CN108715863A - A kind of tumor-targeting carrier pcTERT and its construction method and its application - Google Patents

Abstract

The present invention relates to a kind of tumor-targeting carrier pcTERT and its construction method and its applications, belong to genetic engineering field.The carrier is based on pcDNA3.1 plasmids, the hTERT promoter hTERT promoters of the optimum length obtained with screening substitute its original CMV promoter, different types of tumoricidal gene order can be connected behind, recombinant vector is transfected into variety classes tumour cell using transfection reagent, achieves certain tumor-killing effect in vivo and in experiment in vitro.It is a kind of effectively for foreign gene tumor locus targeted expression non-virus carrier, be expected in the gene therapy of tumour realize application.

Description

A kind of tumor-targeting carrier pcTERT and its construction method and its application

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of promoter containing hTERT is used for cancer target base Because of the carrier, vector construction and its application for the treatment of.

Background technology

The incidence of malignant tumour persistently increases in recent years, it has also become threatens the second major class disease of human health, full generation There are about 13% numbers to die of malignant tumour every year on boundary, and also there is 60% or more cancer mortality in China.With the deterioration of environment, Operating pressure increases, the mode of bad life and world population ages, this number will also continue to rise, estimate according to WHO, To between the year two thousand thirty, new cancer cases give house by by 15,500,000 of 11,300,000 in 2007 surges to the year two thousand thirty within 2007 Front yard and society cause irreparable damage.And clinically therapies and means such as traditional operation, radiation and chemotherapy at present Its effect is unsatisfactory.

Operation and the treatment that radiotherapy is locality, it is limited to the recurrence and transferance of tumour, although chemotherapy is systemic Treatment means, but be the absence of targeting, the injury of human health cell be difficult to avoid that, Murray JC publish an article report Only 2%~5% reaches tumor locus, is absorbed by normal structure more than 90% drug, causes prodigious toxic side effect.Therefore it develops The tumor therapeutic agent of targeting is very urgent.

The magnetic target therapy drug research of tumour is concentrated mainly on following several respects at present：1, it is directed to and tumor cell differentiation The key enzyme (protein tyrosine kinase, aromatizing enzyme, Topoisomerase etc.) for being proliferated relevant intracellular signal transduction pathway is target Point, selectively acting is in efficient, less toxic, high specificity the micromolecular compound of specific target site.2, antibody target medicine.It is profit Tumor surface related antigen or special receptor specific recognition are improved to which drug is directly directed to tumour cell with monoclonal antibody The effect of drug, reduces toxicity of the drug to the circulatory system and other positions.According to its structure, anti-tumor monoclonal antibody class can To be divided into monoclonal antibody therapeutics for tumor and anti-tumor monoclonal antibody conjugate.Wherein monoclonal antibody therapeutics for tumor energy It is attached to tumour cell, cell death is led to by direct antigen-antibody reaction.Anti-tumor monoclonal antibody conjugate, also known as Immune conjugate, by ingredient (chemicals, radionuclide, photosensitizer, enzyme etc.) 2 moieties of monoclonal antibody and " attack " tumour It constitutes.3, targeting drug delivery sys tems.It can pass through special carrier or the action specificity of modification group using the systemic drug Ground is concentrated on target area.These special carriers include liposome, nanoparticle, micella and nanocapsule etc., modification group include antibody, Glycoprotein, lipoprotein, transferrins, polypeptide, folic acid etc..4, targeting gene therapy.It will be controlled using special carrier system Treating gene target importing tumour cell is the research hotspot of domestic and international neoplasm targeted therapy at present, and is probably dashed forward One of the field that broken property achievement promotes the well-being of mankind.

In order to realize targeting gene therapy, need by target gene by certain means orientation import specific tissue, Cell makes it be expressed in certain specific histocytes, to accurately eliminate tumour cell, while not injuring normal cell, Or reduce damage to normal cell to the greatest extent, to mitigate the side effect come due to foreign gene lead-in zone, this is in addition to fixed Outside limited tumor locus is treated, the target gene used in treatment is also required to there is the targeting of transfer and in its turn The expression etc. of energy targeting after shifting.Go deep into to study on the carrier, it has been found that single pass carrier is difficult to meet targeting Needs, and can be very good to fill up the deficiency with single carrier for the research of tumor-specific promoters.Normal cell It is interior due to and do not contain the specific transcriptional activity factor, thus the therapeutic gene imported in these cells is not expressed.About Specificity promoter reports more have：

Tyrosinase (Tyrosinase, Tyr) promoter：Tyr shows as specifically being expressed in melanoma cells, base Reason Tyr promoters are expressed by specifically mode.Such as Swoboda uses Try promoters and cell cycle dependant Suppressor (cyclin-dependent kinase inhibitor, CDKN2A) combines, and realizes gene in murine melanoma In it is specific expressed.

