CN108715863B

CN108715863B - Tumor targeting vector pcTERT and construction method and application thereof

Info

Publication number: CN108715863B
Application number: CN201810543497.XA
Authority: CN
Inventors: 马杰; 周超; 赵通鉴; 魏雪晨; 周孟瑒; 陆元花; 宋卓瑶; 王孝娟; 赵畅
Original assignee: Jilin University
Current assignee: Saipu Biotechnology (Changchun) Co.,Ltd.
Priority date: 2018-05-30
Filing date: 2018-05-30
Publication date: 2020-09-11
Anticipated expiration: 2038-05-30
Also published as: CN108715863A

Abstract

The invention relates to a tumor targeting vector pcTERT, a construction method and application thereof, belonging to the field of genetic engineering. The vector is based on pcDNA3.1 plasmid, replaces the original CMV promoter with the hTERT promoter of the human telomerase reverse transcriptase promoter with the optimal length obtained by screening, can be connected with different tumor killing gene sequences, uses a transfection reagent to transfect the recombinant vector to different tumor cells, and obtains certain tumor killing effect in vivo and in vitro experiments. An effective non-viral vector for targeted expression of exogenous genes at tumor sites is expected to be applied to gene therapy of tumors.

Description

Tumor targeting vector pcTERT and construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a vector containing an hTERT promoter and used for tumor targeted gene therapy, vector construction and application thereof.

Background

The incidence of malignant tumors has been increasing continuously in recent years, and the malignant tumors become the second major disease threatening human health, about 13% of people die of malignant tumors every year worldwide, and more than 60% of cancer mortality rate in China. With the deterioration of the environment, the increase of the working pressure, the bad life style and the aging of the world population, the number will continue to rise, and according to the estimation of the WHO, between 2007 and 2030, new cancer cases will increase from 1130 ten thousand cases in 2007 to 1550 ten thousand cases in 2030, which causes irreparable loss to families and society. But the effect of the current traditional treatment methods and means such as surgery, radiotherapy, chemotherapy and the like is difficult to satisfy clinically.

Surgery and radiotherapy are local treatments, the recurrence and metastasis effects on tumors are limited, chemotherapy is a systemic treatment means, but lacks targeting property, the damage to human healthy cells is difficult to avoid, the Murray JC publication reports that only 2% -5% of drugs reach tumor parts, more than 90% of drugs are absorbed by normal tissues, and great toxic and side effects are caused. Therefore, the development of targeted tumor treatment drugs is very urgent.

At present, the research of the targeting therapeutic drugs for tumors mainly focuses on the following aspects: 1. aiming at key enzymes (protein tyrosine kinase, aromatizing enzyme, topoisomerase and the like) of a cell signal transduction path related to the differentiation and proliferation of tumor cells as targets, the small molecular compound selectively acts on a specific target and has high efficiency, low toxicity and strong specificity. 2. The antibody targets the drug. The monoclonal antibody is used for identifying the specificity of tumor surface related antigen or specific receptor, so that the medicine is directly guided to tumor cells, the curative effect of the medicine is improved, and the toxicity of the medicine to the circulatory system and other parts is reduced. According to their structure, anti-tumor monoclonal antibodies can be classified into anti-tumor monoclonal antibody drugs and anti-tumor monoclonal antibody conjugates. Wherein the anti-tumor monoclonal antibody drug is capable of binding to tumor cells and causing cell death by a direct antigen-antibody reaction. An antineoplastic monoclonal antibody conjugate, also called immunoconjugate, is prepared from the monoclonal antibody and the 2 parts of the components (chemical medicines, radionuclides, photosensitizers, enzymes, etc.) for attacking tumor. 3. A targeted drug delivery carrier system. The system can be used for specifically concentrating the drug at a target site through the action of a special carrier or a modifying group. The special carriers comprise liposomes, nanoparticles, micelles, nanocapsules and the like, and the modifying groups comprise antibodies, glycoproteins, lipoproteins, transferrin, polypeptides, folic acid and the like. 4. Targeted gene therapy. The target introduction of the therapeutic gene into tumor cells by using a specific vector system is a research hotspot of the tumor target treatment at home and abroad at present, and is also one of the fields which are likely to obtain breakthrough results to benefit human beings.

