CN117417962A - Construction method and application of multi-target oncolytic adenovirus - Google Patents
Construction method and application of multi-target oncolytic adenovirus Download PDFInfo
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Abstract
The invention provides a construction method and application of a multi-target oncolytic adenovirus, and belongs to the field of genetic engineering medicines. The invention constructs a modified shuttle vector by inserting an hTERT promoter, an E1A gene region, a 55kD E1B related fragment and a p53 gene into the shuttle vector, modifies a skeleton vector of adenovirus with deleted E1 and E3 regions to obtain a modified adenovirus vector, and expresses the modified shuttle vector and the modified adenovirus vector after co-transfecting cells to obtain the multi-target oncolytic adenovirus. The multi-target oncolytic virus has strong killing power to tumor cells and high safety to normal cells, can effectively inhibit the growth of tumors in tumor model mice, and has good clinical application prospect.
Description
Technical Field
The invention belongs to the field of genetic engineering medicaments, and particularly relates to a construction method and application of a multi-target oncolytic adenovirus.
Background
Oncolytic viruses are tumor killing viruses with replication capacity, can invade tumor cells through cell surface receptor molecules, and have tumor treatment potential. An effective strategy of the existing oncolytic virus treatment is to reconstruct oncolytic viruses with specificity, target specific receptors overexpressed in tumor cells, invade the viruses into the tumor cells and perform subsequent functions.
Oncolytic virus mediated gene therapy has high killing efficiency on tumor cells, good specificity, high safety, small side effect and low cost, and is an emerging tumor immunotherapy mode. Oncolytic viral therapy is not a complete decade, however, its greatest challenge is still how to improve the safety and effectiveness of the product.
The Chinese patent with application number 201010621084.2 discloses a construction scheme for artificially modifying human adenovirus type 5 (Ad 5) and a specific application of a novel oncolytic adenovirus construct obtained by using the scheme in tumor treatment, and belongs to the technical field of medical genetic engineering. The recombinant adenovirus construct is obtained by PCR amplification fixed-point deletion, enzyme digestion, connection, cloning, homologous recombination, transfection, adenovirus monoclonal purification and other technologies, and is characterized in that: the Ad5 genome E1A conserved sequence 2 (CR 2) region lacks 27 bases; 29477-29714nt of E3 region ADP gene is deleted; meanwhile, the full-length coding sequence (1131 bp) of the HSV-TK gene is inserted into the deletion region. The construct is a novel oncolytic adenovirus vector with higher tumor selective replication capacity, and has unique practical value in the biological treatment of tumors by utilizing the suicide gene effect of HSV-TK and the dual killing effect of oncolytic viruses on dissolving tumor cells. But have limited tumor killing ability.
Chinese patent application number 201910462073.5 discloses: recombinant oncolytic viruses, and a preparation method, application and medicaments thereof relate to the field of biotechnology. The disclosed recombinant oncolytic viruses contain the following exogenous elements in their genomic sequence: (1) A first expression cassette comprising a first promoter and a first interfering RNA expression sequence; (2) a target sequence; and (3) a second expression cassette. The propagation or replication of the recombinant oncolytic virus is regulated by exogenous elements inserted in the genome sequence thereof, and by virtue of the regulation of these exogenous elements, the recombinant oncolytic virus can selectively propagate or replicate in different types of cells, and by virtue of the selective propagation or replication, a second cell, i.e., a target cell (e.g., a tumor cell), can be selectively killed without causing damage to a first cell, i.e., a non-target cell (e.g., a normal cell). The prepared recombinant tumor virus has unstable effect and further improved tumor killing capacity.
Disclosure of Invention
In order to solve the problems, the invention utilizes an Ad5 adenovirus vector with the replication defect of AdMax to carry out genetic engineering modification, and obtains the oncolytic virus which utilizes multiple mechanisms to improve the safety and the effectiveness of the oncolytic virus.
The invention respectively modifies adenovirus skeleton vector (pBHGloxDeltaE 1,3Cre is taken as an example) and shuttle vector (pDC 316 is taken as an example), and uses the two modified vectors to co-transfect expression cells (293 cells are taken as an example) to prepare the oncolytic adenovirus.
The backbone vector pBHGloxDeltaE 1,3Cre of the AdMax system lacks the E1 gene and E3 gene of Ad5 adenovirus, so adenovirus prepared by cotransfecting 293 cells with shuttle vectors such as pDC316 does not have replication capacity, i.e. the characteristics of oncolytic virus are lost. The invention restores the replication capacity of the virus and enhances the high efficiency and safety of oncolytic viruses in tumor killing effect through the following transformation.
In one aspect, the invention provides a method for constructing a multi-target oncolytic adenovirus.