Survivin (survivin) promoter：Survivin belongs to inhibition apoptotic proteins, with tumour-specific.Survivin Promoter can be by mediating nucleotide probe to play a role in the treatment of liver cancer, can also be by connecting tumour downstream Gene is killed, plays the role of effectively killing tumour cell by starting the killing gene expression in tumour cell.

Carcinomebryonic antigen (carcino-embryonic antigen, CEA) promoter：CEA it is specific expressed in colorectal cancer, The tumor tissues such as breast cancer.Have been reported that display, the adenovirus that tumor suppressor gene is loaded with using CEA promoter regulations can be killed effectively Colon cancer cell.

Human telomerase reverse transcriptase (human telomerase reverse transcriptase, hTERT) promoter： Telomere is the end structure positioned at Eukaryotic chromosome linear DNA molecule, in end of chromosome in graininess is expanded, by one The non-transcribed sequences of Duan Chongfu are combined with multiple protein to be collectively constituted, and effect is to maintain chromosome integrality and control cell week Phase.By the research to people and transgenic mouse diseases associated with senescence it can be found that telomere is to the amplification of cell and survives Vital effect, there may be aging or the Pathologicals of cancer when telomere generates dysfunction.Telomere Extend or shorten and is adjusted by Telomerase.The activity of Telomerase is suppressed with cell differentiation.

In recent years, effect of the Telomerase played in cell immortality and cancer occur attracts wide attention, 85% High expression is all presented in above human tumour tissue, and has no expression in most of normal somatic cells, so that Telomerase Become the novel targets of oncotherapy as tumour-specific markers object.And the study found that hTERT promoter transcriptions activity with it is huge Cell virus (Cytomegalovirus, CMV) promoter is closer to, this makes the carrier of hTERT promoter regulations become tool Have and is generally applicable in, acts on sensitive tumor-specific promoters.At present and have no non-viral using hTERT promoter direct constructions Recombinant vector and the application for obtaining high efficient expression result.

Invention content

A kind of tumor-targeting carrier pcTERT of present invention offer and its construction method and its application.

The technical solution adopted by the present invention is that：Include the following steps：

(1) using pcDNA3.1+ as template, sense primer is designed：

5 '-CTGACGCGTGGAGTTCCGCGTTAC-3 ' and

Downstream primer：5 '-CGCGCTAGCCAAAACAAACTCCCATTG-3 ' are reacted through PCR, obtain pcDNA3.1+ matter Grain in cmv enhancer segments, and make its 5 ' end with the sites MluI and 3 ' end carry the sites NheI；

(2) using human gene group DNA as template, sense primer is designed：

5 '-ATAACGCGTACCCTGGGAGCGCGAGCGGC-3 ', and

Downstream primer：5 '-CGCGCTAGCCAAAACAAACTCCCATTG-3 ' are reacted through PCR, and it is 378bp to obtain length HTERT promoter fragments, and make its 5 ' end with the sites NheI and 3 ' end carry III sites Hind；

(3) respectively through MluI and NheI double digestions, T4DNA connects the enhancing sub-piece for obtaining pcDNA3.1+ carriers and PCR It connects enzyme to be attached to obtain intermediate 1, intermediate carrier 1 and length is used into Nhe I and Hind respectively for the promoter fragment of 378bp After III digestion, T4DNA ligases are attached to obtain recombinant vector pcTERT.

By melittin (Melittin) sequence with tumor-killing effect, Pseudomonas Exotoxin (PEA) sequence, noxa Sequence and puma sequences are connected into recombinant vector respectively, four kinds of 5 ' ends of segments difference with III restriction enzyme sites of Hind and 3 ' ends carry I restriction enzyme sites of Xba.