In order to realize targeted gene therapy, it is necessary to introduce a target gene into a specific tissue or cell by a certain means or to express the target gene in some specific tissue cells, thereby eliminating tumor cells accurately without damaging normal cells, or minimizing damage to normal cells to reduce side effects due to introduction of foreign genes. With the progress of vector research, people find that the targeting requirement is difficult to be met by a single vector, and the research on tumor specific promoters can well fill up the defect of using a single vector. Since normal cells do not contain the specific transcription activator, the therapeutic gene introduced into these cells is not expressed. There are many reports on specific promoters:

tyrosinase (Tyrosinase, Tyr) promoter: tyr appears to be specifically expressed in melanoma cells, and its gene is expressed by the Tyr promoter in a specific manner. Specific expression of the gene in mouse melanoma was achieved using the Try promoter in conjunction with a cell cycle-dependent suppressor (CDKN 2A) as by Swoboda et al.

Survivin (survivin) promoter: survivin belongs to an apoptosis-inhibiting protein, which is tumor-specific. The survivin promoter can play a role in treating the liver cancer through a mediated nucleotide probe, and can also play a role in effectively killing tumor cells by connecting a tumor killing gene at the downstream of the survivin promoter and starting the expression of the killing gene in the tumor cells.

Carcinoembryonic antigen (CEA) promoter: CEA is specifically expressed in tumor tissues such as colorectal cancer, breast cancer and the like. Reports show that the adenovirus carrying the cancer suppressor gene regulated by the CEA promoter can effectively kill colon cancer cells.

Human telomerase reverse transcriptase (hTERT) promoter: telomeres are terminal structures of linear DNA molecules of chromosomes of eukaryotes, are in expanded granular shapes at the ends of the chromosomes, are formed by combining a repeated non-transcribed sequence and a plurality of proteins, and have the functions of maintaining the integrity of the chromosomes and controlling the cell cycle. It has been found through studies of human and transgenic mouse senescence-associated diseases that telomeres play a crucial role in cell expansion and survival, and may contribute to senescence or pathological manifestations of cancer when telomeres become dysfunctional. Lengthening or shortening of telomeres is regulated by telomerase. Telomerase activity is inhibited as cells differentiate.

In recent years, the role of telomerase in cell immortalization and carcinogenesis attracts extensive attention, and the telomerase is highly expressed in more than 85% of human tumor tissues and is not expressed in most normal somatic cells, so that the telomerase serving as a tumor specific marker becomes a new target for tumor treatment. In addition, researches show that the transcriptional activity of the hTERT promoter is relatively close to that of a Cytomegalovirus (CMV) promoter, so that a vector regulated by the hTERT promoter becomes a tumor specific promoter with universal applicability and sensitive action. At present, the application of directly constructing a non-viral recombinant vector and obtaining a high-efficiency expression result by using the hTERT promoter is not seen.

Disclosure of Invention

The invention provides a tumor targeting vector pcTERT, a construction method and application thereof.

The technical scheme adopted by the invention is as follows: comprises the following steps:

(1) taking pcDNA3.1+ as a template, designing an upstream primer:

5'-CTGACGCGTGGAGTTCCGCGTTAC-3' and

a downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3', obtaining a cmv enhancer fragment in pcDNA3.1+ plasmid through PCR reaction, and leading the 5 'end to be provided with MluI site and the 3' end to be provided with NheI site;

(2) designing an upstream primer by taking human genome DNA as a template:

5'-ATAACGCGTACCCTGGGAGCGCGAGCGGC-3', and

a downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3', obtaining an hTERT promoter fragment with the length of 378bp through PCR reaction, and leading the 5 'end to be provided with an NheI site and the 3' end to be provided with a Hind III site;

(3) the pcDNA3.1+ vector and the enhancer fragment obtained by PCR are subjected to MluI and NheI double enzyme digestion respectively, T4DNA ligase is used for connecting to obtain an intermediate 1, the intermediate 1 and the promoter fragment with the length of 378bp are subjected to enzyme digestion respectively by NheI and Hind III, and the T4DNA ligase is used for connecting to obtain the recombinant vector pcTERT.

