CN101475936B

CN101475936B - EDRF gene fragment

Info

Publication number: CN101475936B
Application number: CN200810157774XA
Authority: CN
Inventors: 谢书阳; 王萍玉; 焦飞; 李尊岭; 李强; 李有杰; 岳真
Original assignee: Binzhou Medical College
Current assignee: Binzhou Medical College
Priority date: 2008-10-17
Filing date: 2008-10-17
Publication date: 2011-04-06
Anticipated expiration: 2028-10-17
Also published as: CN101475936A

Abstract

The invention discloses an EDRF gene fragment and its expression vector, and the experiment proves that the vector can stabilize excessive alpha-globin peptide chain, the invention reduces the sediment and reduces the harm of the forming of alpha-inclusionbody by excess alpha-chain, mitigates the anemia symptoms of beta<654>-mediterranean anemia mice, testifies the value of EDRF gene expression inerythrocytic series and disease treatment, and provides a new idea for the treatment of mediterranean anemia.

Description

A kind of EDRF gene fragment

Technical field

The invention belongs to molecular biology and gene therapeutics field, be specifically related to a kind of EDRF gene fragment for the treatment of beta Thalassemia and relevant expression vector thereof and scientific research, application clinically.

Background technology

Thalassemia is because certain or some protein chain synthesis rates reduce and cause that some peptide chains lack, other peptide chains increase relatively, the imbalance of quantity of peptide chains resulted occurs, the hemolytic anemia that causes.Beta Thalassemia (be called for short β ground poor) wherein, because the β chain synthesizes and subtracts scarcely, the α chain is too much relatively, unnecessary α chain formation α inclusion body.In the red corpuscle of differentiation and maturation not, α chain inclusion body can make cytolemma sustain damage, and is wherein destroyed in marrow greatly.Though part red corpuscle energy maturation also is released in the peripheral blood, but because α chain inclusion body is attached on the erythrocyte membrane, make the stiff of red corpuscle change, this red corpuscle makes by microcirculation, easily is torn, extract inclusion body, form the teardrop shaped red corpuscle, and this red blood cell life span is short, easily destroyed, produce symptoms such as anaemia, deposition of iron, splenomegaly, patient's physical and mental health in serious harm.Therefore, reduce the harm that the deposition of alpha globin causes, the recovery and the treatment of diseases of red corpuscle physiological function had vital role.

Red system differentiation correlation factor (EDRF) has another name called the hemoglobin alpha stabilize proteins, is a kind of protein at the erythron high expression level, and is closely related with the generation of erythron.Recent study confirms that further EDRF is the hemoglobin alpha molecular chaperones, and it can combine with hemoglobin alpha, but can not combine with beta globin or tetrameric hemoglobin body.EDRF can keep the stability of hemoglobin alpha, keeps the hemoglobin alpha solubility, weakens the destruction of alpha globin deposition cell membrane.As seen EDRF can stable alpha oxyphorase, reduce the harm that too much hemoglobin alpha causes, when EDRF with after hemoglobin alpha combines, iron ion on the protoheme is combined with the Histidine of far-end and near-end, certain change takes place in the structures such as α spiral that cause hemoglobin alpha, and this structural change can suppress separating of the generation of active oxonium ion ROS and protoheme and alpha globin effectively.Said process is the mechanism of EDRF stable alpha oxyphorase, the effect of protection cell injury.

Before the present invention, the poor treatment in β ground mainly contains pharmacological agent, and it is synthetic not enough to induce the fetal type oxyphorase to increase with the substituted beta globin gene as hydroxyurea, butyric acid etc.; Bone marrow transplantation therapy (but lacking effective donor); Therapeutic transgene (as the beta-globin gene); The method of the unusual β chain of Antisense gene therapy etc.But above-mentioned method is defectiveness all, can not reach satisfactory therapeutic effects.And the invention is intended to found a kind of new methods of treatment, use EDRF and stablize too much alpha globin peptide chain, reduce its deposition, purpose is the harm that alleviates unnecessary α chain formation α inclusion body, the value of proof EDRF genetic expression in erythron and disease treatment is for thalassemic treatment provides new thinking.Using the method that this new carrier carries out the poor treatment in β ground does not appear in the newspapers.