The construction method comprises the following steps:
(1) Inserting an hTERT promoter, an E1A gene region, a 55kD E1B related fragment and a p53 gene into the shuttle vector to construct an improved shuttle vector, wherein: the E1A gene region is an E1A gene lacking a CR2 region fragment; the 55kD E1B related fragment comprises an E1B gene deleted 19kD fragment and an E1B gene promoter region, wherein the E1B gene deleted 19kD fragment is SEQ ID NO.1;
(2) Modifying the skeleton vector of adenovirus with E1 and E3 deleted to obtain modified adenovirus vector:
replacing Knob and Shaft sequences on Ad5 adenovirus Fiber molecules with Knob and Shaft sequences on Ad35 adenovirus; inserting hGM-CSF gene into E3 deletion region of skeleton carrier;
(3) The shuttle vector and adenovirus vector are transformed to co-transfect cells for expression.
Specifically, in the step (1):
the hTERT promoter is SEQ ID NO.2;
the CR2 region fragment is SEQ ID NO.3;
the promoter region of the E1B gene is SEQ ID NO.4;
the sequence of the p53 gene region is SEQ ID NO.5.
Preferably, in the step (1): the E1A gene region is SEQ ID NO.6.
Preferably, in the step (1): the shuttle vector was pDC316. Indeed, the choice of shuttle vector by the person skilled in the art is not limited to the type preferred in the present invention, and can be used to perform the function of the shuttle vector.
The Knob and Shaft sequences on the Ad35 adenovirus in the step (2) are SEQ ID NO.7.
The hGM-CSF gene in the step (2) is SEQ ID NO.8.
The step of modifying the skeleton vector of the adenovirus with deleted E1 and E3 regions in the step (2) comprises the following steps:
1) Constructing a vector containing Cre enzyme recognition sites, resistance screening markers and Knob and Shaft functional regions on Ad35 type virus Fiber genes, and obtaining positive clones through resistance screening;
2) Homologous recombination is carried out by using escherichia coli expressing Cre recombinase, and the added screening mark is knocked out;
3) The hGM-CSF gene was inserted into the E3 deletion region of the backbone vector.
In the step 1): resistance selection markers include resistance genes and promoters; the Cre enzyme recognition site is a LoxP site.
Preferably, in the step 1): loxP sequence is shown in SEQ ID NO. 9.
In the step 3): the hGM-CSF gene is co-inserted with AD5 framework sequence, and the insertion enzyme cutting site is PacI.
In some embodiments, the modification of the shuttle vector of step (1) may comprise the steps of:
inserting a human telomerase reverse transcriptase hTERT promoter region and an E1A gene region regulated by the human telomerase reverse transcriptase hTERT promoter region, wherein the sequences are respectively shown as SEQ ID NO.2 and SEQ ID NO.6;
inserting a deletion 19kD E1B gene regulated by E1A and a promoter region of the E1B gene, namely a 55kD E1B related fragment; SEQ ID NO.4-SEQ ID NO.1 (representing the connection of the two);
deletion of the CR2 segment (24 bp sequence) on the E1A gene by homologous recombination; CR2 segment is shown as SEQ ID NO.3;
the p53 gene was inserted into the vector at the multiple cloning site. The specific sequence is shown in SEQ ID NO.5.
The transformation mode has the following effects:
(1) The specific replication capacity of oncolytic viruses in tumor cells is further enhanced by utilizing the characteristic that a tumor specific promoter is utilized to specifically express an E1A gene and the protein with a24 bp sequence deleted from the E1A gene can not be combined with RB molecules;
(2) The apoptosis of tumor cells induced by oncolytic viruses is enhanced by deleting the 19kD molecule of the E1B gene.
The shuttle vector of the present invention further comprises:
the p53 gene was inserted at the multiple cloning site. p53 is a tumor suppressor gene. In all malignant tumors, mutations in this gene occur in more than 50%. Overexpression of wild-type p53 in tumor cells can block tumor cell cycle, promote apoptosis and inhibit angiogenesis of tumors, thereby exerting the cancer-inhibiting function of p53.
In the modification of the shuttle vector, the invention is mainly realized by a eukaryotic cell one-step homologous recombination method or a proper enzyme digestion and enzyme ligation method.
Preferably, the plasmid map of the engineered shuttle vector pDC316 of the invention is fig. 1: inserting an E1A gene and inserting a tumor specific promoter hTERT sequence upstream of the E1A on the basis of a shuttle vector PDC316, inserting a gene which is regulated by the E1A and lacks a 55kD-E1B gene with 19kD and a transcription regulation region (promoter) thereof, and deleting 24bp basic groups on a CR2 gene of the E1A; the p53 gene is inserted into the multiple cloning site of the vector, and the expression of the gene is regulated by a CMV promoter. The shuttle vector was designated pDC316-hTERT-del24E1A-55kDE1B-p53.
In some embodiments, the modification of the backbone vector pbhgloxΔe1,3Cre of adenovirus in step (2) mainly comprises two positions:
1) Replacing Knob and Shaft sequences on the Ad5 adenovirus Fiber molecule with Knob and Shaft sequences on Ad35 adenovirus;
2) The hGM-CSF gene is inserted into the E3 deletion region by homologous recombination, and the expression of hGM-CSF is regulated by a promoter endogenous to genome, the promoter sequence of which is shown in SEQ ID NO. 10.
