CN102876696A - Preparation method and application of tissue in situ high-expression ILK (integrin linked kinase) protein carrier - Google Patents

Abstract

The invention relates to a preparation method and application of a tissue in situ high-expression ILK (integrin linked kinase) protein carrier. According to the technical scheme of the invention, an expression vector comprises the gene for coding human ILK protein, and the gene sequences comprise the nucleotide sequence showed as the SEQ ID No. 1 and the nucleotide sequence showed as the SEQ ID No. 2. The invention overcomes the defects that the patients taking the medicament for treating chronic heart failure can still be inevitably subjected to the end-stage heart failure and the CRT (cardiac resynchronization therapy) is extremely expensive and can only take effect on the patients with asynchronous right and left ventricular contraction. The medicament transfects specifically in the focal tissue, has high transfection efficiency, has minimal toxicity to the infected cells, is easy to operate and causes a few complications. The CD45 positive cells in the myocardium of the ILK treatment group are reduced significantly so that the high-expression ILK can inhibit the inflammatory cell from infiltrating to the local tissues. The medicament can not lead to the specific damage, the formation of the tumor and the deformity and is non-toxic.

Description

Preparation method and the application thereof of tissue in situ high expression level ILK protein carrier

Technical field

The invention belongs to biomedicine field, tissue in situ in particular to allogenic gene is expressed, and the expression that utilizes foreign gene is played the effect of chronic heart failure disease, particularly preparation method and the application thereof of tissue in situ high expression level ILK protein carrier.

Background technology

Chronic heart failure is mainly lost with the myocardial cell, and the inharmonious of residual myocardial cell's function deterioration, myocardial hypertrophy and angiogenesis is feature.And the chronic heart failure disease is stubborn problem relatively in the present medical treatment always, and its treatment is mainly concerned with relief of symptoms, suppress cardiac remodeling, improve the aspect such as long-term prognosis.Yet at present China has the survival rate of symptom Patients with Cardiac Failure very low, prognosis than most of progressive stage malignant tumour also poor, along with the quickening of aging population process and the rising of the common cardiovascular diseases sickness rate such as hypertension, coronary heart disease, its morbidity is still in continuous rising.

Role of integrin-linking kinase (ILK) is one the serine/threonine protein kitase of function to be arranged, its energy self-phosphorylation, and the many external source substrates of phosphorylation, such as myelin basic protein, integrin beta 1 cytoplasmic domain, protein kinase B (PKB/Akt) and glycogen synthase kinase 3 β (GSK-3 β).ILK in the heart mainly concentrates on rib (costamere) and Z plate.Rib is the zone that the myocardium myo tunica fibrosa links to each other with heart voluntary muscle Z plate, and it is comprised of cytoskeletal protein and the kinase whose complicated albumen network of signal, can connect constriction device and born of the same parents' external environment in the born of the same parents, experiences mechanical stretch stimulation and transduction downstream signal.Studies show that, ILK is by N-terminal ankyrin repeat and PINCH protein binding, by kinases district and β integrin cytoplasmic domain and parvin protein binding, thereby form the ILK-PINCH-parvin complex body, this complex body is the key component of adhesion plaque complex body (FAK), in cardiac mechanical induction, mechanical conductive and keep play a part aspect the heart function very important.In vertebrate research, the ILK gene pure missense mutation of the discovery zebra fishs such as Bendig will cause the carrying out property reduction of heart contraction ability and the serious downward modulation of tractive one response gene ANF, VEGF, cause that heart failure namely occured in embryonic stage zebra fish.In Mammals, the confirmation mouse ILK such as White are essential to normal heart function: the homozygote mouse of ILK disappearance dies from Peri-implantation Process of Embryos; Specificity is eliminated heart ILK gene and will be caused mouse that spontaneous dilated cardiomyopathy occurs in several weeks after birth, and left ventricle generally enlarges companion's fibrosis.Therefore, as the hinge of ILK-PINCH-parvin complex body, ILK plays a part very important keeping cardiac structure and function aspects.

Before the present invention, the medicine of chronic heart failure disease mainly includes: the medicine of the relief of symptoms such as diuretic(s), purple foxglove and beta-blockers, ACE inhibitor, aldosterone antagonists etc. improve prognosis class medicine; In recent years, implantation CRT carried out following cardiac resynchronization therapy and also began utilization in the sub-fraction heart failure patient.At present, the pharmacological agent curative effect is limited, although a lot of patient has given best pharmacological agent, still proceeds to inevitably endstage cardiac insufficiency; And CRT treatment is very expensive, and it is effective only left and right ventricles to be shunk nonsynchronous patient, and the heart failure patient synchronous to most of ventricular systole is invalid.

