CN113201508A

CN113201508A - Recombinant Newcastle disease oncolytic virus and preparation method and application thereof

Info

Publication number: CN113201508A
Application number: CN202110541328.4A
Authority: CN
Inventors: 赵永祥; 钟莉娉
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-05-18
Filing date: 2021-05-18
Publication date: 2021-08-03

Abstract

The invention discloses a recombinant Newcastle disease oncolytic virus, a preparation method and application thereof, and belongs to the field of tumor treatment. The invention constructs the oncolytic newcastle disease virus of the recombinant MMP8 gene, acts on the tumor to replicate, expresses MMP8 protein in the tumor tissue, degrades the extracellular matrix, is beneficial to the local diffusion of the drug in the tumor, leads more circulating immune cells to enter the tumor tissue, induces strong immune response in the tumor part and plays the role of anti-tumor. The recombinant virus provides a new strategy for oncolytic virus treatment and has good application prospect.

Description

Recombinant Newcastle disease oncolytic virus and preparation method and application thereof

Technical Field

The invention belongs to the field of tumor treatment, and particularly relates to a recombinant Newcastle disease oncolytic virus, a preparation method and application thereof.

Background

MMP8, also known as neutrophil collagenase, is the first metallomatrix protease found in neutrophils. With the progress of research, it was found that endothelial cells, stem cells, smooth muscle cells, macrophages, etc., all express MMP 8. MMP8 plays an important role in the proliferation and migration of a variety of cells. In addition to the cleavage of fibrillar collagen (I-III), MMP8 also cleaves or activates chemokines (e.g., LIX), extracellular matrix proteins, cell adhesion proteins, protease inhibitors, growth factors, and the like. One reason for the poor therapeutic effect of the existing solid tumor treatment is that the medicine can not enter the interior of the tumor.

The recombinant Newcastle disease virus is constructed based on an oncolytic Newcastle disease virus Lasota strain, and the recombinant oncolytic virus expresses MMP8 while selectively killing tumor cells on the premise of not influencing oncolytic characteristics; the invention can efficiently express MMP8, and can be secreted into the extracellular environment of tumor cells, thereby triggering the immune response of organisms.

Therefore, the oncolytic newcastle disease virus of the recombinant MMP8 gene is constructed, acts on a tumor to replicate, expresses MMP8 protein in a tumor tissue, degrades an extracellular matrix, is beneficial to local diffusion of a medicament in the tumor, allows more circulating immune cells to enter the tumor tissue, induces strong immune response in the tumor part, and plays an anti-tumor role. The recombinant virus provides a new strategy for oncolytic virus treatment and has good application prospect.

Disclosure of Invention

One of the purposes of the present invention is to provide a recombinant newcastle disease oncolytic virus, which comprises a nucleotide sequence shown as SEQ ID NO. 1.

Further, the recombinant newcastle disease virus is constructed based on an oncolytic newcastle disease virus Lasota strain.

Further, the recombinant newcastle disease virus also comprises an hTERT promoter.

Further, the recombinant newcastle disease virus includes N gene, P gene and NP gene of newcastle disease virus.

The second purpose of the invention is to provide the application of the recombinant Newcastle disease virus in preparing anti-tumor drugs.

Furthermore, the tumor is liver cancer tumor cells.

Furthermore, the liver cancer tumor cell is HepG2 or MHCC 97.

Compared with the prior art, the invention has the following beneficial effects:

the invention constructs the oncolytic newcastle disease virus of the recombinant MMP8 gene, acts on the tumor to replicate, expresses MMP8 protein in the tumor tissue, degrades the extracellular matrix, is beneficial to the local diffusion of the drug in the tumor, leads more circulating immune cells to enter the tumor tissue, induces strong immune response in the tumor part and plays the role of anti-tumor. The recombinant virus provides a new strategy for oncolytic virus treatment and has good application prospect.

Drawings

FIG. 1 is a gel electrophoresis diagram of MMP8 gene amplification in example 1.

FIG. 2 is a gel electrophoresis of the PmeI-digested Lasota vector of example 1.