The transformation mode has the following effects:
because the first step in the invasion of Ad5 adenovirus into cells requires interaction with tumor cells, this interaction is achieved by the recognition of CAR expressed on the tumor surface by the Fiber protein of the virus (coxsackie-adenovirus receptor, CAR), but in some tumor cells recognition and invasion of tumor cells by Ad5 oncolytic adenovirus is restricted due to lack of CAR expression or low expression. Some group B adenoviruses, such as adenovirus type 35, can infect tumor cells through non-CAR receptors, such as CD 46. CD46 is a widely expressed membrane protein. In order to enable oncolytic viruses to generate anti-tumor effects on various tumors, the chimeric adenovirus Fiber molecules are established, and can be combined with CD46 on the surfaces of tumor cells, so that invasion of tumor cells with negative expression of a plurality of CARs is realized.
The skeleton plasmid pBHGloxDeltaE 1,3Cre of the five adenovirus has completely deleted the expression of E3 gene, and the deletion of this segment of gene segment can let us insert exogenous functional gene hGM-CSF. hGM-CSF promotes proliferation of macrophages, monocytes, dendritic cells, and phagocytes by modulating mammalian bone marrow cell production. In immune response, hGM-CSF acting cells are mainly antigen presenting cells APC (antigen presenting cell) to maintain and enhance adaptive immune responses by enhancing the processing and expression of antigens by APCs.
The size of the skeleton plasmid pBHGloxDeltaE 1,3Cre of the five-type adenovirus reaches more than 34kb, so that the technical method for carrying out reconstruction by eukaryotic homologous recombination or enzyme digestion and enzyme ligation is not feasible.
The present invention preferably works by combining techniques of homologous recombination of multiple bacteria.
The pBHGloxDeltaE 1,3Cre modification is specifically as follows:
(1) The Knob and the sh domain on the type 5 adenovirus Fiber molecule was replaced by the Knob and sh domain on the type 35 adenovirus by a two-step homologous recombination technique:
the first step is to replace the Knob and the sh domains on the type 5 adenovirus Fiber molecule by homologous recombination of the SW102 strain, which carries a kanamycin resistance on the viral backbone plasmid and a pair of LoxP sequences-Lox 2272 (SEQ ID No. 9) at both ends of the selection marker. The complete sequence of the kanamycin resistance gene is shown as SEQ ID NO. 11.
And secondly, performing Cre-loxP homologous recombination by using escherichia coli expressing Cre recombinase, and knocking out an added screening marker kanamycin resistance gene, wherein only one segment of Lox2272 sequence is reserved. The viral backbone of the constructed Fiber chimera is abbreviated as Ad5F35 type.
(2) Insertion of hGM-CSF in backbone deleted E3 region was a successful vector constructed by PacI cleavage of Fiber chimera, immediately prior to insertion of hGM-CSF gene into PacI cleavage site on backbone vector by homologous recombination in E.coli BJ 5183.
In some embodiments, the vector plasmid map is fig. 2.
The skeleton carrier pBHGloxDeltaE 1,3Cre of the five adenovirus is used for modification to form chimera of Fiber gene, which is called Ad5F35 for short; the hGM-CSF gene was inserted into the E3 deletion region. The backbone vector was finally designated pBHGloxDeltaE 1,3Cre-hGM-CSF-Ad5F35-Lox2272.
The co-transfection in step (3) is performed by transfecting cells having an expression function, which cells may be selected according to common general knowledge of a person skilled in the art, preferably 293 cells in the present invention. In fact, other cell types are possible, such as A549 cells or 293 cells stably expressing the ADP gene.
In yet another aspect, the invention provides a multi-target oncolytic adenovirus constructed by the aforementioned construction method.
In still another aspect, the invention also provides application of the multi-target oncolytic adenovirus in preparing an antitumor drug.
The invention also protects the tumor-dissolving adenovirus containing the multiple targets in preparing the antitumor drug.
Such tumors include, but are not limited to, digestive system tumors or respiratory system tumors.
Such digestive system tumors include, but are not limited to: esophageal cancer, pancreatic cancer, intestinal cancer, gastric cancer, liver cancer, etc.
The respiratory bone tumors include, but are not limited to: lung cancer, nasopharyngeal cancer, laryngeal cancer.
Preferably, the medicament is for the treatment of intestinal cancer or lung cancer.
Further preferably, the bowel cancer includes, but is not limited to: colorectal cancer, duodenal cancer, preferably colorectal cancer.
The intestinal cancer may be of the bulge type, ulcer type or infiltration type.
Preferably, the drug is a targeted drug.
The medicine also comprises other pharmaceutically acceptable carriers or excipients.
