAD65 virus transfection BMDC and empty virus transfection BMDC, and the collecting cell total protein carries out SDS-PAGE, electric transfer printing pvdf membrane after electrophoresis finishes; Wash film, sealing adds anti-people DcR3 of rabbit or GAD65 polyclonal antibody; Hatched 1 hour for 37 ℃, wash film, add goat anti-rabbit igg again; Hatched 1 hour for 37 ℃, wash film, colour developing.The result sees Fig. 3, and Ad-DcR3/GAD65 virus can be expressed DcR3 and GAD65 in BMDC.

Three, the anti-pancreatic islets transplantation repulsion of recombinant adenovirus Ad-DcR3/GAD65 transfection dendritic cell vaccine ability detects

Get 2 monthly age mellitus susceptibility NOD mouse, be divided into two groups at random: experimental group and control group, experimental group tail vein feed back Ad-DcR3/GAD65 virus transfection dendritic cell vaccine, feed back continuously 3 times, 1 time weekly, import 1 * 10 at every turn ⁶Individual cell; Control group is normally raised.

1, the lymphopoiesis ability detects

Last feeds back 1 week of back; Disconnected neck is put to death two groups of mouse; Get mouse spleen under the aseptic condition; Be milled into cell suspension with 100 eye mesh screens, separate obtaining SPL with conventional Ficoll-Hypaque layering liquid gradient centrifugation, use that to contain volume(tric)fraction be that the RPMI 1640 substratum adjustment cell concn of 10% foetal calf serum is 1 * 10 ⁶Individual/ml, be seeded to 96 orifice plates, every hole 100 μ l establish 3 multiple holes for every group, and it is 1mg/ml to final concentration that every hole adds the GAD65 recombinant protein, and putting temperature is 37 ℃, CO ₂The gas volume mark is 5% interior the cultivation 96 hours of incubator, and it is 1 * 10 that every again hole adds 0.1ml concentration ³3H-thymidine (3H-TdR) solution of μ Ci/L continue to be cultivated 12 hours, with PBS rinsing cell 3 times; Formaldehyde fixed 10 minutes, the volume(tric)fraction of using 4 ℃ of precoolings of temperature again are 5% trichloroacetic acid solution rinsing 3 times, and it is the NaOH solution of 0.3mol/L that every hole adds 0.5ml concentration; 60 ℃ of water-baths 30 minutes are cooled to room temperature, are transferred in the scintillation vial; Add the 5ml scintillation solution; With the flicker number of times (cpm) of liquid scintillation counter counting PM, measure DPM value (reflection cell DNA synthesis rate), replication 3 times.

The result sees Fig. 4; The lymphocytic cpm value of experimental mice is 38000 ± 3100; And the lymphocytic cpm value of control group mice is 54000 ± 6500, confirms that Ad-DcR3/GAD65 virus transfection dendritic cell vaccine can effectively reduce lymphocytic multiplication capacity.

2, lymphocytic emiocytosis cytokine ability detects

Last feeds back 1 week of back; Disconnected neck is put to death two groups of mouse; Get mouse spleen under the aseptic condition; Be milled into cell suspension with 100 eye mesh screens, separate obtaining SPL with conventional Ficoll-Hypaque layering liquid gradient centrifugation, use that to contain volume(tric)fraction be that the RPMI 1640 substratum adjustment cell concn of 10% foetal calf serum is 1 * 10 ⁶Individual/ml, be seeded to 96 orifice plates, every hole 100 μ l establish 3 multiple holes for every group, and it is 1mg/ml to final concentration that every hole adds the GAD65 recombinant protein, and putting temperature is 37 ℃, CO ₂The gas volume mark is 5% interior the cultivation 96 hours of incubator, detects the content of interleukin-22 (IL-2) and interferon-(IFN-γ) with the ELISA method.

The result sees Fig. 5; Lymphocytic IL-2 of experimental mice and IFN-γ content are respectively 51.3 ± 5.7pg/ml and 45.6 ± 4.9pg/ml; And lymphocytic IL-2 and the IFN-γ content of control group mice is respectively 114.8 ± 12.3pg/ml and 99.3 ± 102pg/ml, confirms that Ad-DcR3/GAD65 virus transfection dendritic cell vaccine can effectively reduce the ability of lymphocytic emiocytosis cytokine.

3, NOD mouse onset diabetes rate detects

The glucose level of monitoring experiment group and control group mice.

The result sees Fig. 6, and begin to occur pathoglycemia ages in experimental mice 18 week, and the onset diabetes rate in 30 ages in week is 30%, and control group mice begins to occur pathoglycemia 14 ages in week, and the onset diabetes rate in 30 ages in week is up to 80%; Confirm that Ad-DcR3/GAD65 virus transfection dendritic cell vaccine can effectively postpone the disease time of NOD mouse mellitus, and reduce the sickness rate of mellitus.

