CN103667345B - A kind of Antioncogene magnetic composite nano particle and preparation method - Google Patents

Abstract

The present invention is a kind of Antioncogene magnetic composite nano particle and preparation method, Egr1 and Hsp70 promotor and HSVTK gene is obtained by pcr amplification, cut through enzyme, the step such as connection is connected in pCDNA3.1 plasmid, and be the channel genes HEK293 cell that genophore will build with magnetic nano particle, the expression of quantitative observation HSV-TK gene after radiation, magnetic thermotherapy or the two common induction.The Egr1 promotor of this plasmid is after 2GyX ray induction after 6 hours, and the expression amount of target gene is the highest.Egr1 and Hsp70 promotor all has abduction delivering activity, can in radiation and heat shock respectively and under combined action, induce the expression of goal gene, and stronger to the ability of Primary structure during combined action, for the complex therapy carrying out cancer further provides experimental basis.

Description

A kind of Antioncogene magnetic composite nano particle and preparation method

Technical field

The present invention relates to a kind of radiation as Therapeutic cancer, the structure of double-promoter pCDNA3.1-Egr1-Hsp70-HSVTK gene of heat-inducible and magnetic composite nano particle and preparation thereof.

Background technology

In recent years, many new gene therapy methods (comprising radiation-gene therapy, gene-mistake heat cure) are applied in liver cancer treatment research in succession, and wherein applying the goal gene that more, curative effect comparatively affirms is HSV-TK/GCV system.The goal gene that it adopts is suicide gene (Suicide gene) or claims drug sensitive gene, its treatment principle to make nontoxic prodrug (as GCV etc.) be converted into virose medicine, by mixing cell DNA, block the synthesis of cell DNA, thus optionally kill the tumour cell of fast breeding.But before gene delivery problem does not well solve, it is very difficult for realizing comprehensive therapy of tumor and obtain desirable curative effect.Virus carrier system is most efficient gene transfer method up to now.But due to its limitation (as lacked efficient and directed carrier system, gene enter Controllability etc. in body), the especially existence of safety problem, makes its clinical application be strictly controlled; Although non-viral carrier systems avoids great potential safety hazard, its transfection efficiency not as virus carrier system, is difficult to obtain significant genetic expression always.Therefore, the task of top priority that transgenosis bottleneck has become field of gene how is broken through.

Along with the development of nanotechnology, be that the research of gene transfer vector attracts wide attention with nano particle.The solution gene transfer vector problem that appears as of nanotechnology provides new thinking.Be that the gene therapy molecules such as DNA, RNA are wrapped in nano particle or are adsorbed on its surface by gene transfer vector exactly with nano particle, enter under endocytosis in cell and discharge gene therapy molecule.International and domesticly in succession develop multiple nano gene transfer vector, and towards trend development that is inorganic, organic and biomaterial hydridization.This study group also sets foot in this field earlier, and develops the multiple magnetic nano-carrier such as magnetic Mn-Zn ferrite nano particle, nano-magnetic liposome with independent intellectual property right.Early-stage Study shows that magnetic nano-carrier preparation is simple, inorganization cytotoxicity, be easy to finishing and make it have very high biocompatibility.Magnetic nano-carrier, except having the characteristic of general nano particle, also has superparamagnetism, can under the effect of externally-applied magnetic field, displacement, thus carries out target gene therapy.Moreover, magnetic nanoparticle also can heat up for tumor thermotherapy under additional the action of a magnetic field in magneticinduction.

Chemotherapy combined radiotherapy thermotherapy treatment malignant tumour has started for clinical in the eighties in 20th century, along with going deep into of research, has had the research of darker people to the mechanism of chemotherapy combined radiotherapy thermotherapy treatment malignant tumour.The combined utilization of radiotherapy and thermotherapy, not only can play respective cytotoxicity, and thermotherapy has radiosensitizing effect.Malignant tumour volume is large, and Liu Ti center is in anoxia state, resists radioactive rays, responsive to the oxygen-rich fraction around knurl body, therefore radiotherapy alone to a certain extent after, the gross tumor volume degree of shrinking back slows down or no longer shrinks back.And thermotherapy can the breathing of inhibition tumor cell, anaerobic glycolysis is strengthened under anoxia state, environment acidity is increased, in sour environment, easily activate lysosomal activity, suppress DNA, RNA and albumen synthesis, make cell membrane disruption, skeleton is at random, and cell function is impaired, finally causes cancer cell death.On the contrary, to circulate good cell to tumour peripheral blood, owing to being difficult to reach effective temperature, and curative effect is relatively poor, but the sensitive area of radiotherapy.S phase tumour cell is the strongest to radiotherapy opposing, and the most responsive to thermotherapy.Therefore, radiotherapy and combined with hyperthermia treat the effect of effective in cure complementation and addition.Thermotherapy 30min after radiotherapy carries out, can also inhibition tumor cell to the reparation of sublethal damage caused by radioactive rays and Potentially lethal damage, the oxygen level of tumour can be significantly improved, strengthen the susceptibility of tumour radiotherapy.