The Melittin sequence, the pseudomonas aeruginosa exotoxin (PEA) sequence, the noxa sequence and the puma sequence with tumor killing function are respectively connected into a recombinant vector, and the 5 'end of each of the four fragments is provided with HindIII enzyme cutting site and the 3' end is provided with XbaI enzyme cutting site.

The recombinant vector containing melittin and the recombinant vector containing pseudomonas aeruginosa exotoxin are transfected into a human esophageal cancer TE1 cell line respectively by using a transfection reagent, the recombinant vector containing a noxa sequence and a puma sequence is transfected into a human hepatoma HepG2 cell line respectively by using the transfection reagent, and the detection of morphological change and related molecular level change of tumor cells proves that the tumor targeting vector pcTERT can start the high-efficiency expression of different tumor killing gene sequences in different tumor cells.

Compared with other discovered tumor specific promoters, the hTERT promoter used in the vector has the advantages that the hTERT promoter has large activity difference in normal and tumor cells and small fragment length, is easy to construct, and can be connected with a longer tumor killing fragment sequence in the vector, so that the invention provides a promising method for the application of gene therapy medicaments.

Drawings

FIG. 1 is a schematic diagram of the construction of a tumor targeting vector pcTERT;

FIG. 2 is an electrophoretogram of an enhancer fragment 406bp in length obtained after PCR reaction;

FIG. 3 is an electrophoretogram of a 378bp promoter fragment obtained after PCR reaction;

FIG. 4A is a fluorescent expression profile of cells transfected with human esophageal cancer TE1 using a green fluorescent expression vector containing a 378bp long hTERT promoter;

FIG. 4B is a fluorescent expression profile of cells transfected with human esophageal cancer TE1 using a green fluorescent expression vector containing a 443bp long hTERT promoter;

FIG. 4C is a fluorescent expression profile of cells transfected with human esophageal cancer TE1 using a green fluorescent expression vector containing an 714bp long hTERT promoter;

FIG. 4D is a fluorescent expression profile of cells transfected with human esophageal cancer TE1 using a green fluorescent expression vector containing an hTERT promoter of 1100bp in length;

FIG. 5A is a photograph of non-transfected Het-1a normal esophageal cells under a light microscope;

FIG. 5B is a photograph of the pcTERT recombinant vector transfected Het-1a normal esophageal cells under 48h of a light mirror,

FIG. 5C is a photograph of recombinant vector containing Melittin (pcTERT-Melitin) transfected Het-1a normal esophageal cells for 48h under a microscope;

FIG. 5D is a photograph of recombinant vector containing Pseudomonas aeruginosa exotoxin (pcTERT-PEA) transfected Het-1a normal esophageal cells for 48h under a light microscope;

FIG. 5E is a photograph under a light microscope of untransfected HL7702 normal hepatocytes;

FIG. 5F is a photograph of a pcTERT recombinant vector transfected HL7702 normal hepatocytes under a 48h light microscope;

FIG. 5G is a photograph under a light microscope of 48h after transfection of HL7702 normal hepatocytes with a noxa-containing recombinant vector (pcTERT-noxa);

FIG. 5H is a photograph under a light microscope of a recombinant vector containing puma (pcTERT-puma) transfected with HL7702 normal hepatocytes for 48H;

FIG. 6A is a photograph under a light microscope of untransfected TE1 esophageal cancer cells;

FIG. 6B is a photograph of the pcTERT recombinant vector transfected TE1 esophageal cancer cells under 48h microscope;

FIG. 6C is a photograph of recombinant vector containing Melittin (pcTERT-Melitin) transfected TE1 esophageal cancer cells under 48h microscope;