Summary of the invention

The objective of the invention is to overcome the deficiency in the existing methods of treatment, a kind of EDRF gene fragment of beta Thalassemia and relevant expression vector thereof for the treatment of is provided.

Technical scheme of the present invention is:

(1) screening of useful length EDRF promotor:

1, the clone of different lengths EDRF promotor

The sequence of the promotor of design different lengths is in order to amplification EDRF promotor.Amplification condition is as follows: 94 ℃ of 45s of sex change, annealing 45s, extension 72 ℃ of 60s, totally 32 circulations.Primer sequence and annealing temperature see Table 1, and amplification region is seen Fig. 1.The PCR product carries out electrophoresis, analytical results with 2% sepharose.

2, make up the action effect that different GFP carriers is analyzed promotor

Be used to drive the expression of GFP with the promotor of above-mentioned different lengths, and make up corresponding carrier.At first, obtain the GFP gene by EcoR I and Not I two restriction enzyme sites from pGEP-N1 carrier (Invitrogen), and GFP is connected on pcDNA (3.1+)/ZEO (Invitrogen) carrier, make up the pcDNA-GFP carrier with same restriction enzyme site.Then, above-mentioned promotor through pcr amplification rear clone (Takara) to the pUCm-T carrier, is made up the pUCm-promoter carrier.Cut the pUCm-T carrier by EcoRV and HindIII enzyme and obtain promotor, and use Nru I and the HindIII restriction enzyme site is cloned on the pcDNA-GFP carrier, finally obtain the pcDNA-Pro-GFP carrier.

3, cell cultures and transfection

The NIH3T3 cell is cultivated with the DMEM that contains 15% foetal calf serum with DMEM (GIBICO) culture medium culturing that contains 10% foetal calf serum, the mel cell of mouse, and induces differentiation and maturation with 1.5% DMSO (Sigma).All cells is cultivated with 37 ℃ of 5%CO2 available from cell institute of the Shanghai Chinese Academy of Sciences.

4, the detection of GFP and analysis

Fluoroscopic examination: 72 hours collecting cells after the transfection, with fluorescent microscope (Leica, DMRXA2) observations, NIH3T3 cell wherein, trysinization with 0.25%.

Flow cytometry analysis (FACS): behind the cell harvesting, wash 2 times with PBS, then with flow cytometry analysis result (Becton Dichinson FACS caliber), the efficient of the expression response EDRF promotor by GFP.

(2) structure of pcDNA-EDRF expression vector and therapeutic action thereof

1, with the structure of effective promoters driven EDRF expression vector

By the GFP expression vector that above-mentioned experiment makes up, determine behind the transfectional cell that the most effective promotor length is 372bp, be that primer sequence is according to redesign primer amplification EDRF gene thus:

F4:5 '-GGCCTTGTTATCTTTCTACCT-3 ' (with primers F 3 basically identicals ,-112bp);

R3：5’-TTCT?TGTTTCTGCCACCC-3’(+1168bp)。

The PCR reaction conditions is: 94 ℃ of 60s of sex change, and the 54 ℃ of 60s that anneal extend 72 ℃ of 90s; Totally 30 circulations.The PCR product is identified with 1.5% sepharose, and order-checking.

The EDRF gene and the promotor thereof of amplification are connected on pUCm-T (Takara) carrier, made up the pUCm-EDRF carrier, then, cut the pUCm-EDRF carrier by EcoRV and BamHI enzyme and obtain EDRF gene and promotor thereof, and use NruI and the BamHI restriction enzyme site is cloned on pcDNA (3.1+)/ZEO carrier (Invitrogen), finally obtain the pcDNA-EDRF expression vector.

2, people EDRF is in the therapeutic action to the beta Thalassemia mouse of the intravital expression of mouse and pcDNA-EDRF carrier

In order to study the therapeutic action of EDRF gene, find after handling mouse, this carrier can be in the normal mouse body effectively expressing people's the EDRF factor, and this protein expression can improve β ⁶⁵⁴The Anemia of the poor mouse in-ground, this result of study is to β ⁶⁵⁴The treatment of-ground anaemia has important value.