Heat shock protein 70 (heat shock protein 70) promotor (Hsp70promoter) has heat-inducible, and its promoter sequence can induce downstream gene expression after heat effect (as 42 DEG C-45 DEG C).IsomotoH etc. utilize the feature of heat effect induced heat shock protein (heat stress protein) high expression level, suicide gene is combined with Hsp70 promotor, to build after corresponding adenovirus carrier in transfered cell, high expression level suicide gene after heat effect, then give Cymevan killer cell, in vitro study shows remarkable lethal effect.This novel method is called as gene-mistake heat cure.This seminar constructs a kind of novel nano magnetic genophore, by itself and P1730OR (mammalian expression vector, coding β-gal, by Heat shock protein 70 promoters driven) combine be prepared into matrix material, can effectively be warming up to 43 DEG C under magneticinduction heating condition, β-gal expression intensity obviously increases along with the rising of temperature.On this basis, this group constructs the pHsp-HSV-TK/As of heat-inducible promoter regulation and control ₂o ₃composite magnetic nanoparticle, induces expression of suicide gene under magneticinduction heating condition, and and As ₂o ₃(white arsenic) acting in conjunction killing hepatoma cell.Because this carrier is inorganic non-virus carrier, itself specific magneticinduction intensification temperature control characteristic in addition, can make gene-mistake thermal treatment method safer, stable, also more convenient practicality.

Radiation-gene therapy (radio-genetic therapy), also known as radiation-burn combined injury, be about to that there is the regulating and controlling sequence of radiation-induced property and the coupling of therapeutic gene phase, the inducible gene expression when implementing local radiotherapy/nucleic internal radiation to tumour, causes the double treatment of ray and gene pairs tumour.Like this, can relatively reduce equivalent irradiation dose on the one hand, alleviate the damage of normal tissue; The localization and expression of tumor-killing gene is realized on the other hand by the partial irradiation of ray.Utilize the radiosensitivity of Egr-1 Gene Promoter sequence and the research of radiation-gene therapy carried out achieves challenging achievement, but the method be still faced with exist in gene therapy specificity, security, the gene expression efficiency low time is short, cytotoxicity is little, after transfer genetic expression lack the problems such as effective control measures.And, because the ubiquitous anaerobic environment of solid tumor also can make the induced activity of radiation promotor reduce, have impact on curative effect and the application of this therapy.

Interestingly, research finds that the number by increasing promotor can improve the expression level of goal gene, the method of more employing is that two single promotors series connection are formed double-promoters, and this be how to improve the key issues such as the expression of goal gene and spatiotemporal database in solution gene therapy process to provide a new strategy.The discovery display double-promoter of the people such as J Finn effectively can improve the gene expression dose of non-virus carrier, therefore has more important clinical value.It may be that multiple cis-acting elements influences each other on regulatory gene transcriptional level, coefficient result that research thinks that double-promoter starting power strengthens.Accordingly, this research is imagined: expression radiation promotor and heat-inducible promoter being combined to form double-promoter to strengthen induction and regulatory gene, on the basis of nucleic internal radiation and combined with hyperthermia, radiation-gene therapy, gene-mistake heating therapy and nanotechnology are organically combined, have complementary advantages, likely form a kind of new tumour comprehensive gene therapy: radiation-overheated-gene therapy.And thermotherapy can strengthen the blood confession of tumor by local, improve oxygen supply, thus play the sensitization of radiation to tumour cell treatment under weary oxygen environment.

Target is established in this research: expression radiation promotor and heat-inducible promoter being combined to form sensitive promotor to strengthen induction and regulatory gene, on the basis of radiation and combined with hyperthermia, radiation-gene therapy, gene-mistake heating therapy and nanotechnology are organically combined, have complementary advantages, likely form a kind of new tumour comprehensive gene therapy: radiation-overheated-gene therapy.And thermotherapy can strengthen the blood confession of tumor by local, improve oxygen supply, thus play the sensitization of radiation to tumour cell treatment under weary oxygen environment.