4, islet transplantation detects lifetime

Islet cells separates and purifying: after the dislocation of C57BL/6J mouse cervical vertebra is put to death; The use volume(tric)fraction is 75% alcohol solution dipping sterilization, opens the abdominal cavity under the aseptic condition, exposes common bile duct; Ligation courage ductus pancreaticus is at duodenal opening under dissecting microscope; Along common bile duct retroperfusion Hank ' s liquid (containing collagenase P and the concentration that concentration is 0.1g/L is the DNase-I of 10mg/L), treat that pancreas expands after, extract pancreatic tissue immediately; Put in the Hank ' s liquid of 4 ℃ of precoolings of temperature and clean 2 times, be cut into 0.5～1mm ³Fritter, the Hank ' s liquid that adds 4 ℃ of precoolings of temperature immediately stopped digestion to becoming cotton-shaped in 10～15 minutes in 37 ℃ of temperature vibration digestion; With Hank ' s liquid washing 2 times; Cell precipitation is resuspended with Hank ' s liquid, adds massfraction more successively and be 84%, 67%, 50% Histopaque, centrifugal 10 minutes of 1500g; The cell precipitation of 84%～67% and 67%～50% aspect of absorption; With Hank ' s liquid washing 2 times, use DMEM substratum (containing glucose, the volume(tric)fraction that concentration is 5.6mmol/L is that 10% foetal calf serum, penicillium mould and the concentration that concentration is 100000U/L are the Streptomycin sulphate of 100mg/L) resuspended again, the islet cells counting is carried out in sampling.

Islet cell transplantation: 1 week after experimental group and control group NOD mouse detect pathoglycemia respectively, with 5 * 10 ⁵The islet cell transplantation of individual C57BL/6J mouse detects mouse blood sugar every day to the peritonaeum of NOD mouse, when glucose level returns to 11.1mM, think successful transplantation, investigates the lifetime of two groups of mouse islet transplantations.

The result is as shown in Figure 7; The mean survival time (MST) of experimental mice islet transplantation is 11 days; And the mean survival time (MST) of control group mice islet transplantation is 4 days, confirms that Ad-DcR3/GAD65 virus transfection dendritic cell vaccine can effectively prolong the lifetime of NOD mouse islet transplantation.

Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although through invention has been described with reference to the preferred embodiments of the present invention; But those of ordinary skill in the art should be appreciated that and can make various changes to it in form with on the details, and the spirit and scope of the present invention that do not depart from appended claims and limited.

Sequence table

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ccgtgccgcc?gagacagccc?cacgacgtgt?ggcccgtgtc?caccgcgcca?ctacacgcag 240

ttctggaact?acctggagcg?ctgccgctac?tgcaacgtcc?tctgcgggga?gcgtgaggag 300

gaggcacggg?cttgccacgc?cacccacaac?cgtgcctgcc?gctgccgcac?cggcttcttc 360

gcgcacgctg?gtttctgctt?ggagcacgca?tcgtgtccac?ctggtgccgg?cgtgattgcc 420

ccgggcaccc?ccagccagaa?cacgcagtgc?cagccgtgcc?ccccaggcac?cttctcagcc 480

agcagctcca?gctcagagca?gtgccagccc?caccgcaact?gcacggccct?gggcctggcc 540

ctcaatgtgc?caggctcttc?ctcccatgac?accctgtgca?ccagctgcac?tggcttcccc 600

ctcagcacca?gggtaccagg?agctgaggag?tgtgagcgtg?ccgtcatcga?ctttgtggct 660

ttccaggaca?tctccatcaa?gaggctgcag?cggctgctgc?aggccctcga?ggccccggag 720

ggctggggtc?cgacaccaag?ggcgggccgc?gcggccttgc?agctgaagct?gcgtcggcgg 780

ctcacggagc?tcctgggggc?gcaggacggg?gcgctgctgg?tgcggctgct?gcaggcgctg 840

cgcgtggcca?ggatgcccgg?gctggagcgg?agcgtccgtg?agcgcttcct?ccctgtgcac 900

tga 903

<210>4

<211>24

<212>DNA

< 213>artificial sequence

<220>

< 223>primers F 2

<400>4

gatcggatcct?accgtagag?gccc 24

<210>5

<211>29

<212>DNA

< 213>artificial sequence

<220>

< 223>primer R2

<400>5

acgcgtcgac?aatatttaga?acaggttcc 29

<210>6

<211>1758

<212>DNA

< 213>homo sapiens (homo sapiens)