Beneficial effect of the present invention: the present invention has screened effective EDRF promotor, express with effective promoters driven EDRF, make up effective EDRF expression vector, and prove by experiment, this carrier can be stablized too much alpha globin peptide chain, reduces its deposition, the harm that alleviates unnecessary α chain formation α inclusion body, the value of proof EDRF genetic expression in erythron and disease treatment is for thalassemic treatment provides new thinking.

Below in conjunction with drawings and Examples the present invention is done and to explain.

Description of drawings

Fig. 1: different primer amplification EDRF genes and promotor synoptic diagram;

Wherein: F1-F4: forward primer; R1-R3: reverse primer;

CAC: exons 1 starting point; ATG: translation starting point;

Fig. 2: different lengths promoters driven GFP expression vector establishment synoptic diagram;

Fig. 3: pcDNA-EDRF makes up synoptic diagram;

Fig. 4: different lengths promotor and vector construction are identified figure;

Wherein: A, pcr amplification different lengths promotor result

M:100bp marker marker;

Swimming lane lane1-5: five different lengths promotors:

697bp，496bp，484p，372bp，283bp；

B, a kind of pcDNA-Pro-GFP carrier and pcDNA-EDRF carrier enzyme are cut evaluation figure swimming lane Lane1-2:pcDNA-Pro-GFP carrier (promotor length is 372bp);

Lane1:Not I restriction analysis: product length is 4800bp, 1152bp;

Lane2:Not I+Hind III restriction analysis: product length is 4800bp, 772bp, 380bp;

Swimming lane Lane3-4:pcDNA-EDRF carrier;

Lane3:Hind III restriction analysis: product length is 5410bp, 669bp;

Lane4:Not I restriction analysis: product length is 4800bp, 1280bp;

Fig. 5: the expression (100X) of fluorescent microscope observation GFP in cell;

Wherein: A:GFP expresses in the NIH3T3 cell;

B:GFP expresses in mel cell;

I, II, III, IV, V, VI: be respectively GFP in control group, 496bp promotor group, 283bp promotor group, 697bp promotor group, 484bp promotor group, 372bp promotor group;

Fig. 6: the EDRF gene that detects the people is expressed in mouse Mel cell;

Wherein: swimming lane Lane1,3: handle Mel result with pcDNA (3.1+)/ZEO control vector;

Lane2,4: handle Mel result with the pcDNA-EDRF control vector;

Fig. 7: people EDRF gene is at the intravital expression analysis of mouse (RT-PCR);

Wherein: swimming lane Lane1: handle normal mouse with pcDNA (3.1+)/ZEO control vector;

Lane2: with pcDNA-EDRF vehicle treated normal mouse;

Lane3: handle β-thalassemia mouse with pcDNA (3.1+)/ZEO control vector;

Lane4,5: with pcDNA-EDRF vehicle treated β-thalassemia mouse;

Fig. 8: with the expression of elisa assay EDRF/AHSP;

Wherein: A: the normal mouse group

Normal mouse (healthy mice, n=5): handle normal mouse with pcDNA (3.1+)/ZEO control vector;

Exp-1, Exp-2, Exp-3: three with pcDNA-EDRF vehicle treated normal mouse;

B: β-thalassemia mouse group

β-thalassemia mouse (654-mice): handle β-thalassemia mouse with pcDNA (3.1+)/ZEO control vector;

Exp-1-654, Exp-2-654, Exp-3-654: three with pcDNA-EDRF vehicle treated β-thalassemia mouse;

Fig. 9: beta Thalassemia mouse blood smear is analyzed the change of blood cell shape;

Wherein: A: β-thalassemia mouse: handle β-thalassemia mouse with the pcDNA-EDRF control vector;

B: β-thalassemia mouse: handle β-thalassemia mouse with pcDNA (3.1+)/ZEO control vector;

C: normal mouse;

Figure 10: the change analysis of beta Thalassemia mouse treatment back Hb and RBC;

Wherein: the change of A:Hb oxyphorase (n=5);

B:RBC RBC number purpose changes (n=5).