The invention has the beneficial effects that:
1. the tumor specific hTERT promoter is inserted, the 24bp sequence on E1A is deleted, and the virus is specifically replicated in RB protein mutated tumor cells, so that the safety is improved;
the vector lacks a 19KD protein on the E1B gene, so that the induction capability of the oncolytic virus to apoptosis is enhanced; expressing p53 and hGM-CSF cytokine, improving the immune regulation of oncolytic virus to organism; the Fiber gene of the type 5 adenovirus is changed into a virus which is chimeric with the type 35 adenovirus Fiber, the way of infecting tumor cells by the virus is changed, and the infection capacity of the CAR expression negative tumor cells is enhanced through the action of CD 46. The above operation has an effect of enhancing the effectiveness.
2. The oncolytic adenovirus prepared by the invention has good killing and inhibiting effects on tumor cells, is superior to the prior art, and has high safety on normal cells.
3. The oncolytic adenovirus can effectively inhibit the growth of tumors in a tumor model mouse body, and has good clinical application prospect.
Drawings
FIG. 1 is a block diagram of a successfully constructed shuttle vector pDC316-hTERT-del24E1A-55kDE B-p53.
FIG. 2 is a diagram of the structure of a modified pBHGloxDeltaE 1,3Cre-hGM-CSF-AD5F35-Lox2272.
FIG. 3 is a diagram of the genome architecture of the multi-target oncolytic adenovirus E10K-1A.
FIG. 4 PCR identification of the genome of the multi-target oncolytic adenovirus E10K-1A.
FIG. 5 expression detection of the p53 gene carried by oncolytic adenovirus E10K-1A in tumor cells.
FIG. 6 expression detection of hGM-CSF gene carried by oncolytic adenovirus E10K-1A in tumor cells.
FIG. 7 in vitro killing effect of oncolytic adenovirus E10K-1A on tumor cells and normal cells.
FIG. 8 effect of oncolytic adenovirus E10K-1A on inhibiting proliferation of human colon carcinoma SW620 nude mice engrafting.
FIG. 9 is a graph showing the effect of oncolytic adenovirus E10K-1A on inhibiting proliferation of human non-small cell lung carcinoma NCI-H1299 nude mice transplantation tumor.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
"hTERT" refers to human telomerase reverse transcriptase. The promoter, i.e., the hTERT promoter, can transcriptionally regulate the E1A gene region.
In the present invention, "SW102" is Escherichia coli having a recombinant enzyme incorporated therein.
EXAMPLE 1 construction of pDC316-hTERT-del24E1A-55kDE1B-p53
(1) The following sequence was inserted on pDC 316:
hTERT promoter: SEQ ID NO.2;
E1A Gene region: SEQ ID NO.6;
55kD E1B related fragment: SEQ ID NO.4-SEQ ID NO.1.
Wherein, hTERT refers to human telomerase reverse transcriptase, and the promoter thereof is hTERT promoter.
The E1A gene region is transcriptionally regulated by the hTERT promoter.
The 55kD E1B-related fragment includes a deletion of the 19kD E1B gene (E1B-55 kD, SEQ ID NO. 1) and a promoter region (E1B gene promoter region, SEQ ID NO. 4) on the viral genome that regulates transcription of 55kD-E1B, the deletion of the 19kD E1B gene being regulated by E1A.
The specific operation is as follows:
extracting genomic DNA of HepG2 cells, and carrying out PCR amplification to obtain a core promoter sequence of hTERT, wherein the N end of the sequence is provided with a homologous arm on the left side of an XbaI enzyme cutting site of a pDC316 vector; the amplification primers were hTERT-F-pDC316 (SEQ ID NO. 12) and hTERT-R (SEQ ID NO. 13).
Extracting RAN from 293 cells, carrying out reverse transcription to obtain a cDNA sequence, and amplifying a required E1A gene sequence, wherein the N end of the sequence is provided with a homology arm with the C end of an hTERT promoter, the C end of the sequence is provided with a homology arm with an E1B gene promoter region, and amplification primers are E1A-F (SEQ ID NO. 14) and E1A-R (SEQ ID NO. 15);
synthesizing 55kD E1B related fragment (E1B gene Promoter region and E1B-55kD sequence), further amplifying by PCR, wherein the 55kD E1B related fragment E1B gene Promoter region and the C end of the 55kD-E1B sequence are provided with homologous arms on the right side of the XbaI enzyme cutting site of the pDC316 vector, and the amplifying primers are E1B-55kD-Promoter-F (SEQ ID NO. 16) and E1B-55kD-R-pDC316R (SEQ ID NO. 17);
the linearized pDC316 vector (purchased from Microbix Biosystems Inc, cat# PD-01-64) was obtained by XbaI cleavage, and the hTERT promoter and E1A gene and 55kD E1B-related fragment (55 kD-E1B-promoter-gene fragment) were simultaneously and homologously recombined and integrated into the XbaI cleavage site of the pDC316 vector using the Norwegian incorporated's integrated ultrafast cloning kit (cat# C115-01).