In sum, although existing multiclass medicine is used to chronic heart failure, overall result for the treatment of is still limited, therefore, and medicine and method that heart failure treatment field makes new advances in the urgent need to exploitation.

Summary of the invention

One object of the present invention just is to overcome defects, and a kind of tissue in situ high expression level ILK protein carrier is provided.

Technical scheme of the present invention is:

Tissue in situ high expression level ILK protein carrier, its technical characteristics are that described expression vector comprises the gene of the human ILK albumen of encoding.

The gene of the human ILK albumen of wherein said coding on human chromosomal particular location for being positioned at Chromosome:11; NC_000011.9, the concrete nucleotides sequence of this gene are listed in that its gene name is called in the human genome database (NCBI and Ensebl genomic): ILK is for the ID of this gene in the NCBI gene pool number: 3611; Be for ID in the Ensebl gene pool number: ENSG00000166333.

Wherein be for NCBI gene pool ID number: 3611 gene order represents with SEQ ID No.1 in the present invention; In the Ensebl gene pool be for ID number: the gene order of ENSG00000166333 represents with SEQ IDNo.2 in the present invention.This two fragment genes sequence all can be expressed human ILK albumen (containing 452 amino acid), the expressed equal indifference of its structure and function of ILK albumen.

Another technical scheme of the present invention is:

The application of tissue in situ high expression level ILK protein carrier, its technical characteristics are that tissue in situ high expression level ILK protein carrier adopts intravascular injection preparation, subcutaneous injection preparation and focus organ local organization injection formulations.

Wherein said biological virus (virus) carrier system is selected from adenovirus, adeno-associated virus and slow virus carrier system, preferred adenovirus system.Above-mentioned virus carrier system all can obtain by commercial the purchase.For example among the present invention preferred adenovirus system can select ask must smart Biochemics Inc. (Qbiogene.inc) AdEasy ^TM

The present invention also has a technical scheme to be:

Tissue in situ high expression level ILK protein carrier preparation method, its major technique step is:

(1) by the human cDNA library wild-type ILK cDNA that increases, and ILK cDNA upstream and downstream primer introduce respectively ScaI and XhoI site with subclone to pShuttle, form shuttle plasmid pShuttle-ILK, the PCR product is confirmed through dna sequencing;

(2) select the positive recombinant plasmid pShuttle-ILK of 1.0g, after Pme I linearizing, be transformed into and contain the efficient competence E.coli BJ5183 of pAdEasy-1,37 ℃ of overnight incubation in containing kantlex LB nutrient agar;

(3) through homologous recombination, produce the AdEasy plasmid of restructuring in the BJ5183;

(4) homologous recombination plasmid pAd-ILK confirms: select the part clone and place 3-5ml to contain 37 ℃ of the LB substratum of kantlex, the 225-250rpm shaking culture is spent the night, and extracts in a small amount pAd-ILK.Pac I enzyme cutting method is adopted in the evaluation of pAd-ILK, and pAd-ILK is 0.8% agarose-TAE gel electrophoresis after Pac I enzyme is cut;

(5) with 7 * 10 ⁶The HEK293A cell is laid on the 100mm culture dish, and cell converges rate and reaches 80% during transfection;

(6) first with cell of OptiMEM substratum flushing, add 5ml OptiMEM substratum in each culture dish;

(7) get 15 μ g recombinant plasmid pAd-ILK, be transfected into the HEK293A cell behind Pac I linearization for enzyme restriction, transfection reagent adopts Lipofectamine2000 (Invitrogen company);

(8) understand progression of infection by the cytopathic effect of observing transfectional cell, collecting cell after transfection 7-10 days obtains Ad-ILK virus for 3 times through the dry ice multigelation and preserves liquid;

(9) unloaded adenovirus Ad-null similarity method by unloaded shuttle plasmid pShuttle-CMV and pAdeasy-1 restructuring preparation, gets tissue in situ high expression level ILK protein carrier.

A further object of the present invention has provided a kind of plasmid vector system and this plasmid vector system application in preparation Cardiovarscular medicine of carrying the gene order of the human ILK of coding.Described plasmid (plasmid) carrier system is selected from PBR322, PUC, PGEM or Minicycle series, wherein preferred minicycle; Above-mentioned pUC pUC all can obtain by commercial the purchase.For example preferred Minicycle series plasmid can be available from system biological scientific company (System Biosciences, Inc) among the present invention.