Embodiment

Below be example with the screening of useful length EDRF promotor, the structure and the therapeutic action thereof of pcDNA-EDRF expression vector, for the present invention is further elaborated.These examples are aimed at the explanation that the present invention does, rather than limitation of the present invention.

Among the present invention, among the above and the following embodiment, employed technology, comprise gene sequencing, pcr amplification and detection, cell transfecting, vector construction equimolecular biology techniques, and cell cultures, detection technique etc., unless stated otherwise, be the known routine techniques of those skilled in the art; Employed plant and instrument, reagent, plasmid and carrier, clone etc., only this specification sheets is dated especially, is that the research of general this area and technician can obtain by public approach.

Embodiment 1, the screening of useful length EDRF promotor

1, the clone of different lengths EDRF promotor

The sequence of the promotor of design different lengths is cloned the EDRF gene promoter from the human gene group DNA, and primer sequence sees Table 1, and the gene region of concrete amplification is seen Fig. 1.Amplification condition is as follows: 94 ℃ of 45s of sex change, annealing 45s (concrete annealing temperature sees Table 1), extension 72 ℃ of 60s, totally 32 circulations.The PCR product carries out electrophoresis, analytical results with 2% sepharose.

The sequence of the promotor of design different lengths is in order to amplification EDRF promotor.Amplification condition is as follows: 94 ℃ of 45s of sex change, annealing 45s, extension 72 ℃ of 60s, totally 32 circulations.The PCR product carries out electrophoresis with 2% sepharose, analytical results, and having obtained length respectively is the promotor of 697bp, 496bp, 484bp, 372bp and 283bp, sees Fig. 4 A.Order-checking correct (Shanghai Bo Shang Bioisystech Co., Ltd), result's correct (summary).

2, make up the action effect that different GFP carriers is analyzed promotor

Be used to drive the expression of GFP with the promotor of above-mentioned different lengths, and make up corresponding carrier.At first, obtain the GFP gene by EcoR I and Not I two restriction enzyme sites from pGEP-N1 carrier (Invitrogen), and GFP is connected on pcDNA (3.1+)/ZEO (Invitrogen) carrier, make up the pcDNA-GFP carrier with same restriction enzyme site.Then, above-mentioned promotor through pcr amplification rear clone (Takara) to the pUCm-T carrier, is made up the pUCm-promoter carrier.Cut the pUCm-T carrier by EcoRV and HindIII enzyme and obtain promotor, and use NruI and the HindIII restriction enzyme site is cloned on the pcDNA-GFP carrier, finally obtain the pcDNA-Pro-GFP carrier and be used for research (Fig. 2).Further cloned the pcDNA-Pro-GFP carrier.Cut (Not I, Not I+Hind) by two groups of enzymes and identify that the result is correct, see Fig. 4 B.

3, the detection of cell cultures and transfection and GFP and analysis

Found that: in NIH3T3 cell and Mel cell, it is best that the long promotor (-116～+ 256 districts of EDRF gene) of 372bp drives the effect that GFP expresses, fluorescence intensity is the brightest, positive cell maximum (Fig. 5 A, B).FACS further analyzes discovery, in mel cell, and the promotor group that 372bp is long, the positive rate of GFP cell (11.2 ± 0.6) % is apparently higher than other length promoters driven groups; But in non-red corpuscle NIH3T3 cell, the promotor group that 372bp is long, be (31.0 ± 0.7%) in the positive rate mel cell of GFP cell, comparing difference with other groups is not very big, and above-mentioned difference illustrates that this promoters driven expression of gene has certain tissue specificity (table 2).

The structure and the therapeutic action thereof of embodiment 2::pcDNA-EDRF expression vector

1, drives the expression vector establishment of EDRF with above-mentioned effectively start

R3：5’-TTCT?TGTTTCTGCCACCC-3’(+1168bp)；

The EDRF gene and the promotor thereof of amplification are connected on pUCm-T (Takara) carrier, made up the pUCm-EDRF carrier, then, cut the pUCm-EDRF carrier by EcoRV and BamHI enzyme and obtain EDRF gene and promotor thereof, and use NruI and the BamHI restriction enzyme site is cloned on pcDNA (3.1+)/ZEO carrier (Invitrogen), the final pcDNA-EDRF expression vector that obtains is used for research (Fig. 3), cut evaluation with HindIII and two kinds of enzymes of NotI, result correct (Fig. 4 B), sequencing result is seen sequence table.