Homologous recombination reaction system:
| component (A) | Dosage of |
| Linearization vector pDC316 | 100ng |
| hTERT promoter fragment | 10ng |
| E1A gene fragment | 20ng |
| 55 kD-E1B-promoter-gene fragment | 40ng(E1B) |
| 2×ClonExpress Mix | 2μL |
| ddH 2 O | Supplement to 10. Mu.L |
Recombination reaction conditions, 50 ℃,15min; then cooling to 4 ℃ or immediately placing on ice, then transforming DH5 alpha competent cells, coating an ampicillin resistance plate for PCR screening and sequencing to obtain the shuttle vector of pDC316-hTERT-E1A-55kD E1B. The linearized shuttle vector was obtained by KasI cleavage.
(2) The 24bp sequence of the CR2 region on the E1A gene was deleted by homologous recombination (SEQ ID NO. 3).
The specific operation is as follows: to delete the 24bp sequence of the CR2 region of the E1A gene, two sequences of E1A were first amplified with two pairs of primers, named Delta24E1A-a (amplification primer Delta24E1A-F: SEQ ID NO.18; delta24E1A-a-R: SEQ ID NO. 19) and Delta24E1A-C (amplification primer Delta24E1A-C-F: SEQ ID NO.20; delta24E1A-C-R: SEQ ID NO. 21), respectively. The 24bp sequence of the CR2 section is deleted at the joint of the two sequences, then the two PCR sequences and a shuttle vector pDC316-hTERT-E1A-55kD-E1B which is tangentially subjected to KasI enzyme are subjected to homologous recombination through a rapid recombination kit (Nuo-vone, cat# C115-01), and a new shuttle vector named pDC316-hTERT-del24E1A-55kDE B is obtained after screening. Homologous recombination reaction system:
| component (A) | Dosage of |
| Linearization shuttle vector | 100ng |
| Delta24E1A-A | 10ng |
| Delta24E1A-C | 20ng |
| 2×ClonExpress Mix | 2μL |
| ddH 2 O | Supplement to 10. Mu.L |
Recombination reaction conditions, 50 ℃,15min; cooling to 4 ℃ or immediately placing on ice, transforming DH5 alpha competent cells, coating an ampicillin resistance plate for PCR screening and sequencing, and finally determining that the shuttle vector pDC316-hTERT-del24E1A-55kDE B is successfully constructed. EcoRI and SalI were digested simultaneously to obtain linearized vectors.
(3) Construction of the shuttle vector pDC316-hTERT-del24E1A-55kD-E1B-p53
The specific operation is as follows: the p53 gene sequence (SEQ ID NO. 5) was synthesized with EcoRI and SalI cleavage sites at both ends. The clone after sequence digestion is inserted between EcoRI and SalI of pDC316-hTERT-del24E1A-55kD-E1B multicloning sites (i.e. connected with the linearization vector obtained in the step (2)), the modified shuttle vector is named pDC316-hTERT-del24E1A-55kDE B-p53, and the plasmid map is shown in figure 1.
EXAMPLE 2 construction of pBHGloxDeltaE 1,3Cre-hGM-CSF-Ad5F35-lox2272
The Knob and the leaf functional region on the type 5 adenovirus Fiber molecule is replaced by the Knob and the leaf functional region on the type 35 adenovirus by a two-step homologous recombination technology:
(1) The first step is to replace the Knob and the sh functional regions on the Fiber molecule of the type 5 adenovirus with the Knob and the sh functional regions on the Ad35 adenovirus by homologous recombination of SW102 bacteria, and the step carries a kanamycin resistance screening mark on the virus skeleton plasmid, and a pair of LoxP sequences-Lox 2272 are arranged at two ends of the screening mark.
The specific operation is as follows: one sequence was synthesized including the Knob and the shift functional region (SEQ ID NO. 7) on the Ad35 type virus Fiber gene, one Lox2272 sequence (SEQ ID NO. 9), the ampicillin promoter regulating kanamycin expression (SEQ ID NO. 22), the kanamycin resistance gene (SEQ ID NO. 11) and the other Lox2272 sequence (SEQ ID NO. 9). The N end of the segment sequence is a 50bp homology arm (SEQ ID NO. 23) with the Tail sequence of the AD5 type virus Fiber gene, and the C end is a 300bp homology arm (SEQ ID NO. 24) with the C end framework genome of the AD5 type virus Fiber gene. PCR amplification primers (AD 35F: SEQ ID NO.25; AD35R: SEQ ID NO. 26) are designed through homologous arms at two ends, and a full-length sequence fragment (SEQ ID NO. 27) to be inserted into the Ad5 type framework is obtained through PCR amplification. 100ng of this fragment was co-electrotransferred to SW102 competent cells (Minghuav, B98002) with 20ng of pBHGloxΔE1,3Cre vector (i.e., ad 5-type backbone, purchased from Microbix Biosystems Inc, PD-01-64), then recovered by adding 1mL of antibiotic-free LB medium for 2h, screening with kanamycin-resistant plates and sequencing, and finally obtaining a successfully-modified backbone vector designated pBHGloxΔE1,3Cre-Ad5F35-lox2272-kana-lox2272. The modified skeleton vector replaces the corresponding sequence on the Ad5 type adenovirus Fiber gene with the Knob and Shaft sequences on the Ad35 type adenovirus Fiber gene, but also introduces the kanamycin resistance gene, the promoter for regulating the transcription and LoxP sequences at the two ends, so that redundant gene sequences need to be knocked out continuously.