The recombinant vector containing melittin and the recombinant vector containing Pseudomonas Exotoxin are transfected respectively using transfection reagent The recombinant vector of sequence containing noxa and puma sequences is transfected human liver cancer by human esophagus cancer TE1 cell lines respectively using transfection reagent HepG2 cell lines pass through the detection to tumor cell morphology variation and the variation of relevant molecule level, it was demonstrated that tumor-targeting Carrier pcTERT can start different tumoricidal gene orders high efficient expression in different tumour cells.

It is an advantage of the current invention that compared to other tumor-specific promoters having found, the hTERT that is used in carrier Promoter has in normal, tumour cell that activity difference is big and fragment length is small is easy to carry out vector construction, and in this carrier Longer tumor-killing fragment sequence can be connected, therefore the present invention provides for the application of gene therapy medicament a kind of having very much foreground Method.

Description of the drawings

Fig. 1 is tumor-targeting carrier pcTERT structure schematic diagrames；

Fig. 2 is that the length obtained after PCR reacts is the enhancer fragment electrophoretic figure of 406bp；

Fig. 3 is that the length obtained after PCR reacts is the promoter fragment electrophoretogram of 378bp；

Fig. 4 A are thin using the green fluorescence expression vector transfection human esophagus cancer TE1 of the hTERT promoters of the length containing 378bp Born of the same parents' luciferase expression figure；

Fig. 4 B are thin using the green fluorescence expression vector transfection human esophagus cancer TE1 of the hTERT promoters of the length containing 443bp Born of the same parents' luciferase expression figure；

Fig. 4 C are thin using the green fluorescence expression vector transfection human esophagus cancer TE1 of the hTERT promoters of the length containing 714bp Born of the same parents' luciferase expression figure；

Fig. 4 D are the green fluorescence expression vector transfection human esophagus cancer TE1 using the hTERT promoters of the length containing 1100bp Cell fluorescence expression figure；

Fig. 5 A are photos under the Het-1a normal esophageal cell light microscopics without transfection；

Fig. 5 B are photos under light microscopic after pcTERT recombinant vectors transfection Het-1a normal esophageal cells 48h,

Fig. 5 C are after the recombinant vector (pcTERT-Melittin) containing melittin transfects Het-1a normal esophageal cells 48h Photo under light microscopic；

Fig. 5 D are recombinant vector (pcTERT-PEA) the transfection Het-1a normal esophageal cells 48h containing Pseudomonas Exotoxin Photo under light microscopic afterwards；

Fig. 5 E are photos under the HL7702 normal liver cell light microscopics without transfection；

Fig. 5 F are photos under light microscopic after pcTERT recombinant vectors transfection HL7702 normal liver cells 48h；

Fig. 5 G are shone under light microscopic after recombinant vector (pcTERT-noxa) the transfection HL7702 normal liver cells 48h containing noxa Piece；

Fig. 5 H are shone under light microscopic after recombinant vector (pcTERT-puma) the transfection HL7702 normal liver cells 48h containing puma Piece；

Fig. 6 A are photos under the TE1 esophageal cancer cell light microscopics without transfection；

Fig. 6 B are photos under light microscopic after pcTERT recombinant vectors transfection TE1 esophageal cancer cells 48h；

Fig. 6 C are after recombinant vector (pcTERT-Melittin) the transfection TE1 esophageal cancer cells 48h containing melittin under light microscopic Photo；

Fig. 6 D are light after recombinant vector (pcTERT-PEA) the transfection TE1 esophageal cancer cells 48h containing Pseudomonas Exotoxin Photo under mirror；

Fig. 6 E are photos under the HepG2 liver cancer cells light microscopics without transfection；

Fig. 6 F are photos under light microscopic after pcTERT recombinant vectors transfection HepG2 liver cancer cells 48h；

Fig. 6 G are photos under light microscopic after recombinant vector (pcTERT-noxa) the transfection HepG2 liver cancer cells 48h containing noxa；

Fig. 6 H are photos under light microscopic after recombinant vector (pcTERT-puma) the transfection HepG2 liver cancer cells 48h containing puma；

Fig. 7 A are to use pcTERT, and pcTERT-Melittin, pcTERT-PEA recombinant plasmids transfect human esophagus cancer respectively TE1 cells for 24 hours, cells survival rate testing result after 48h, 72h；