Summary of the invention

Technical problem: the invention provides one and can be used for radiation, heat shock sensitive promotor strengthens the Antioncogene magnetic composite nano particle of inducing and regulation and control Antioncogene is expressed, and provides a kind of preparation method of above-mentioned Antioncogene magnetic composite nano particle simultaneously.

Technical scheme: the method preparing Antioncogene magnetic composite nano particle of the present invention, comprises the steps:

1) with pIRES-CMVE-Egr1p eukaryon expression plasmid for template, carry out polymerase chain reaction, obtain radiation-induced promoter fragment, by its subclone on pCDNA3.1-EGFP plasmid, obtain pCDNA3.1-Egr1-EGFP eukaryon expression plasmid;

2) to be loaded with the plasmid of warm start and suicide gene for template, carry out polymerase chain reaction, obtain warm start sub-pieces section and Antioncogene fragment;

3) by Antioncogene fragment subclone to step 1) in the pCDNA3.1-Egr1-EGFP eukaryon expression plasmid that obtains, replace EGFP, obtain pCDNA3.1-Egr1-HSV-TK eukaryon expression plasmid;

4) by warm start sub-pieces section subclone on pCDNA3.1-Egr1-HSV-TK eukaryon expression plasmid, obtain pCDNA3.1-Egr1-Hsp70-HSVTK eukaryon expression plasmid;

5) by the Mn of Polyethylenimine load _0.5zn _0.5fe ₂o ₄nanoparticle and pCDNA3.1-Egr1-Hsp70-HSV-TK eukaryon expression plasmid mass ratio are 20:1 ~ 50:1, the two is diluted with serum free medium respectively, room temperature places more than 5 minutes, then the diluting soln of the two is mixed evenly, incubated at room temperature more than 30 minutes, obtain pEgr1-Hsp70-HSV-TK/PEI-MZF-NPs mixture, be Antioncogene magnetic composite nano particle.

Oncogene magnetic composite nano particle of the present invention prepares according to the method described above.

Beneficial effect: the present invention compared with prior art, has the following advantages:

1, enzyme is cut, the qualification result that checks order shows often to walk and all obtain object plasmid, through the goal gene molecular weight of pcr amplification acquisition and the identical of expectation, insertion vector correct position, order-checking finds that Insert Fragment sequence is without change, and opening code-reading frame is correct, obtains double-promoter plasmid pEgr1-Hsp70-HSV-TK.Sequencing result is specifically shown in accompanying drawing (Fig. 1-4).

2, Mn prepared by chemical coprecipitation is improved _0.5zn _0.5fe ₂o ₄magnetic nano particle is brown powder, high-resolution electron microscopy (High Resolution Electron Microscopy, HREM) show nanoparticle size comparatively evenly, electron density is high, particle diameter is (Fig. 5) between 30nm-40nm; Energy spectrum analysis (EDS) confirms containing Mn, Zn, Fe and O four kinds of elements in the MZF nanoparticle of preparation, and the mol ratio of Mn, Zn, Fe is about 1:1:4 (Fig. 6).Fourier infrared spectrum (fourier transform infrared spectroscopy, FTIR) characterizes display :-NH appears in the infrared spectrogram of particle after modifying ₂with-CH ₂-feature small peak, demonstrate PEI and can produce absorption (Fig. 7) at particle surface.And external intensification experiment display, the MZF magnetic nano particle of 10mg/mL and the magnetic fluid of PEI-MZF composite nano-granule, under the AMF effect of same intensity, can rise to 43 DEG C after 40Min and keep stable, can be used for thermotherapy (Fig. 8).

3, in the research carrying out radiosensitive promotor efficiency, under first the HEK293 cell of Egr1-Hsp70-HSV-TK/PEI-MZF mixture is placed in particle accelerator by transfection, give the roentgen radiation x of 2Gy dosage, then observe the expression of TK gene.Collecting cell when 1h, 6h, 12h, 24h, 48h after treatment, the relative expression quantity detecting TK gene with QPCR finds, after induction, namely the expression amount of 1h, TK gene starts to raise, and 6h is raised to maximum (Fig. 9).Each group of fluorescence intensity is respectively 1.86 ± 0.21 (1h), 4.53 ± 0.29 (6h), 3.72 ± 0.30 (12h), 1.31 ± 0.19 (24h) and 0.33 ± 0.15 (48h).Find after comparing result, before 6h, the prolongation in time of the TK expression intensity under Egr1 promoters driven and increasing, reduces afterwards gradually.If get most high expression level compared with minimum expression level, when 6h, 5.8 times when the expression of TK is 48h, 2.35 times when being 1h, 1.19 times when being 12h.When after 48h, its expression amount drops to 1h level thereupon 2/5.After radiation-induced, the expression amount of TK is improved, and other organize P < 0.05 compared with 48h group; The expression amount of 6h group TK compares P < 0.05 with 24h with 48h group, the results are shown in Figure 9.