FIG. 6D is a photograph of recombinant vector containing Pseudomonas aeruginosa exotoxin (pcTERT-PEA) transfected TE1 esophageal cancer cells under 48h microscope;

FIG. 6E is a photograph of untransfected HepG2 hepatoma cells under a light microscope;

FIG. 6F is a photograph of the pcTERT recombinant vector transfected HepG2 liver cancer cell under a 48h microscope;

FIG. 6G is a photograph of HepG2 liver cancer cells transfected with a recombinant vector containing noxa (pcTERT-noxa) for 48h under a light mirror;

FIG. 6H is a photograph of recombinant vector containing puma (pcTERT-puma) transfected HepG2 hepatocarcinoma cell for 48H under a light mirror;

FIG. 7A shows the results of cell survival rate measurements after transfection of human esophageal cancer TE1 cells with pcTERT, pcTERT-Melitin, and pcTERT-PEA recombinant plasmids for 24h, 48h, and 72h, respectively;

FIG. 7B shows the results of cell survival rate measurements after 24h, 48h and 72h of transfection of human normal esophageal Het-1a cells with pcTERT, pcTERT-Melitin and pcTERT-PEA recombinant plasmids, respectively;

FIG. 7C shows the results of cell survival assays after transfection of human hepatoma HepG2 cells with pcTERT, pcTERT-noxa, and pcTERT-puma recombinant plasmids for 24h, 48h, and 72h, respectively;

FIG. 7D shows the results of cell survival assays after transfection of human normal liver HL7702 cells with pcTERT, pcTERT-noxa, and pcTERT-puma recombinant plasmids for 24h, 48h, and 72h, respectively;

FIG. 8 is a graph showing the tumor growth of a nude mouse injected with Melittin-containing recombinant plasmid (pcTERT-Melittin) after construction of a nude mouse HepG2 transplanted tumor model;

FIG. 9A is a graph showing the results of protein caspase 3 extraction from tumor tissue and Western blot analysis;

FIG. 9B is a graph showing the results of extracting tumor tissue protein and analyzing the protein clear caspase 3 by Western blotting;

FIG. 9C is a graph showing the results of extracting tumor tissue protein and analyzing the protein cyclin D1 by Western blotting.

Detailed Description

Example 1 construction of the desired primer design

(1) Designing a primer, designing an enzyme cutting site at each of two ends of CMV enhancer of a pcDNA3.1+ vector, so that the 5 'end of the primer is provided with an Mlu I site and the 3' end of the primer is provided with an Nhe I site:

an upstream primer: 5'-CTGACGCGTGGAGTTCCGCGTTAC-3'

A downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

(2) Designing primers, designing a restriction enzyme site at each end of the hTERT promoter, so that the 5 'end of the primers is provided with an Nhe I site, and the 3' end of the primers is provided with a Hind III site:

promoter primer with length of 378bp

An upstream primer: 5'-ATAACGCGTACCCTGGGAGCGCGAGCGGC-3'

A downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

Promoter primer with length of 443bp

An upstream primer: 5'-ATAGCTAGCACCCTGGGAGCGCGAGCGGC-3'

A downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

714bp long promoter primer

An upstream primer: 5'-ATAACGCGTGTGTCAAGGAGCCCAAGTC-3'

A downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

Promoter primer with length of 1100bp

An upstream primer: 5'-ATAACGCGTCTGTCCTGCGGTTGTG-3'

A downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3'

The promoter with the length of 378bp, the promoter with the length of 443bp, the promoter with the length of 714bp and the promoter with the length of 1100bp are reported in the literature.

(3) Designing primers, and designing enzyme cutting sites at two ends of melittin respectively, so that the 5 'end of the melittin is provided with HindIII enzyme cutting sites, and the 3' end of the melittin is provided with XbaI enzyme cutting sites:

an upstream primer: 5'-TATAAGCTTGCCACCATGGTCGCC-3'

A downstream primer: 5'-TGCTCTAGATTATCACTTGGCCTTGGCC-3'

(4) Designing primers, and designing a restriction enzyme site at each end of an exotoxin sequence of pseudomonas aeruginosa to ensure that the 5 'end of the exotoxin sequence has a Hind III restriction enzyme site and the 3' end of the exotoxin sequence has an Xba I restriction enzyme site:

an upstream primer: 5'-ATTAAGCTTGCCACCATGGTCGGCTACCACGGC-3'

A downstream primer: 5'-TGCTCTAGATAACGAGGGAATCACCACG-3' are provided.