(2) And secondly, performing Cre-LoxP homologous recombination by using escherichia coli expressing Cre recombinase, and knocking out the added screening mark. The specific operation is as follows: 50ng of pBHGloxDeltaE 1,3Cre-Ad5F35-lox2272-kana-lox2272 are electrotransformed into escherichia coli competent cells expressing Cre recombinase, colony PCR screening identification is carried out after ampicillin plates are coated, and sequencing is carried out, so that a kanamycin resistance gene, a promoter for regulating transcription of the kanamycin resistance gene and a skeleton vector with a lox2272 knocked out are finally obtained, and the skeleton vector is named as pBHGloxDeltaE 1,3Cre-Ad5F35-lox2272 and is called Ad5F35 skeleton vector for short.
(3) The hGM-CSF gene was inserted into the E3 deletion region of the Ad5F35 backbone vector.
This step linearizes the Ad5F35 backbone vector by PacI cleavage (from NEB, R0547V) and the hGM-CSF gene is inserted directly into the backbone vector by homologous recombination after transduction of E.coli competent BJ5183 (from Bio-only, DL 1075S). The specific operation is as follows:
synthesizing a DNA sequence comprising:
1kb homology arm upstream of PacI cleavage site of Ad5F35 backbone vector: SEQ ID NO.28;
an Ad 5-type framework sequence: SEQ ID NO.29; (original Ad5 backbone is self, knocked out during vector construction, thus reconstructed into vector);
hGM-CSF expression Gene: SEQ ID NO.8;
homology arms of 260bp downstream of PacI cleavage site: SEQ ID NO.30.
The primers (hGM-CSF Primer F: SEQ ID NO.31; hGM-CSF Primer R: SEQ ID NO. 32) were designed with this sequence to amplify fragments, and the PacI enzyme-tangential Ad5F35 backbone vector was co-transferred into E.coli competent BJ5183, and after ampicillin plating, PCR clone identification and sequencing analysis were performed to finally obtain a modified backbone vector having the hGM-CSF gene inserted at the deletion position of E3 of the Ad5F35 vector, designated pBHGloxΔE1,3Cre-hGM-CSF-Ad5F35-lox2272.
Vector construction was complete and sequencing was correct. The final plasmid map is FIG. 2.
EXAMPLE 3 packaging, preparation and identification of Multi-target oncolytic adenoviruses
(1) The 293 cells were co-transfected with the 2 vectors constructed in example 1 and example 2 to prepare multi-target oncolytic adenoviruses by the following method:
HEK293 cells are co-transfected with the constructed shuttle vector pDC316-hTERT-del24E1A-55kDE B-p53 (modified shuttle vector for short) and chimeric skeleton vectors pBHGloxdelta E1,3Cre-hGM-CSF-Ad5F35-lox2272 (modified adenovirus vector for short) to package oncolytic viruses. Day before transfection, 5X 10 5 Inoculating HEK293 cells into 6cm culture dish with DMEM+10% FBS, and placing at 37deg.C with 5% CO 2 Cell culture in incubatorAnd (5) culturing overnight.
The next day when the cell density reached around 80% the fresh 10% fbsdem medium was changed and culture continued for 2 hours. Co-transfection was performed with 3.2. Mu.g of the engineered adenovirus vector and 0.8. Mu.g of the engineered shuttle vector using the TurboFect transfection reagent (ThermoFisher, cat. R0533) according to the instructions for use. The following day after transfection, the grown cells were passaged into T25 cell flasks and continued to be cultured with DMEM medium containing 5% fbs, and transferred into T75 cell flasks after the cells were grown. During the period, part of the culture medium is added, and cells are toxic about 20 days, and the cells are in grape shape and start to fall off from the wall of the culture flask in a large amount. After eluting the toxic cells with the culture solution, 500g is centrifuged for 10 minutes, the supernatant is discarded, the cells are resuspended with 2mL PBS, and the cells are repeatedly frozen and thawed three times in a refrigerator at-80 ℃ and a water bath at 37 ℃. The supernatant containing the virus was collected and stored for later use by centrifugation at 12000g for 10 minutes. The oncolytic virus obtained was designated as E10K-1A and FIG. 3 is a schematic representation of the E10K-1A genome structure.