4, to study gene transcript expression situation after the induction of different radiation dose, under the HEK293 cell of Egr1-Hsp70-HSVTK/PEI-MZF mixture is placed in particle accelerator by transfection, give 0Gy respectively, 1Gy, 2Gy, the roentgen radiation x of 4Gy, 8Gy, 16Gy dosage, collecting cell during 6h after treatment, the relative expression quantity detecting TK gene with QPCR finds: fluorescence intensity is respectively 0.47 ± 0.04,0.56 ± 0.02,2.78 ± 0.12,1.95 ± 0.05,1.81 ± 0.09 and 1.67 ± 0.18.After find, the highest (see photo) of expression amount during 2Gy.When 1Gy, the TK expression intensity under Egr1 promoters driven starts to increase along with the increase of radiation dose, slightly reduces afterwards.If get most high expression level compared with minimum expression level, when 2Gy, 5.91 times when the expression of TK is 0Gy, 4.96 times when being 1Gy.When 2Gy even 8 thereupon, its expression amount of 16Gy drops to 2Gy level 0.7.Each radiation group is compared with non-raying group, and the expression amount of TK is improved, and P < 0.05; P < 0.05 (Figure 10) compared with expression amount and other 5 groups of 2Gy induction group TK.

5, the difference of single, double promotor to gene regulating efficiency is studied, the Egr1-HSVTK by transfection respectively, different treatment condition are given: the roentgen radiation x of 2Gy dosage under the HEK293 cell of Hsp70-HSVTK and Egr1-Hsp70-HSVTK is placed in linear accelerator, the radiation carrying out magnetic thermotherapy and 2Gy under being placed in alternating magnetic field adds magnetic thermotherapy dual function and does not give any induction, then observes the expression of TK gene.Collecting cell after process, the relative expression quantity detecting TK gene with QPCR finds, after giving heat shock, the dual induction of radiation, the expression amount of TK gene is the highest, 2.4 times when not giving any induction, 1.68 times during tailored radiation induction, 1.4 times (Figure 11) when being simple thermal induction.When the expression amount of TK is a little more than tailored radiation during simple thermal induction, be about its 1.18 times.Induction group with do not induce compared with group, the expression amount of TK is improved, and P < 0.05; Compared with expression amount and other three groups of dual induction group TK, P < 0.05, the results are shown in Figure 11.

6, RT-PCR detects the integrative gene expression of TK gene in pEgr1-Hsp70-HSVTK transfectional cell

The pEgr1-Hsp70-HSV-TK plasmid of double-promoter is imported HEK293 by Magnetofection method, and after radiation, heating induction, PCR method detects the integration of TK gene.Afterwards with HSV-TK gene for template design primer, through pcr amplification, product size is 469bp.Extract RNA after post transcription cloning, the SMMC7721 cell of transfection all occurs positive band.And the cell of untransfected does not detect that TK gene respective strap only detects internal reference GAPDH (Figure 12).Confirm that pEgr1-Hsp70-HSVTK can also express by Successful transfection SMMC-7721 liver cancer cell within it.

The present invention obtains a kind of pEgr1-Hsp70-HSV-TK plasmid, is by radiosensitive promotor Egr1 and the coupling of temperature-sensitive promotor Hsp70 phase, forms radiation, heat shock double-promoter, is connected to Antioncogene HSV-TK upstream.Utilize heat shock and the dual induction regulating controlling of radiation, to accomplishing to be controlled by goal gene at suitable position, reasonable time, even suitable horizontal expression.Adopt Antioncogene magnetic composite nano particle prepared by the inventive method, on the basis of radiation and combined with hyperthermia, radiation-gene therapy, gene-mistake heating therapy and nanotechnology are organically combined, the effect of gene therapy is farthest played, and reduce toxic side effect as far as possible, likely form a kind of new tumour comprehensive gene therapy.