Example 2 PCR reaction System and reaction parameters for obtaining fragments of recombinant vector

(1) Cmv enhancer PCR reaction

(2) hTERT prompter PCR reaction:

(3) melittin PCR reaction:

(4) pseudomonas aeruginosa exotoxin fragment PCR reaction

After PCR reaction, a cmv enhancer fragment is obtained, as shown in FIG. 2 (M is DNA marker; lane 1 is cmvenenhancer fragment), and an hTERT promoter fragment with the length of 378bp is obtained, as shown in FIG. 3 (M is DNA marker; lane 1 is hTERT promoter fragment). The two fragments are used for the subsequent construction of recombinant plasmids containing melittin, pseudomonas aeruginosa exotoxin, noxa and puma.

Example 3 promoter optimal Length Screen

The method comprises the steps of using pEGFP-N1 plasmid as a template, obtaining a green fluorescent protein gene (EGFP) fragment through PCR reaction, enabling two ends of the EGFP fragment to have HindIII and Xba I enzyme cutting sites, connecting the fragment into a recombinant vector to obtain an EGFP recombinant plasmid, finding out that the fluorescence expression of the plasmid connected with a 378bp promoter is strongest (figure 4A), transfecting cells with the recombinant EGFP plasmid containing the 378bp promoter to obtain the EGFP recombinant plasmid, finally selecting a 378bp hTERT promoter for constructing the recombinant vector, and naming the constructed vector as TERpcT.

Example 4 detection of hTERT-initiating ability of recombinant plasmid transfected cells containing tumor killer gene

The steps of transfecting human esophageal cancer TE1 cells and normal human esophageal cells Het-1a respectively by using recombinant plasmids containing melittin and pseudomonas aeruginosa exotoxin with tumor killing effects, transfecting human hepatoma cells HepG2 and human normal hepatic cells HL7702 by using recombinant plasmids containing noxa and puma are as follows:

(1) one day before transfection, the cells are transferred to a 12-hole plate, so that the cell confluence rate reaches about 65% in the next day;

(2) the cells were replaced with fresh DMEM medium (containing 10% standard fetal bovine serum) before transfection;

(3) mu.l of Opti-MEM per well diluted 4. mu.g of plasmid;

(4) mu.l of Opti-MEM diluted 2. mu.l liposomes 2000 per well;

(5) uniformly mixing the diluent obtained in the steps (3) and (4), and standing at room temperature for 15 min;

(6) transfection Complex addition12 mesh plate, 37 ℃, 5% CO₂Culturing in an incubator;

as shown in FIGS. 5A-5H, the cell morphology of non-tumor cells Het-1a, HL7702 did not change significantly after transfection. As shown in fig. 6A to 6H, the hTERT promoter promotes the expression of the tumor killing genes Melittin and PEA in the tumor cell TE1, and the hTERT promoter promotes the expression of the tumor killing genes noxa and puma in the tumor cell HepG2, so that the tumor cell shows a relatively obvious apoptotic cell morphology, and shows that more cells with shrinkage, deformation and shedding appear, the cell transmittance is poor, and the intercellular connection disappears.