2, detect the method that EDRF expresses

RT-PCR: the extraction of total RNA is according to test kit explanation carrying out (Gentra company, the total RNA extraction reagent box), the cDNA synthetic system is as follows: total RNA of 1.5 μ g, 4 μ l dNTP (10mmol/L), 0.5 μ loligo-dT (0.5mg/ml), 1 μ l reversed transcriptive enzyme (RT, New England), DEPC H ₂O to 20 μ l.In 70 ℃ of 10min; 2min on ice; 37 ℃ of synthetic cDNA of 1h.

EDRF gene amplification primer is:

Forward: 5 '-CGCAGGATTGAAGGAGTTC-3 ';

Oppositely: 5 '-GTGTCAGGGTAGAGTGGC-3 '.

β-actin cDNAs primer (reference gene) is:

Forward: 5 '-CTACAATGAGCTGCGTG TGGC-3 ';

Oppositely: 5 '-CAGGTCCAGACGCAGGATGGC-3 '.

Reaction conditions is: 94 ℃ of 45s of sex change, 57 ℃ of 45s of annealing, extension 72 ℃ of 45s, totally 28 circulations.

Enzyme linked immunosorbent assay (ELISA) detects: detailed process is summarized as follows: with the hemocyte cracking of mel cell and mouse, in wrapping by the enzyme plate endoperidium of anti-mouse, the monoclonal antibody (Santa Cruz Biotechnology) that adds the anti-people EDRF of goat then respectively, two anti-(IgG of the anti-goat of rabbit, Abcam) and substrate etc.With microplate reader (EL _x800) in OD _450nmWavelength detects.

3, people EDRF is in the intravital expression of mouse

For study the pcDNA-EDRF expression vector can be in the mouse body effectively expressing, our purifying pcDNA-EDRF plasmid (method see Promega Kit explanation), at first we have verified the expression effect of carrier at cell levels, and RT-PCR analyzes and finds that EDRF can express (Fig. 6) efficiently in the MEL of mouse.Then 5 normal mouses are experimentized, 30-35ug pcDNA-EDRF plasmid is dissolved in the 800ul artificial sera, the tail vein injection mouse detects the mouse peripheral blood after 7 days, observe the result of genetic expression.RT-PCR finder's EDRF gene can be expressed (Fig. 7 .lane2) in the mouse blood cell.The elisa assay discovery, in the mouse body that pcDNA-EDRF handles, the about 4mg/ml of EDRF expression amount (Fig. 8).

4, the pcDNA-EDRF carrier is to the therapeutic action of beta Thalassemia mouse

In order to observe the therapeutic action of pcDNA-EDRF carrier to beta Thalassemia, we are again to 5 β ⁶⁵⁴The poor mouse in-ground experimentizes, and injected dose and method are the same.β wherein ⁶⁵⁴The poor mouse in-ground is available from U.S. JacksonLaboratory (JAX).This mouse model utilizes gene targeting, knocks in people's Disease-causing gene (beta globin genes of aberrant splicing), has replaced the beta globin genes of mouse.A coding people's the aberrant splicing beta globin peptide chain and the beta globin peptide chain gene of an encoding murine are arranged in the heterozygote mouse cell of this model, the aberrant splicing beta globin peptide chain of energy expressing human, showing the typical β poor patient's in ground Anemia, is a kind of poor modeling tool in β ground of studying well.