Accompanying drawing explanation

Fig. 1 is for adopting comparison software: the pCDNA3.1-Egr1-EGFP order-checking comparison chart that Dnassit 2.0 obtains.

Fig. 2 is for adopting comparison software: the pCDNA3.1-Egr1-HSV-TK order-checking that Dnassit 2.0 obtains is than the first part of p-figure.

Fig. 3 is for adopting comparison software: the second section of the pCDNA3.1-Egr1-HSVTK order-checking comparison chart that Dnassit 2.0 obtains.

Fig. 4 is for adopting comparison software: the pCDNA3.1-Egr1-HSP70-EGFP order-checking comparison chart that Dnassit 2.0 obtains.

Fig. 5 is the PEI-Mn of 10mg/mL _0.5zn _0.5fe ₂o ₄with Mn _0.5zn _0.5fe ₂o ₄external magneticinduction heating curve figure.

Fig. 6 is Mn _0.5zn _0.5fe ₂o ₄magnetic nano particle high-resolution electron microscopy figure.

Fig. 7 is Mn _0.5zn _0.5fe ₂o ₄energy spectrum analysis figure.

Fig. 8 is for modifying front and back Mn _0.5zn _0.5fe ₂o ₄the FTIR collection of illustrative plates of nanoparticle.

Fig. 9 is relative expression quantity schematic diagram Figure 10 of the radiation-induced rear different time TK gene of 2Gy is the relative expression quantity schematic diagram that different radiation dose induces rear TK gene.

Figure 11 is that different inductive condition affects schematic diagram to TK expression amount.

Figure 12 is HSV-TK amplified production.

Embodiment

Below by embodiment the present invention program done and illustrate further.

Embodiment 1:

The preparation method of Antioncogene magnetic composite nano particle of the present invention:

1) with the eukaryon expression plasmid (i.e. pIRES-CMVE-Egr1p eukaryon expression plasmid) with radiation-induced promotor for template, carry out polymerase chain reaction, obtain radiation-induced promoter fragment (i.e. CMVE-Egr1 fragment), its subclone to be commercially loaded with on the plasmid (i.e. pCDNA3.1-EGFP plasmid) of fluorescin to a kind of, to obtain the radiation-induced eukaryon expression plasmid (i.e. pCDNA3.1-Egr1-EGFP eukaryon expression plasmid) being loaded with fluorescin.Wherein, pIRES-CMVE-Egr1p eukaryon expression plasmid is the eukaryon expression plasmid being loaded with radiation promotor, pIRES refers to a kind of eucaryon plasmid that two kinds of genes can be allowed simultaneously to express, synthesized by west biotech firm the earliest and apply, there is no corresponding Chinese at present, CMVE refers to cytomegalovirus promoter, and Egr1p refers to radiation-induced promotor.PCDNA3.1-EGFP plasmid is a kind of plasmid being loaded with fluorescin, and pCDNA3.1 is the title of a conventional gene clone plasmid, is synthesized the earliest and apply by west biotech firm, and do not have corresponding Chinese at present, EGFP refers to green fluorescent protein.In pCDNA3.1-Egr1-EGFP eukaryon expression plasmid, Egr1 refers to radiation-induced promotor.

The amplification of CMVE-Egr1 promoter fragment:

PCR primer with the CMVE-Egr1 fragment in pIRES-CMVE-Egr1p sequence for stencil design.

Primer sequence is as follows:

Egr1-f：

5’-CCG CTCGAG CG ACGCGT GATCTTCAATATTGGCCATTAGC-3’

---MluI：CG ACGCGT

---XhoI：CCG CTCGAG

Egr1-r：

5’-CCG CTCGAG CTA GCTAGC CCAAGTTCTGCGCGCTGGGAT-3’

---NheI：CTA GCTAGC

---XhoI：CCG CTCGAG

Product：Length＝1117+17+18＝1152bp,GC％＝52.3,Ta＝55.8

Pcr amplification obtains the CMVE-Egr1 fragment of 1152bp size.

The structure of pCDNA3.1-Egr1-EGFP plasmid and qualification:

With Mlu I and Nhe I double digestion pCDNA3.1-EGFP plasmid, the CMVE-Egr1 fragment reclaimed in the first step is connected to carrier, builds pCDNA3.1-Egr1-EGFP.Choose positive colony, extract plasmid in a small amount, carry out double digestion and order-checking qualification.