Example 5 cell proliferation assay after transfection of cells with recombinant plasmids containing tumor killer genes

(1) Taking cells in logarithmic growth phase, digesting conventionally, blowing off cells to be in single cell state, inoculating to 96-well plate at 50000/ml, 100 μ l/well, setting 4 multiple wells per group, at 37 deg.C and 5% CO₂Culturing in a cell culture box overnight;

(2) transfecting a human esophageal cancer TE1 cell and a normal esophageal cell Het-1a by using pcTERT-Melitin (Melittin recombinant plasmid), pcTERT-PEA (pseudomonas aeruginosa exotoxin recombinant plasmid) and pcTERT respectively; transfecting human liver cancer HepG2 cells and normal liver cells HL7702 by using pcTERT-noxa, pcTERT-puma and pcTERT, and putting the cells back into the incubator for continuous culture;

(3) plates treated at different time points were removed and 10. mu.l of CCK-8 solution was added to each well. Placing the mixture back into the incubator to continue culturing for 1h, and detecting the absorbance of each hole at 450nm by using an enzyme-labeling instrument;

(4) the survival rate of the cells was calculated by the following formula (OD value of experimental group/OD value of control group × 100%);

the recombinant vector connected with the tumor killing effect gene has strong inhibition effect on the proliferation of a human esophageal cancer TE1 cell (figure 7A) and a human liver cancer HepG2 cell (figure 7C) respectively, and basically has no inhibition effect on the proliferation of a normal esophageal cell Het-1a (figure 7B) and a normal liver cell HL7702 (figure 7D). The recombinant vector has the function of promoting the subsequent gene expression with tumor specificity.

Example 6 in vivo study of the Effect of recombinant vectors ligated with melittin sequences on tumors

HepG2 cells are inoculated to the armpit of a nude mouse to establish a HepG2 transplantation tumor model, and the pcTERT-Melitin vector is found to have a certain inhibition effect on tumor growth by intratumoral injection administration. The method comprises the following steps:

(1) the HepG2 cells in a 10cm culture dish grow and converge to 80%, and the cells are collected by conventional digestion;

(2) the culture medium was aspirated and the physiological saline resuspended the cells to a density of 5 × 10⁷Per mL, 200ul of cell suspension was injected left axillary;

(3) tumor size 80mm³On the left and right, the groups were divided into 4 groups of physiological saline, 4 groups of pcTERT and 4 groups of pcTERT-Melitin. Injecting transferrin cation liposome compound into tumor. The injection is injected once every three days and three times in total;

(4) the mouse activity status and the tumor growth condition are observed and recorded.

Through the research of in vivo experiments, the plasmid constructed by connecting the recombinant vector with the melittin sequence with the tumor killing effect can be stably expressed in vivo and has the effect of inhibiting the growth of tumor tissues. As shown in FIG. 8, the tumor growth was significantly inhibited, and as shown in FIGS. 9A to 9C, the expression level of the apoptosis-related protein clear-caspase 3 in the tumor cells was increased, and the expression level of the cycle-related protein cyclinD1 was decreased.

The above description is related to embodiments of the present invention, and the description is specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for constructing a tumor targeting vector pcTERT is characterized by comprising the following steps:

(1) taking pcDNA3.1+ as a template, designing an upstream primer:

5'-CTGACGCGTGGAGTTCCGCGTTAC-3' and

5'-CGCGCTAGCCAAAACAAACTCCCATTG-3' as downstream primer, PCR reaction to obtain 406bp length cmv enhancer fragment in pcDNA3.1+ plasmid with MluI site in 5 'end and NheI site in 3' end;

secondly, the human genome DNA is used as a template, and an upstream primer is designed:

5'-ATAACGCGTACCCTGGGAGCGCGAGCGGC-3', and

a downstream primer: 5'-CGCGCTAGCCAAAACAAACTCCCATTG-3', obtaining an hTERT promoter fragment with the length of 378bp through PCR reaction, and leading the 5 'end to be provided with a NheI site and the 3' end to be provided with a HindIII site;

(3) the pcDNA3.1+ vector and the cmv enhancer fragment obtained by PCR are subjected to MluI and NheI double enzyme digestion respectively, T4DNA ligase is used for connecting to obtain an intermediate 1, the intermediate 1 and the hTERT promoter fragment with the length of 378bp are subjected to NheI and HindIII enzyme digestion respectively, and then the T4DNA ligase is used for connecting to obtain the recombinant vector pcTERT.