The present invention also provides a kind of preparation method of biological virus carrier system who carries the gene order of the human ILK of coding.Particularly the method comprises the steps:

1, searches ILK gene position (Chromosome:11 by human genome database (Ensebl genomic and NCBI); NC_000011.9), the gene name is called: ILK is for the ID of this gene in the NCBI gene pool number: 3611; Be for ID in the Ensebl gene pool number: ENSG00000166333.

2, utilize humanized's gene library to clone the mRNA fragment of ILK gene, and identify through order-checking.

3, prepare virion by three carrier methods well known in the art.

4, after recombinating successfully, identifying virus obtains at last recombinant viral vector system with the ILK gene through amplification and purifying again.

The cardiovascular disorder that the present invention puts down in writing refers to the chronic heart failure that a variety of causes causes.

Advantage of the present invention and effect are medicine in the transfection of lesion tissue internal specific, and transfection efficiency is high, and be minimum to the cytotoxicity that infects simultaneously, operate simpler, few intercurrent disease etc.

Other concrete advantages of the present invention and effect will go on to say below.

Embodiment

Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.The experimental technique of unreceipted actual conditions among the embodiment, usually according to normal condition, or the condition of advising according to manufacturer.

Embodiment 1:

Carry the preparation of the Adenovirus Transfection system of ILK gene:

By the human cDNA library wild-type ILK cDNA that increases, and ILK cDNA upstream and downstream primer introduce respectively ScaI and XhoI site with subclone to pShuttle, form shuttle plasmid pShuttle-ILK, the PCR product is confirmed (Shanghai Ying Jun Bioisystech Co., Ltd) through dna sequencing.Select the positive recombinant plasmid pShuttle-ILK of 1.0g, after the PmeI linearizing, be transformed into and contain the efficient competence E.coli BJ5183 of pAdEasy-1,37 ℃ of overnight incubation in containing kantlex LB nutrient agar.Through homologous recombination, produce the AdEasy plasmid (pAd-ILK) of restructuring in the BJ5183.For being confirmed to be homologous recombination plasmid pAd-ILK, to select part clone and place 3-5ml to contain 37 ℃ of the LB substratum of kantlex, the 225-250rpm shaking culture is spent the night.Extract in a small amount pAd-ILK.The PacI enzyme cutting method is adopted in the evaluation of pAd-ILK, and pAd-ILK is 0.8% agarose-TAE gel electrophoresis after the PacI enzyme is cut.

With about 7 * 10 ⁶The HEK293A cell is laid on the 100mm culture dish, and cell converges rate and reaches 80% during transfection.With cell of OptiMEM substratum flushing, add 5ml OptiMEM substratum in each culture dish first.Get 15 μ g recombinant plasmid pAd-ILK, be transfected into the HEK293A cell behind the PacI linearization for enzyme restriction, transfection reagent adopts Lipofectamine2000 (Invitrogen company).Understand progression of infection by the cytopathic effect of observing transfectional cell.Collecting cell after transfection 7-10 days obtains Ad-ILK virus for 3 times through the dry ice multigelation and preserves liquid.Unloaded adenovirus Ad-null similarity method is by unloaded shuttle plasmid pShuttle-CMV and pAdeasy-1 restructuring preparation.

Whether can express ILK albumen in order to identify the ILK recombinant adenovirus, Ad-ILK or the Ad-null adenovirus of taking a morsel preserved the liquid rotaring redyeing 293 cell, adopt the cell pyrolysis liquid lysing cell after 48 hours, extract cell protein, the BCA method detects protein concentration, and Western blot method detects ILK protein expression situation.

The HEK293A cell cultures of 4xT-75 culturing bottle is converged rate to individual layer 90-100%, add enough Ad-ILK or Ad-null cells infected, inner cell presented CPE in 48 hours.When CPE is close to when finishing, collect the cell that infects, through 3 multigelations virus is released in the supernatant liquor.Collect supernatant, through ViraBind adenovirus purification kit purification Ad-ILK or Ad-null.The OD260 method is adopted in the measuring and calculating of adenovirus titre behind the purifying.The OD260 method is namely measured virion in the absorbancy at 260nm place, measures to represent virus titer by DNA, and 1 OD value is equivalent to 1.1 * 10 ¹²Individual virion.