Fig. 7 B are to use pcTERT, and pcTERT-Melittin, pcTERT-PEA recombinant plasmids distinguish transfected with human normal esophageal Het-1a cells for 24 hours, cells survival rate testing result after 48h, 72h；

Fig. 7 C are to use pcTERT, and pcTERT-noxa, it is thin that pcTERT-puma recombinant plasmids transfect human liver cancer HepG2 respectively Born of the same parents for 24 hours, cells survival rate testing result after 48h, 72h；

Fig. 7 D are to use pcTERT, and pcTERT-noxa, pcTERT-puma recombinant plasmids distinguish transfected with human normal hepatocytes HL7702 cells for 24 hours, cells survival rate testing result after 48h, 72h；

Fig. 8 is recombinant plasmid (pcTERT- of the intratumor injection containing melittin after structure nude mice HepG2 Transplanted tumor models Melittin), tumor growth curve figure；

Fig. 9 A are extraction tumor tissues albumen, and detected by Western blot analyzes 3 result figures of albumen caspase；

Fig. 9 B are extraction tumor tissues albumen, and detected by Western blot analyzes 3 result figures of albumen cleaved caspase；

Fig. 9 C are extraction tumor tissues albumen, and detected by Western blot analyzes albumen cyclinD1 result figures.

Specific implementation mode

Embodiment 1 builds required design of primers

(1) design primer respectively designs a restriction enzyme site at the both ends CMV enhancer of pcDNA3.1+ carriers, makes it 5 ' end with I sites Mlu and 3 ' end carry I sites Nhe：

Sense primer：5'-CTGACGCGTGGAGTTCCGCGTTAC-3'

Downstream primer：5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

(2) design primer respectively designs a restriction enzyme site at the both ends hTERT promoter, its 5 ' end is made to carry Nhe I Point and 3 ' end carry III sites Hind：

Length is the promoter primer of 378bp

Sense primer：5'-ATAACGCGTACCCTGGGAGCGCGAGCGGC-3'

Downstream primer：5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

Length is the promoter primer of 443bp

Sense primer：5'-ATAGCTAGCACCCTGGGAGCGCGAGCGGC-3'

Downstream primer：5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

Length is the promoter primer of 714bp

Sense primer：5'-ATAACGCGTGTGTCAAGGAGCCCAAGTC-3'

Downstream primer：5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

Length is the promoter primer of 1100bp

Sense primer：5'-ATAACGCGTCTGTCCTGCGGTTGTG-3'

Downstream primer：5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

The promoter and length that promoter that promoter that the length is 378bp, length are 443bp, length are 714bp There is reported in literature for the promoter of 1100bp.

(3) design primer respectively designs a restriction enzyme site at melittin both ends, its 5 ' end is made to carry III restriction enzyme sites of Hind And 3 ' ends carry I restriction enzyme sites of Xba：

Sense primer：5'-TATAAGCTTGCCACCATGGTCGCC-3'

Downstream primer：5'-TGCTCTAGATTATCACTTGGCCTTGGCC-3'

(4) design primer respectively designs a restriction enzyme site at Pseudomonas Exotoxin sequence both ends, its 5 ' end is made to carry III restriction enzyme sites of Hind and 3 ' end carry I restriction enzyme sites of Xba：

Sense primer：5'-ATTAAGCTTGCCACCATGGTCGGCTACCACGGC-3'

Downstream primer：5'-TGCTCTAGATAACGAGGGAATCACCACG-3'.

PCR reaction systems and response parameter when embodiment 2 obtains each segment of recombinant vector

(1) cmv enhancer PCR react

(2) hTERT prompter PCR react：

(3) melittin PCR reacts：

(4) Pseudomonas Exotoxin segment PCR reacts

Cmv enhancer segments are obtained after PCR reacts, (M is DNA marker as shown in Figure 2；No. 1 swimming lane is cmv Enhancer segments), length is the hTERT promoter fragments of 378bp, and (M is DNA marker as shown in Figure 3；No. 1 swimming lane is HTERT promoter fragments).It carries out subsequently containing melittin using above-mentioned two segment, Pseudomonas Exotoxin, noxa and puma weights The structure of group plasmid.