Fig. 1 is the Sequencing chromatogram of pCDNA3.1-Egr1-EGFP, and by its sequence and the comparison of Egr1 template sequence, known Egr1 sequence is entirely true, shows that pCDNA3.1-Egr1-EGFP successfully constructs.(Sequence0:Egr1 promoter sequence (CMVE sequence excludes) Sequence 1:pCDNA3.1-Egr1-EGFP-EGFP-N-3.ab1 reverse complementary sequence)

2) to be loaded with the plasmid (i.e. pD3SX-Hsp70-HSVTK plasmid) of warm start and suicide gene for template, carry out polymerase chain reaction, obtain warm start sub-pieces section (i.e. Hsp70 fragment) and Antioncogene fragment (i.e. HSV-TK fragment).PD3SX-Hsp70-HSVTK plasmid is the plasmid being loaded with warm start and suicide gene, pD3SX be refer to a kind of with stress the plasmid vector of promotor, synthesized by west biotech firm the earliest and apply, there is no corresponding Chinese at present, Hsp70 refers to warm start, and HSVTK refers to the Antioncogene in suicide gene and this patent.

2.1) pcr amplification of HSV-TK fragment

The pD3SX-HSP70-HSVTK plasmid preserved with this room is template, pcr amplification HSV-TK fragment.

Primer pair:

HSV-TK-f：

5’-G GAATTC GCC ACC ATGGCCTCGTACCCCGGCCAT-3’(EcoRI)

(GCC ACC is Kozak sequence, and reinforcing gene expression is used)

HSV-TK-r：

5’-CCG CTCGAG TCAGTTAGCCTCCCCCATCT-3’(XhoI)

Product：Length＝1131+13+9＝1153bp,GC％＝65.4,Ta＝59.7

Amplification obtains the HSV-TK fragment of 1153bp.

2.2) pcr amplification Hsp70 fragment

With pD3SX-Hsp70-HSV-TK plasmid for template, pcr amplification Hsp70 fragment.

Primer pair:

Hsp70-f：

5’-CCC AAGCTT CTCGAGGCGCGTCCTCAGA-3’(HindIII)

Hsp70-r：

5’-G GAATTC GGTCGACTAGAGAGCTTCTT-3’(EcoRI)

Product：Length＝418+9+7＝434bp,GC％＝70.6,Ta＝58

Obtain the Hsp70 promoter fragment that size is 434bp.

3) by Antioncogene fragment subclone to step 1) in the radiation-induced pCDNA3.1-Egr1-EGFP eukaryon expression plasmid that obtains, replace fluorescin fragment (i.e. EGFP), obtain radiation-induced Antioncogene eukaryon expression plasmid (i.e. pCDNA3.1-Egr1-HSV-TK eukaryon expression plasmid);

3.1) by HSV-TK fragment subclone in pCDNA3.1-Egr1-EGFP, build pCDNA3.1-Egr1-HSV-TK

Reclaim HSV-TK fragment according to preceding method, then with EcoRI, XhoI for subcloning sites, HSV-TK fragment is built up in pCDNA3.1-Egr1-EGFP, replaces original EGFP, build pCDNA3.1-Egr1-HSV-TK.

After conversion, several clones of picking, extract plasmid, carry out PCR qualification.

Primer pair:

HSV-TK-f：

5’-G GAATTC GCC ACC ATGGCCTCGTACCCCGGCCAT-3’

HSV-TK-r：

5’-CCG CTCGAG TCAGTTAGCCTCCCCCATCT-3’

Product：Length＝1153bp,GC％＝65.4,Ta＝59.7

1. pCDNA3.1-Egr1-HSV-TK is sent to order-checking, sequencing primer is Egr1-f, BGH-r, because HSV-TK Gene sequence comparison is long, so need to check order from two ends respectively, secondary to be checked order the result obtained respectively and the comparison of HSV-TK template sequence, known sequence is entirely true, Fig. 2 is 5 '-3 ' sequencing result comparison chart, Fig. 3 is 3 '-5 ' sequencing result comparison diagram (Sequence 0:HSVTK sequence, Sequence 1:pCDNA3.1-Egr1-HSVTK-Egr1-f.ab1 sequence, comparison software: Dnassit 2.0).Show that pCDNA3.1-Egr1-HSV-TK successfully constructs.

4) by warm start sub-pieces section subclone on pCDNA3.1-Egr1-HSV-TK eukaryon expression plasmid, obtain radiation, heat shock double-promoter induction Antioncogene eukaryon expression plasmid (i.e. pCDNA3.1-Egr1-Hsp70-HSV-TK eukaryon expression plasmid).