FIG. 3 is a gel electrophoresis of the recombinant Newcastle disease virus vector NDV-MMP8 in example 1.

FIG. 4 is a gel electrophoresis chart of RT-PCR verified recombinant Newcastle disease virus NDV-MMP8 target gene fragment in example 2.

FIG. 5a is the viability assay of HepG2 in example 3.

FIG. 5b shows the results of the activity assay for MHCC97 in example 3.

FIG. 6a is a graph of tumor volume change and Kaplan-Meier survival for the mouse HepG2 transplanted tumor model of example 3.

FIG. 6b is a Kaplan-Meier survival graph of the mouse transplanted tumor model in example 3.

Detailed Description

Example 1 construction of recombinant viral genome plasmid carrying MMP8 Gene

1. Amplification of MMP8 fragment carrying NDV homologous sequence and hTERT promoter

The coding sequence of MMP8 protein, i.e., the CDs region, was found from NCBI and synthesized by Biotechnology engineering (Shanghai) GmbH, and constructed onto pcDNA3.1+ cloning vector.

MMP8(SEQ ID NO.1)：

ATGCAACAAATACCTCAAGAGAAGTCAATTAATGACTACCTGGAAAAGTTCTACCAATTACCAAGCAACCAGTATCAGTCTACAAGGAAGAATGGCACTAATGTGATCGTTGAAAAGCTTAAAGAAATGCAGCGATTTTTTGGGTTGAATGTGACGGGGAAGCCAAATGAGGAAACTCTGGACATGATGAAAAAGCCTCGCTGTGGAGTGCCTGACAGTGGTGGTTTTATGTTAACCCCAGGAAACCCCAAGTGGGAACGCACTAACTTGACCTACAGGATTCGAAACTATACCCCACAGCTGTCAGAGGCTGAGGTAGAAAGAGCTATCAAGGATGCCTTTGAACTCTGGAGTGTTGCATCACCTCTCATCTTCACCAGGATCTCACAGGGAGAGGCAGATATCAACATTGCTTTTTACCAAAGAGATCACGGTGACAATTCTCCATTTGATGGACCCAATGGAATCCTTGCTCATGCCTTTCAGCCAGGCCAAGGTATTGGAGGAGATGCTCATTTTGATGCCGAAGAAACATGGACCAACACCTCCGCAAATTACAACTTGTTTCTTGTTGCTGCTCATGAATTTGGCCATTCTTTGGGGCTCGCTCACTCCTCTGACCCTGGTGCCTTGATGTATCCCAACTATGCTTTCAGGGAAACCAGCAACTACTCACTCCCTCAAGATGACATCGATGGCATTCAGGCCATCTATGGACTTTCAAGCAACCCTATCCAACCTACTGGACCAAGCACACCCAAACCCTGTGACCCCAGTTTGACATTTGATGCTATCACCACACTCCGTGGAGAAATACTTTTCTTTAAAGACAGGTACTTCTGGAGAAGGCATCCTCAGCTACAAAGAGTCGAAATGAATTTTATTTCTCTATTCTGGCCATCCCTTCCAACTGGTATACAGGCTGCTTATGAAGATTTTGACAGAGACCTCATTTTCCTATTTAAAGGCAACCAATACTGGGCTCTGAGTGGCTATGATATTCTGCAAGGTTATCCCAAGGATATATCAAACTATGGCTTCCCCAGCAGCGTCCAAGCAATTGACGCAGCTGTTTTCTACAGAAGTAAAACATACTTCTTTGTAAATGACCAATTCTGGAGATATGATAACCAAAGACAATTCATGGAGCCAGGTTATCCCAAAAGCATATCAGGTGCCTTTCCAGGAATAGAGAGTAAAGTTGATGCAGTTTTCCAGCAAGAACATTTCTTCCATGTCTTCAGTGGACCAAGATATTACGCATTTGATCTTATTGCTCAGAGAGTTACCAGAGTTGCAAGAGGCAATAAATGGCTTAACTGTAGATATGGCTAA