Subsequently, PCR identification of genes related to the genome of oncolytic virus E10K-1A was performed. The specific operation is as follows: 50 mu L E K-1A virus solution is taken, 2 mu L of proteinase K is added, and the virus genome is released by digestion for 30min at 50 ℃, and the gene sequence of E1A/p53/hGM-CSF is amplified by using the virus genome as a template for PCR. The identification primers used were as follows:
AD-E1A-F：SEQ ID NO.33；
AD-E1A-R：SEQ ID NO.34；
AD-p53-F：SEQ ID NO.35；
AD-p53-R：SEQ ID NO.36；
AD-hGMCSF-F：SEQ ID NO.37；
AD-hGMCSF-R：SEQ ID NO.38。
the PCR result is subjected to agarose gel electrophoresis, and the result shows that the single target band can be amplified, and the fragment size is correct. The specific electrophoresis results are shown in FIG. 4.
Identification of inserted foreign genes p53 and hGM-CSF expression on oncolytic virus: after infection of tumor cells A549 and SW620 (purchased from Wohaze life technologies Co., ltd.) with MOI 0.05 oncolytic virus E10K-1A, cell culture supernatants were collected 72 hours, and the expression of hGM-CSF was detected by ELISA (Shanghai Biyun biotechnology Co., ltd., hGM-CSF Elisa kit (cat. PG 355)); the oncolytic virus E10K-1AMOI was taken as 2 to infect H1299 tumor cells (p 53 negative cells, purchased from the Marinozier life technologies Co., ltd.) and after 24 hours, the p53 protein expression was detected by Western-Blot (p 53 antibody was purchased from Cell Signaling Co., ltd., product No. 2524T) after Cell lysis, and as a result, both exogenous genes were able to be normally expressed in tumor cells as shown in FIG. 5 and FIG. 6.
Example 4 functional analysis of oncolytic Virus E10K-1A on tumor cells and mouse tumor models
(1) Specific killing inhibition of tumor cells by oncolytic adenovirus E10K-1A:
the killing effect of oncolytic virus E10K-1A on various tumor cells and normal cells was studied by CCK8 experiments and compared with ADv-p53 virus in which a non-replicating adenovirus type 5 was used as a vector to express p53 (trade name: produced by Shenzhen Siebold Gene technology Co., ltd.).
Taking logarithmic growth phase of A549, SW620 and H1299 tumor cells and normal BJ cells 1×10 4 Cell/well inoculation 96-well cell culture plate, cell adherence is good after 24 hours, according to the cell inoculation number of each well in the 96-well plate and the titer of each oncolytic virus, oncolytic virus E10K-1A or ADv-p53 virus is used for infecting various cell lines with different infection complex (MOI 0,0.01,0.02,0.05,0.1,0.2,0.5,1), 3 compound wells are made for each MOI, and no virus is added to a blank group. After the cells are added with the toxin, the 96-well plate is placed into a cell culture box for continuous culture. After 72h of virus infection, 10 μl of CCK-8 solution was added to each well of a 96-well plate, the plate was further placed in an incubator for 2 hours, the 96-well plate was removed, absorbance at 450nm of each well cell was measured with a microplate reader, cell viability was calculated (cell viability was calculated by dividing absorbance of the test group by absorbance of the control group), and a cell viability curve was drawn.
As shown in FIG. 7, the oncolytic virus E10K-1A has good killing and inhibiting effects on the three tumor cells, is closely related to the virus infection titer, and the E10K-1A is obviously better than the killing effect of ADv-p53 virus on the tumor cells. For normal BJ cells, the killing effect of oncolytic viruses is lower than that of tumor cells under the same MOI, and the good safety is shown.
(2) E10K-1A mouse animal model experiment for resisting human colorectal adenocarcinoma
15 healthy, pure-bred male BALB/C nude mice of 4 weeks old were purchased from Zhuhai Bai Tong Biotechnology Co., ltd., pass No.44822700013148. 200 μl of a suspension of human colorectal adenocarcinoma cells SW620 in logarithmic growth phase was injected subcutaneously into the back of BALB/c nude mice. 7 days after inoculation when the tumor reached an average of 100mm 3 The cells were randomly divided into 3 groups (PBS control group, E10K-1A group, ADv-P53 group) of 5 cells each. The 2 virus treatment groups were given corresponding intratumoral multiple injections at 2.95X10 each dose 7 pfu/100. Mu.L, once every other day, 5 total injections; the blank group was injected with PBS simultaneously, 100. Mu.L each. After treatment, the short diameter and long diameter of the tumor are measured periodically with a digital vernier caliper every day, and the long diameter is multiplied by the short diameter 2 X 0.5 calculate tumor volume and plot tumor growth curves.
The experiment starts from the 7 th day after SW620 inoculation, and the observation shows that the E10K-1A oncolytic virus administration group and the ADv-P53 administration group can obviously inhibit the proliferation of tumors relative to the PBS control group on the 21 st day, and the E10K-1A can obviously differ from the PBS group in the tumor size from the 11 th day until the inhibition rate of the tumor proliferation reaches about 50 percent on the 21 st day; whereas the inhibition of tumor proliferation was significantly different in the E10K-1A oncolytic virus administration group compared to the ADv-P53 administration group. This demonstrates that oncolytic virus E10K-1A is effective in inhibiting colorectal tumor growth (FIG. 8).