Work as β ⁶⁵⁴After the poor mouse in-ground was used the pcDNA-EDRF vehicle treated, the blood smear analysis was found, β ⁶⁵⁴Target cell of the poor mouse in-ground and different in nature total cellular score obviously lower, from original 50% be reduced to about 38.4 ± 2.3% (p＜0.05, Fig. 9); The amount of oxyphorase rises to 7.93 ± 0.2g/dL from 7.1 original ± 0.3g/dL; Since therapeutic action, the compensatory hematopoiesis of body, and red corpuscle number RBC rises to 6.4 * 106/ μ l (Figure 10) from 5.5 * 106/ original μ l.And control group β ⁶⁵⁴The poor mouse in-ground is handled with control vector pcDNA (3.1+)/ZEO, and different in nature cell and target type total cellular score, hematological indices do not have obvious variation.This result of treatment, may with EDRF at β ⁶⁵⁴The poor mouse expression in vivo in-ground has relation, has proved the therapeutic action of carrier.RT-PCR (Fig. 7, lane4,5) and ELISA (Fig. 8) detected result are supported this result of treatment.

Form:

Table 1: with the promotor of primer amplification different lengths

Table 2: flow cytometer detects the expression of GFP positive cell

SEQ ID NO:1 sequence table-order-checking table

<>plasmid

<>(1)..(1500)

<>1

gcttgaccga?caattgcatg?aagaatctgc?ttagggttag?gcgttttgcg?ctgcttcgat 60

cccatgggcg?gcgcctgcag?accaggtctg?gccttgttat?ctttctacct?tcagggaagc 120

ctctcccttc?ttctcctccc?tagaatgacc?tatcaccctc?cttcaggacc?tagatgcagg 180

gcgtttctat?cttaggctga?cacttgactc?cttgcctaca?tctatagctt?ggcacagaga 240

gattcacgca?ccctcaagag?tgtgggtgag?acatatacag?cctgttagac?ctgaaggtga 300

gcccaacctg?ggaaaatcgt?gacctcagag?cagggcagga?tgtgaaaggt?tttggaaagg 360

agatagccct?gcagggcagg?agggattttt?aaggggagga?agtgggttgg?gggaaatacc 420

cagtgaggag?ggaaacagat?atgtaaattc?tacccttttc?tctacccagg?cagatggctc 480

ttcttaaggc?caataaggat?ctcatttccg?caggattgaa?ggagttcagc?gttctgctga 540

atcagcaggt?gagtccaagc?tttccatttc?aaaggactgg?cctggagact?ggggggtgcc 600

ggggaagtgg?aggaagagga?taattggagc?tggtgaagta?atggtggagt?tgatggaaac 660

aacgagagac?acgggataca?atgcagagga?aagagaatgt?gagagttggt?gctgtggcac 720

tattctagta?ttcccgaatc?ccattgctga?ccacattctc?ccttgccaac?tgcctctccg 780

caacccccca?ggtcttcaat?gatcctctcg?tctctgaaga?agacatggtg?actgtggtgg 840

aggactggat?gaacttctac?atcaactatt?acaggcagca?ggtgacaggg?gagccccaag 900

agcgagacaa?ggctctgcag?gagcttcggc?aagagctgaa?cactctggcc?aaccctttcc 960

tggccaagta?cagggacttc?ctgaagtctc?atgagctccc?gagtcaccca?ccgccctcct 1020

cctagctcag?ggacccagcc?cctcctctct?gagaaactct?gaccttcatg?tccttaggct 1080

gtgctcctgc?cactctaccc?tgacacctca?ataaagacca?gtgctggttt?tgttggactt 1140

cctggcttct?ctttcatggt?ctgtcctgac?atgtggaggc?actcggtccc?tttccttccg 1200

ccctccttct?tagatatgct?cttggaagct?taattcctta?cagcagaggt?tagcagcctt 1260

gagtgaccac?cgggcagcga?ttctaaacta?aaacaggctg?aacgagggcc?aggaaggcgg 1320

gactagagag?agtgcagagc?tgcaggggag?ggggtggcag?aaacaagaaa?agactggaga 1380

tctggatcca?ctagtccagt?gtggtggaat?tctgcagata?tccagcacag?tggcggccgc 1440

tcgagtctag?agggcccgtt?taaacccgct?gatcagcctc?gactgtgcct?tcta 1500

Claims

1. EDRF gene fragment, its sequence is shown in SEQ ID NO:1.

2. the expression vector of an EDRF gene fragment is characterized in that, this expression vector has the described gene fragment of claim 1.