Upstream primer (SEQ ID NO. 2):

5’-CCAAGGTCCAACTCTGTTTAAACTTAGAAAAAATACGGGTAGAAGTGCCACCgacccccgggtccg-3’；

downstream primer (SEQ ID NO. 3):

5’-ATTGCCGCTTGGGTTTAAACttaTTACTTGTACAGCTC-3’。

for PCR, Vazyme method was used

Max Master Mix high fidelity DNA polymerase completes fragment amplification, and the PCR system is shown in Table 1:

TABLE 1

The PCR amplification procedure is shown in table 2:

TABLE 2

2. Recovery of amplified fragments

Gel recovery of PCR products was carried out using Gel Extraction Kit Gel recovery Kit from OMEGA, as shown in FIG. 1, the Gel recovery method is described in detail in the relevant instructions, and the target fragments were finally recovered by elution with 30. mu.L of sterile, enzyme-free water.

Pme I enzyme digestion of Newcastle disease virus attenuated strain Lasota vector (linearization of NDV vector)

The enzyme digestion was carried out using the Pme I restriction enzyme from New England Biolabs (NEB) at 37 ℃ for 2h, as shown in Table 3:

TABLE 3

4. Recovery of enzyme-cleaved NDV vector fragments

Gel recovery of PCR products was carried out using Gel Extraction Kit (OMEGA corporation), the Gel electrophoresis results of PmeI enzyme-digested Lasota vector are shown in FIG. 1, the Gel recovery method is described in detail in the relevant description, and finally the linearized NDV fragment was recovered by elution with 30. mu.L of sterile, enzyme-free water.

5. Homologous recombination of target fragment and linearized NDV vector

Homologous recombination adopts a Clonexpress II One Step Cloning Kit single-fragment rapid Cloning Kit of Vazyme company, the reaction temperature is 37 ℃, the reaction time is 0.5h, and the reaction system is shown in Table 4:

TABLE 4

6. Transformation of competent cells with homologous recombination products

Competent cells used for transformation were TransStbl3 chemical company Cell from TransGen Biotech, and the transformation method is described in the specification.

7. Extraction and identification of ligation products

The colony of the plate is picked and put on a shaker in an Amp (+) LB culture medium, the shaking culture is carried out for 12h at 30 ℃ and 180rpm, the Escherichia coli is extracted by adopting a DNAPlasmid Mini Kit I plasmid miniprep Kit I type of OMEGA company, the specific method is described in the specification, and finally 30 mu L of sterile enzyme-free water is used for eluting and recovering the plasmid and carrying out gel electrophoresis verification, and the result is shown in figure 3.

And (3) carrying out gel electrophoresis verification after plasmid PCR on the complete insert by using a base redesign primer on the NDV vector at the upstream/downstream of the target fragment, and sending a PCR product to a biological engineering (Shanghai) corporation for sequencing verification of the target fragment.

NDV vector upstream primer (SEQ ID NO. 4):

5’-GTTAGATGCAGCCGGGTCG-3’；

NDV vector downstream primer (SEQ ID NO. 5):

5’-AATGGGCAGAATCAAAGTA-3’。

the PCR system is shown in Table 5:

TABLE 5

The PCR amplification procedure is shown in table 6:

TABLE 6

Example 2 rescue of recombinant Newcastle disease Virus

1. Preparation of the plasmid of interest and helper plasmid

The bacterium liquid of the plasmid containing the target fragment, the N fragment, the P fragment and the NP fragment is subjected to shake bacterium mass amplification, and a fast endo free plasmid maxi Kit of Vazyme company is used for carrying out mass extraction on the plasmid, and the specific method is described in the specification.

Preparation of BSR cells

BSR cells were cultured in complete DMEM medium (10% fetal bovine serum, 1% penicillin streptomycin solution) containing 0.5mg/ml g418 and screened for three generations, and then seeded into 6-well plates until the cells grew to 70%.