(3) E10K-1A anti-human non-small cell lung cancer mouse animal model experiment
15 healthy, pure-bred male BALB/C nude mice of 4 weeks old were purchased from Zhuhai Bai Tong Biotechnology Co., ltd., pass No.44822700013576. 200 mu L of NCI-H1299 cell suspension of human non-small cell lung cancer in logarithmic growth phase is injected into the back of BALB/c nude mice subcutaneously. About 4 weeks after inoculation when the tumor reaches an average of 100mm 3 The cells were randomly divided into 3 groups (PBS control group, E10K-1A group, ADv-P53 group) of 5 cells each. Administration of corresponding intratumoral multiple injections to 2 viral treatment groupsEach dose was 2.95×10 7 pfu/100. Mu.L, once every other day, 5 total injections; the blank group was injected with PBS simultaneously, 100. Mu.L each. After treatment, the short diameter and long diameter of the tumor are measured periodically with a digital vernier caliper every day, and the long diameter is multiplied by the short diameter 2 X 0.5 calculate tumor volume and plot tumor growth curves.
Experimental observation shows that the therapeutic effect from the 1 st to 19 th of administration shows that E10K-1A has a significant difference in tumor size relative to the PBS group at the 5 th day after the first administration, and the inhibition rate of tumor proliferation reaches about 90% at the 19 th day; the ADv-P53 administration group showed a significant difference in tumor size from the PBS group at day 5, and the inhibition rate of tumor proliferation reached about 70% by day 19, and showed a significant difference in inhibition effect on tumor proliferation. It was also found that the E10K-1A-administered group showed a significant difference in inhibition of tumor proliferation relative to ADv-P53-administered group. This demonstrates that oncolytic virus E10K-1A is effective in inhibiting the growth of non-small cell lung cancer (FIG. 9).
Claims (14)
1. A method for constructing a multi-target oncolytic adenovirus, comprising:
(1) Inserting an hTERT promoter, an E1A gene region, a 55kD E1B related fragment and a p53 gene into the shuttle vector to construct an improved shuttle vector, wherein: the E1A gene region is an E1A gene lacking a CR2 region fragment; the 55kD E1B related fragment comprises an E1B gene deleted 19kD fragment and an E1B gene promoter region, wherein the E1B gene deleted 19kD fragment is SEQ ID NO.1;
(2) Modifying the skeleton vector of adenovirus with E1 and E3 deleted to obtain modified adenovirus vector:
replacing Knob and Shaft sequences on Ad5 adenovirus Fiber molecules with Knob and Shaft sequences on Ad35 adenovirus; inserting hGM-CSF gene into E3 deletion region of skeleton carrier;
(3) The shuttle vector and adenovirus vector are transformed to co-transfect cells for expression.
2. The method according to claim 1, wherein in the step (1), the hTERT promoter is SEQ ID No.2; the CR2 region fragment is SEQ ID NO.3; the sequence of the p53 gene region is SEQ ID NO.5.
3. The construction method according to claim 2, wherein the E1A gene region in the step (1) is SEQ ID NO.6.
4. The method of claim 3, wherein the shuttle vector in step (1) is pDC316.
5. The method according to claim 1, wherein the Knob and Shaft sequences on the Ad35 adenovirus in step (2) are SEQ ID No.7.
6. The construction method according to claim 5, wherein hGM-CSF gene in the step (2) is SEQ ID NO.8.
7. The construction method according to claim 1, wherein the step of engineering the backbone vector of the adenovirus with deleted E1 and E3 regions in step (2) comprises:
1) Constructing a vector containing Cre enzyme recognition sites, resistance screening markers and Knob and Shaft functional regions on Ad35 type virus Fiber genes, and obtaining positive clones through resistance screening;
2) Homologous recombination is carried out by using escherichia coli expressing Cre recombinase, and the added screening mark is knocked out;
3) The hGM-CSF gene was inserted into the E3 deletion region of the backbone vector.
8. The method according to claim 7, wherein in the step 1): resistance selection markers include resistance genes and promoters; the Cre enzyme recognition site is a LoxP site.
9. The method according to claim 7, wherein in the step 3): the hGM-CSF gene is co-inserted with AD5 framework sequence, and the insertion enzyme cutting site is PacI.
10. The method according to claim 8, wherein in the step 1): loxP sequence is shown in SEQ ID NO. 9.
11. A multi-target oncolytic adenovirus constructed by the construction method of any one of claims 1-10.
12. The use of the multi-target oncolytic adenovirus of claim 11 in the preparation of an anti-tumor drug.
13. The use according to claim 12, wherein the neoplasm is a solid tumor, including breast cancer, liver cancer, gall bladder cancer, stomach cancer, colon cancer, lung cancer, prostate cancer, lymphoma, colorectal cancer, ovarian cancer, cervical cancer, cholangiocarcinoma, esophageal cancer, renal cancer, glioma, melanoma, pancreatic cancer, bladder cancer or head and neck cancer.
14. An antitumor agent comprising the multi-target oncolytic adenovirus of claim 11.
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