3 promoter optimization length of embodiment screens

Using pEGFP-N1 plasmids as template, green fluorescence protein gene (EGFP) segment is obtained by the reaction through PCR, and make its two End carries I restriction enzyme site of Hind III and Xba, which is connected into recombinant vector and obtains EGFP recombinant plasmids, finds to be connected with Length is that the plasmid encoding luciferase expression of 378bp promoters is most strong (Fig. 4 A), contains the recombination EGFP plasmids that length is 378bp promoters Luciferase expression intensity is most strong after transfectional cell, and the hTERT promoters that final choice length is 378bp are used to build recombinant vector, And the carrier built is named as pcTERT.

The recombinant plasmid transfected cell detection hTERT of 4 gene containing tumor-killing of embodiment starts ability

Using the melittin and Pseudomonas Exotoxin recombinant plasmid acted on containing tumor-killing, human esophagus cancer is transfected respectively TE1 cells and normal person esophageal cells Het-1a, just containing the Transfected Recombinant Plasmid human liver cancer cell HepG2 of noxa and puma and people Steps are as follows by normal liver cell HL7702：

(1) day before transfection, cell, which reaches 12 orifice plates, makes second day cell confluency rate up to 65% or so；

(2) cell replaces fresh DMEM culture solutions (containing 10% standard fetal calf serum) before transfecting；

(3) 4 μ g plasmids are diluted per 100 μ l Opti-MEM of hole；

(4) 2 μ glipo2000 liposomes are diluted per 100 μ l Opti-MEM of hole；

(5) by (3), the dilution mixing of (4) step, it is stored at room temperature 15min；

(6) 12 orifice plates of transfection composite addition, 37 DEG C, 5%CO₂It is cultivated in incubator；

As shown in Fig. 5 A~5H, significant change does not occur for cellular morphology after transfection by non-tumor cell Het-1a, HL7702. As shown in Fig. 6 A~6H, hTERT promoters start tumor-killing gene M elittin, PEA and are expressed in tumour cell TE1, HTERT promoters start tumor-killing gene noxa, puma and are expressed in tumour cell HepG2 so that tumour cell is shown Obvious apoptotic cell form shows as the cell for more shrinkages occurred, having deformed and falling off, and cell light transmittance is poor, Cell tracking disappears.

Cell Proliferation detects after the recombinant plasmid transfected cell of 5 gene containing tumor-killing of embodiment

(1) cell of logarithmic growth phase, conventional digestion, it is in unicellular to dispel cell, with 50000/ml, 100 μ l/ Hole is inoculated in 96 orifice plates, and every group sets 4 multiple holes, in 37 DEG C, 5%CO₂Overnight incubation in cell incubator；

(2) pcTERT-Melittin (melittin recombinant plasmid), pcTERT-PEA (Pseudomonas Exotoxin weights are used respectively Group plasmid) and pcTERT transfection human esophagus cancer TE1 cells and normal esophageal cell Het-1a；Using pcTERT-noxa, PcTERT-puma and pcTERT transfection human hepatoma HepG2 cells and normal liver cell HL7702, put back in incubator and continue to train It supports；

(3) culture plate for taking out processing different time points, 10 μ l CCK-8 solution are added per hole.It puts back in incubator and continues 1h is cultivated, microplate reader detects the absorbance per hole at 450nm；

(4) survival rate=experimental group OD values/control group OD value × 100% of cell is calculated as follows；

It is connected with the recombinant vector of tumor-killing effect gene, respectively to human esophagus cancer TE1 cells (Fig. 7 A) and human liver cancer The proliferation of HepG2 cells (Fig. 7 C) has stronger inhibiting effect, to normal esophageal cell Het-1a (Fig. 7 B) and normal liver cell The basic unrestraint effect of proliferation of HL7702 (Fig. 7 D).Illustrate that there is recombinant vector the startup subsequent gene of tumour-specific to express Effect.