4.1) pCDNA3.1-Egr1-Hsp70-HSV-TK is built

Reclaim Hsp70 fragment, with HindIII, EcoRI for subcloning sites, Hsp70 fragment is built up in pCDNA3.1-Egr1-EGFP (single promotor contrast), build pCDNA3.1-Egr1-Hsp70-EGFP.

After conversion, several clones of picking, extract plasmid, carry out PCR qualification.

Primer pair:

Hsp70-f：

5’-CCC AAGCTT CTCGAGGCGCGTCCTCAGA-3’(HindIII)

Hsp70-r：

5’-G GAATTC GGTCGACTAGAGAGCTTCTT-3’(EcoRI)

Product：Length＝418+9+7＝434bp,GC％＝70.6,Ta＝58

Amplification obtains the Hsp70 fragment of 434bp.

1. pCDNA3.1-Egr1-Hsp70-EGFP is sent to order-checking, sequencing primer is Egr-f, by its sequence and the comparison of HSV-TK template sequence, known sequence is entirely true, show that pCDNA3.1-Egr1-HSV-TK successfully constructs Fig. 4 (Sequence 0:HSP70 sequence, Sequence 1:pCDNA3.1-Egr1-HSP70-EGFP checks order .ab1, comparison software: Dnassit 2.0).

4.2) pCDNA3.1-Egr1-Hsp70-HSV-TK eukaryon expression plasmid is built

With HindIII, EcoRI for subcloning sites, by Hsp70 fragment from subclone pCDNA3.1-Egr1-Hsp70-EGFP on pCDNA3.1-Egr1-HSV-TK, after being transformed into DH5 α, several clones of picking, extract plasmid, carry out enzyme and cut qualification.

First cut qualification with HindIII, EcoRI enzyme, cut out the little band of an about 450bp, be shown to be correct clone.

Choose and can cut to obtain the plasmid of 450bp band, further with the qualification of MluI single endonuclease digestion, the little band of an about 1.6K can be cut out.

5) by the Mn of Polyethylenimine load ₀₅zn _0.5fe ₂o ₄antioncogene eukaryon expression plasmid (the i.e. pCDNA3.1-Egr1-Hsp70-HSV-TK eukaryon expression plasmid) mass ratio that nanoparticle and radiation, heat shock double-promoter are induced is 20:1, the two is diluted with serum free medium respectively, room temperature places more than 5 minutes, then the diluting soln of the two is mixed evenly, incubated at room temperature more than 30 minutes, obtain the double-promoter plasmid magnetic composite nano grain (i.e. pEgr1-Hsp70-HSV-TK/PEI-MZF-NPs mixture) of radiation heat-inducible, be Antioncogene magnetic composite nano particle.Wherein, pEgr1-Hsp70-HSV-TK refers to the double-promoter plasmid of radiation heat-inducible, and PEI refers to Polyethylenimine, and MZF refers to Mn ₀₅zn _0.5fe ₂o ₄, NPs is the abbreviation of English nanoparticles, represents nanoparticle.

Magnetic genophore PEI-Mn _0.5zn _0.5fe ₂o ₄the preparation of nanoparticle and Characteristics Detection:

Adopt the chemical coprecipitation of improvement, its basic step is as follows: be the stoicheiometry of 0.5:0.5:2 according to the mol ratio of Mn:Zn:Fe, takes raw material ZnSO ₄7H ₂o, FeSO ₄7H ₂o, MnSO ₄h ₂o, pours speed lapping cup pulverize into after mixing, take the amount of required NaOH in the ratio of n (M): n (OH –)=1:2.4 (M represents all metal ions), to pour in above-mentioned powder and to mix, adding appropriate distilled water, constantly stirring into pasty state to reacting completely, after room temperature places 12h, 80 DEG C of dryings, be ground into powder in mortar presoma, puts into retort furnace 400 DEG C of roasting 1h, embathe after naturally cooling with hot distilled water, filter, use BaCl ₂detect until without after white precipitate in filtrate, with dehydrated alcohol drip washing one time, 60 DEG C of dry for standby.Weigh Mn _0.5zn _0.5fe ₂o ₄4g puts into 100mL deionized water and is made into the magnetic fluid that massfraction is 4%, and after ultrasonic disperse, magnetic resolution abandons supernatant.Precipitation is resuspended in PBS liquid, and slowly add a certain amount of PEI after ultrasonic disperse process and fully mix, then room temperature magnetic agitation 3h, reacts fully, and forms the Mn that stable PEI modifies _0.5zn _0.5fe ₂o ₄.Be separated from solution by composite magnetic particle with magnetic separation method, and use distilled water repetitive scrubbing, last washing with alcohol once, obtains the MZF nanoparticle of finishing, deposits in drying basin stand-by after drying.The magnetic Nano material of preparation is close to circular, and Electronic Speculum figure is shown in Fig. 5, and energy spectrum analysis finds that the mol ratio of Mn, Zn, Fe meets 1; 1:4 (see Fig. 6), magneticsubstance before and after modifying is placed in alternating magnetic field, carry out intensification experiment to detect, the two rises to 43 ° and keeps stable (Fig. 7) after 40min, the nanoparticle infared spectrum that Polyethylenimine modifies front and back is shown in Fig. 8, adds several distinctive small peak after modification.