<400>6

atggcatctc?cgggctctgg?cttttggtct?ttcgggtcgg?aagatggctc?tggggattcc 60

gagaatcccg?gcacagcgcg?agcctggtgc?caagtggctc?agaagttcac?gggcggcatc 120

ggaaacaaac?tgtgcgccct?gctctacgga?gacgccgaga?agccggcgga?gagcggcggg 180

agccaacccc?cgcgggccgc?cgcccggaag?gccgcctgcg?cctgcgacca?gaagccctgc 240

agctgctcca?aagtggatgt?caactacgcg?tttctccatg?caacagacct?gctgccggcg 300

tgtgatggag?aaaggcccac?tttggcgttt?ctgcaagatg?ttatgaacat?tttacttcag 360

tatgtggtga?aaagtttcga?tagatcaacc?aaagtgattg?atttccatta?tcctaatgag 420

cttctccaag?aatataattg?ggaattggca?gaccaaccac?aaaatttgga?ggaaattttg 480

atgcattgcc?aaacaactct?aaaatatgca?attaaaacag?ggcatcctag?atacttcaat 540

caactttcta?ctggtttgga?tatggttgga?ttagcagcag?actggctgac?atcaacagca 600

aatactaaca?tgttcaccta?tgaaattgct?ccagtatttg?tgcttttgga?atatgtcaca 660

ctaaagaaaa?tgagagaaat?cattggctgg?ccagggggct?ctggcgatgg?gatattttct 720

cccggtggcg?ccatatctaa?catgtatgcc?atgatgatcg?cacgctttaa?gatgttccca 780

gaagtcaagg?agaaaggaat?ggctgctctt?cccaggctca?ttgccttcac?gtctgaacat 840

agtcattttt?ctctcaagaa?gggagctgca?gccttaggga?ttggaacaga?cagcgtgatt 900

ctgattaaat?gtgatgagag?agggaaaatg?attccatctg?atcttgaaag?aaggattctt 960

gaagccaaac?agaaagggtt?tgttcctttc?ctcgtgagtg?ccacagctgg?aaccaccgtg?1020

tacggagcat?ttgaccccct?cttagctgtc?gctgacattt?gcaaaaagta?taagatctgg?1080

atgcatgtgg?atgcagcttg?gggtggggga?ttactgatgt?cccgaaaaca?caagtggaaa?1140

ctgagtggcg?tggagagggc?caactctgtg?acgtggaatc?cacacaagat?gatgggagtc?1200

cctttgcagt?gctctgctct?cctggttaga?gaagagggat?tgatgcagaa?ttgcaaccaa?1260

atgcatgcct?cctacctctt?tcagcaagat?aaacattatg?acctgtccta?tgacactgga?1320

gacaaggcct?tacagtgcgg?acgccacgtt?gatgttttta?aactatggct?gatgtggagg?1380

gcaaagggga?ctaccgggtt?tgaagcgcat?gttgataaat?gtttggagtt?ggcagagtat?1440

ttatacaaca?tcataaaaaa?ccgagaagga?tatgagatgg?tgtttgatgg?gaagcctcag?1500

cacacaaatg?tctgcttctg?gtacattcct?ccaagcttgc?gtactctgga?agacaatgaa?1560

gagagaatga?gtcgcctctc?gaaggtggct?ccagtgatta?aagccagaat?gatggagtat?1620

ggaaccacaa?tggtcagcta?ccaacccttg?ggagacaagg?tcaatttctt?ccgcatggtc?1680

atctcaaacc?cagcggcaac?tcaccaagac?attgacttcc?tgattgaaga?aatagaacgc?1740

cttggacaag?atttataa 1758

<210>7

<211>32

<212>DNA

< 213>artificial sequence

<220>

< 223>primers F 3

<400>7

acgcgtcgac?tcgcgagcgg?ccgcacaact?tt 32

<210>8

<211>32

<212>DNA

< 213>artificial sequence

<220>

< 223>primer R3

<400>8

agaatgcggc?cgcaatattt?agaacaggtt?cc 32

Claims

1.DcR3 with GAD65 double gene coexpression recombinant adenovirus; It is characterized in that: comprising a trick acceptor 3 in the said recombinant adenovirus genome is that DcR3 and L-Glutamic decarboxylase 65 are GAD65 double gene expression box, and said DcR3 and GAD65 double gene expression box comprise CMV promotor, DcR3 total length encoding sox, terminator, internal ribosome entry site IRES, GAD65 total length encoding sox and terminator successively by the upper reaches to downstream.

2. the preparation method of said DcR3 of claim 1 and GAD65 double gene coexpression recombinant adenovirus is characterized in that: may further comprise the steps:

A, RT-PCR amplification DcR3 full-length cDNA;

B, RT-PCR amplification GAD65 full-length cDNA;

3. said DcR3 of claim 1 and the GAD65 double gene coexpression recombinant adenovirus application in the dendritic cell vaccine that the anti-pancreatic islets transplantation of preparation is repelled.