Embodiment 6 connects In vivo study of the recombinant vector to function of tumor of bee venom prime sequences

In nude mice oxter, inoculation HepG2 cells establish HepG2 Transplanted tumor models, are administered, are found by intratumor injection PcTERT-Melittin carriers have certain inhibiting effect to tumour growth.Its step are as follows：

(1) for HepG2 cell growth degree of converging to 80%, conventional digestion collects cell in 10cm culture dishes；

(2) it inhales and abandons culture solution, physiological saline resuspension cell to density to 5 × 10⁷A/mL, it is thin that 200ul is injected in left side oxter Born of the same parents' suspension；

(3) tumor size 80mm³Left and right, divides cage, physiological saline group 4, pcTERT groups 4, pcTERT- at random Melittin groups 4.Intratumor injection transferrins cationic liposome complex.Injection is primary every three days, and co-injection is three times；

(4) mouse active state and tumour growth situation are observed and recorded.

By the research of experiment in vivo it can be found that bee venom prime sequences structure of the recombinant vector connection with tumor-killing effect The plasmid built can stablize expression in vivo, and play the role of inhibiting tumor tissue growth.Tumour growth as shown in Figure 8 Obviously inhibited, 3 expression quantity of apoptosis of tumor cells GAP-associated protein GAP cleaved-caspase as shown in Fig. 9 A~9C increases, week Phase GAP-associated protein GAP cyclinD1 expression quantity declines.

The above be related embodiment of the present invention, the description thereof is more specific and detailed, but can not therefore and be interpreted as Limitation to the scope of the claims of the present invention.It should be pointed out that for those of ordinary skill in the art, not departing from this hair Under the premise of bright design, various modifications and improvements can be made, these are all within the scope of protection of the present invention.Therefore, this hair The protection domain of bright patent should be determined by the appended claims.

Claims (3)

1. a kind of tumor-targeting carrier pcTERT, it is characterised in that obtained by the following steps：

(1) using pcDNA3.1+ as template, sense primer is designed：

5 '-CTGACGCGTGGAGTTCCGCGTTAC-3 ' and

Downstream primer：5 '-CGCGCTAGCCAAAACAAACTCCCATTG-3 ' are reacted through PCR, are obtained in pcDNA3.1+ plasmids Cmv enhancer segments, and make its 5 ' end with the sites MluI and 3 ' end carry the sites NheI；

(2) using human gene group DNA as template, sense primer is designed：

5 '-ATAACGCGTACCCTGGGAGCGCGAGCGGC-3 ', and

Downstream primer：5 '-CGCGCTAGCCAAAACAAACTCCCATTG-3 ' are reacted through PCR, and it is 378bp's to obtain length HTERT promoter fragments, and make its 5 ' end with the sites NheI and 3 ' end carry III sites Hind；

(3) the enhancing sub-piece for obtaining pcDNA3.1+ carriers and PCR is respectively through MluI and NheI double digestions, T4DNA ligases It is attached to obtain intermediate 1, intermediate carrier 1 and length is used into III enzyme of Nhe I and Hind respectively for the promoter fragment of 378bp After cutting, T4DNA ligases are attached to obtain recombinant vector pcTERT.

2. a kind of construction method of tumor-targeting carrier pcTERT as described in claim 1, which is characterized in that including following Step：

(1) using pcDNA3.1+ as template, sense primer is designed：

5 '-CTGACGCGTGGAGTTCCGCGTTAC-3 ' and

Downstream primer：5 '-CGCGCTAGCCAAAACAAACTCCCATTG-3 ' are reacted through PCR, are obtained in pcDNA3.1+ plasmids Cmv enhancer segments, and make its 5 ' end with the sites MluI and 3 ' end carry the sites NheI；

(2) using human gene group DNA as template, sense primer is designed：

5 '-ATAACGCGTACCCTGGGAGCGCGAGCGGC-3 ', and

Downstream primer：5 '-CGCGCTAGCCAAAACAAACTCCCATTG-3 ' are reacted through PCR, and it is 378bp's to obtain length HTERT promoter fragments, and make its 5 ' end with the sites NheI and 3 ' end carry III sites Hind；

(3) the enhancing sub-piece for obtaining pcDNA3.1+ carriers and PCR is respectively through MluI and NheI double digestions, T4DNA ligases It is attached to obtain intermediate 1, intermediate carrier 1 and length is used into III enzyme of Nhe I and Hind respectively for the promoter fragment of 378bp After cutting, T4DNA ligases are attached to obtain recombinant vector pcTERT.

3. a kind of tumor-targeting carrier pcTERT as described in claim 1 is applied in preparing gene therapy medicament.