Embodiment 2:

Experimentation on animals

50 SD rats open respectively and inject adenovirus or the unloaded adenovirus that contains the ILK gene in the chest cardiac muscle after the abdominal injection Zorubicin causes heart failure.Contain in the rat of adenovirus of ILK gene 25 injections, have 10 to occur deadly within 4 weeks, and 25 injections contain the rat of unloaded adenovirus, have 16 death occur.All around, we find by ultrasonic cardiogram, we find by ultrasonic cardiogram, the cardiac ejection fraction for the treatment of group rat (EF), left LVSF (FS) all significantly are better than not treating rat, shrink left chamber, the diastasis internal diameter is significantly less than not treatment group, the prompting left ventricular remodeling be improved significantly.We have detected left chamber dp/dt, left chamber diastasis pressure and serum BNP simultaneously, find that all the treatment group rat significantly is better than not treatment group rat (table 1).

Further pathological examination is found: ILK Adenovirus Transfection group heart collagen deposition obviously reduces than control group; The myocardial cell that the Tunel apoptosis detects apoptosis in the prompting ILK treatment group heart obviously reduces; Use Ki-67 and H3P dyeing, the myocardial cell that can detect heart stalk peripheral region propagation significantly increases (table 2); Transmission electron microscope observing finds that the myocardial cell that autophagy occurs in the ILK treatment group also obviously reduces; Simultaneously we observe that the CD45 positive cell obviously reduces in the ILK treatment group cardiac muscle, and prompting high expression level ILK can infiltrate to local organization by the inflammation-inhibiting cell.

In addition, we have carried out the pathology detection to tissues such as the heart of ILK adenovirus treatment group rat, liver, kidney, lung, muscle, do not find that specific damage and tumour form; Pregnant mouse detects its sub-mouse after carrying out the treatment of ILK adenovirus, does not also find that deformity exists, and the treatment that has further proved us is safe, nontoxic.

Claims (7)

1. tissue in situ high expression level ILK protein carrier is characterized in that described expression vector comprises the gene of the human ILK albumen of encoding.

2. tissue in situ high expression level ILK protein carrier according to claim 1 is characterized in that the gene order of the human ILK albumen of described coding has the nucleotide sequence shown in the SEQ ID No.1.

3. tissue in situ high expression level ILK protein carrier according to claim 1 is characterized in that the gene order of the human ILK albumen of described coding has the nucleotide sequence shown in the SEQ ID No.2.

4. the application of tissue in situ high expression level ILK protein carrier according to claim 1 is characterized in that tissue in situ high expression level ILK protein carrier adopts intravascular injection preparation, subcutaneous injection preparation and focus organ local organization injection formulations.

5. the application of tissue in situ high expression level ILK protein carrier according to claim 4, the preparation that it is characterized in that intravascular injection most preferably is the injection of coronary artery preparation.

6. tissue in situ high expression level ILK protein carrier according to claim 1 is characterized in that the application as treatment medicine in heart failure.

7. tissue in situ high expression level ILK protein carrier preparation method according to claim 1, its step is:

(1) by the human cDNA library wild-type ILK cDNA that increases, and ILK cDNA upstream and downstream primer introduce respectively ScaI and XhoI site with subclone to pShuttle, form shuttle plasmid pShuttle-ILK, the PCR product is confirmed through dna sequencing;

(2) select the positive recombinant plasmid pShuttle-ILK of 1.0g, after the PmeI linearizing, be transformed into and contain the efficient competence E.coli BJ5183 of pAdEasy-1,37 ℃ of overnight incubation in containing kantlex LB nutrient agar;

(3) through homologous recombination, produce the AdEasy plasmid of restructuring in the BJ5183;

(4) homologous recombination plasmid pAd-ILK confirms: select the part clone and place 3-5ml to contain 37 ℃ of the LB substratum of kantlex, the 225-250rpm shaking culture is spent the night, and extracts in a small amount pAd-ILK.The PacI enzyme cutting method is adopted in the evaluation of pAd-ILK, and pAd-ILK is 0.8% agarose-TAE gel electrophoresis after the PacI enzyme is cut;

(5) with 7 * 10 ⁶The HEK293A cell is laid on the 100mm culture dish, and cell converges rate and reaches 80% during transfection;

(6) first with cell of OptiMEM substratum flushing, add 5ml OptiMEM substratum in each culture dish;

(7) get 15 μ g recombinant plasmid pAd-ILK, be transfected into the HEK293A cell behind the PacI linearization for enzyme restriction, transfection reagent adopts Lipofectamine2000 (Invitrogen company);

(8) understand progression of infection by the cytopathic effect of observing transfectional cell, collecting cell after transfection 7-10 days obtains Ad-ILK virus for 3 times through the dry ice multigelation and preserves liquid;

(9) unloaded adenovirus Ad-null similarity method by unloaded shuttle plasmid pShuttle-CMV and pAdeasy-1 restructuring preparation, gets tissue in situ high expression level ILK protein carrier.