The preparation method of Antioncogene magnetic composite nano particle and extracorporeal biology Characteristics Detection thereof:

According to above-mentioned Magnetofection method by pEgr1-Hsp70-HSV-TK channel genes HEK293 cell, after 2GyX line irradiates 1h, 6h, 12h, 24h and 48h, Real-time PCR method detects the difference of TK gene expression amount.HEK293 cell, through the x-ray bombardment of 1Gy, 2Gy, 4Gy, 8Gy, 16Gy various dose, collects each group of cell after 6h, detect the difference of TK gene expression amount..Double-promoter plasmid pEgr1-Hsp70-HSV-TK is imported HEK293 cell by same transfection method, and compare through 2Gy roentgen radiation x associating magnetic thermotherapy, tailored radiation, after simple magnetic thermotherapy process 6h, observes the impact of different inductive condition on TK expression amount.The pEgr1-Hsp70-HSV-TK plasmid of double-promoter is imported SMMC7721 liver cancer cell by Magnetofection method, and after radiation, heating induction, PCR method detects that TK gene is at cell inner expression.

Embodiment 2: basic procedure and step are with embodiment 1, and difference is, step 5) in press the Mn of Polyethylenimine load _0.5zn _0.5fe ₂o ₄antioncogene eukaryon expression plasmid (the i.e. pEgr1-Hsp70-HSV-TK eukaryon expression plasmid) mass ratio that magnetic nanoparticle and radiation, heat shock double-promoter are induced is that 35:1 operates.The assay of final product is the same.

Embodiment 3: basic procedure and step are with embodiment 1, and difference is, step 5) in press the Mn of Polyethylenimine load _0.5zn _0.5fe ₂o ₄antioncogene eukaryon expression plasmid (the i.e. pEgr1-Hsp70-HSV-TK eukaryon expression plasmid) mass ratio that magnetic nanoparticle and radiation, heat shock double-promoter are induced is that 50:1 operates.The assay of final product is the same.

Claims (1)

1. an Antioncogene magnetic composite nano particle, is characterized in that, this composite nanometer particle prepares according to following steps:

1) with pIRES-CMVE-Egr1p eukaryon expression plasmid for template, carry out polymerase chain reaction, obtain radiation-induced promoter fragment CMVE-Egr1, by its subclone on pCDNA3.1-EGFP plasmid, obtain pCDNA3.1-Egr1-EGFP eukaryon expression plasmid;

2) to be loaded with the plasmid of the sub-Hsp70 of warm start and suicide gene HSVTK for template, carry out polymerase chain reaction, obtain warm start sub-pieces section and Antioncogene fragment;

3) by Antioncogene fragment subclone to described step 1) in the pCDNA3.1-Egr1-EGFP eukaryon expression plasmid that obtains, replace EGFP, obtain pCDNA3.1-Egr1-HSV-TK eukaryon expression plasmid;

4) by warm start sub-pieces section subclone to described pCDNA3.1-Egr1-HSV-TK eukaryon expression plasmid, pCDNA3.1-Egr1-Hsp70-HSV-TK eukaryon expression plasmid is obtained;

5) by the Mn of Polyethylenimine load _0.5zn _0.5fe ₂o ₄nanoparticle and pCDNA3.1-Egr1-Hsp70-HSV-TK eukaryon expression plasmid mass ratio are 20:1 ~ 50:1, the two is diluted with serum free medium respectively, room temperature places more than 5 minutes, then the diluting soln of the two is mixed evenly, incubated at room temperature more than 30 minutes, obtain pEgr1-Hsp70-HSV-TK/PEI-MZF-NPs mixture, be Antioncogene magnetic composite nano particle.