3. Co-transfection of a plasmid of interest with a helper plasmid

First, incomplete DMEM medium 1: poxvirus stably expressing T7RNA polymerase was diluted 100 times, 200ul poxvirus-containing medium was added per well to infect the cells for 1h, poxvirus-containing medium was aspirated, and PBS was added to wash 2 times. In addition, the extract 2000Transfection Reagent of Vazyme, and four plasmids of the target fragment plasmid, the N fragment, the P fragment, and the NP fragment were prepared as solution a and solution b using Opti-MEM of Samieheir technologies (China), respectively, and mixed and incubated for 5min, and then solution a and solution b were mixed and incubated for 5 min. 250. mu.L of the mixture and 1750. mu.L of incomplete DMEM were added to each well for transfection, and the incubator was incubated for 6 h. The reagents required for solution a and solution b per 3 wells were prepared as follows:

liquid a: transfection reagent 30. mu. L, Opti-MEM 375. mu.L

b, liquid: plasmid of interest (430 ng/. mu.L), 15.6. mu. L, N fragment plasmid (880 ng/. mu.L), 3.9. mu. L, NP fragment plasmid (520 ng/. mu.L), 6.6. mu. L, L fragment plasmid (550 ng/. mu.L), 3.1. mu. L, Opti-MEM 375. mu.L.

After 6h, the medium was aspirated, and 10. mu.L of cytarabine and 1990. mu.L of DMEM medium containing 5% fetal bovine serum 2mL were added to each well and incubated for 24 h. After 24h, the medium was aspirated and 10. mu.L cytarabine and 1988. mu.L Opti-MEM were added to each well. Cell death was observed and cell culture fluid was collected until the number of cells was less than 20% (about 24 h).

4. Chick embryo allantoic cavity culture and rescue of recombinant Newcastle disease virus NDV-MMP8

After the collected cell culture solution was filtered using a 0.22 μm pore size Syring Filter from Guangzhou Jiete biofiltration Co., Ltd, 400 μ L of the filtered culture solution was inoculated into SPF hatching eggs, and incubation was continued in an incubator. After 24h of inoculation, dead chick embryos are discarded. After 120h of inoculation, the eggs were removed from the incubator and placed in a refrigerator at 4 ℃ overnight. Then, the egg air chamber was knocked off, and allantoic fluid of the chick embryo was collected.

5. Hemagglutination test of recombinant Newcastle disease virus NDV-MMP8, RT-PCR verification and sequencing thereof

Chick embryo allantoic fluid and PBS are mixed according to the proportion of 1: 20 to 1: diluting to 50 μ L volume at 212 times, mixing with 50 μ L PBS containing 1% chicken red blood cell, incubating for 15min, observing blood coagulation titer, and selecting allantoic fluid with positive blood coagulation titer for RT-PCR verification. The allantoic fluid RNA extraction Kit was RNeasy Mini Kit available from QIAGEN, and the RT-PCR Kit was TransGen Biotech

One-Step RT-PCR SuperMix was performed, and the RT-PCR products were gel electrophoresis verified (see FIG. 4) and sent to Guangzhou Huada gene for sequencing verification of the target fragment. The steps for extracting viral RNA from allantoic fluid are detailed in the specification, and the RT-PCR system is shown in Table 7:

TABLE 7

The amplification procedure is shown in table 8:

TABLE 8

Example 3 antitumor Effect of recombinant Newcastle disease Virus

1. In vitro killing effect of recombinant Newcastle disease virus on HepG2

HepG2, MHCC97 cells in DMEM medium (10% fetal bovine serum, 1% penicillin streptomyces, respectively)Vegetable solution) 10⁴Individual cells/well were seeded into 96-well plates. When the cell density reached 70-80%, viruses of different MOI values were added to 96-well plates. CCK8 reagent was added to each well 24, 48, 72, 96 hours post infection, respectively, and at 37 ℃ and 5% CO₂Incubate for 2 hours. Finally, absorbance values were measured at 450nm using a microplate reader, see fig. 5a and 5b, where fig. 5a is the viability assay of HepG2 and fig. 5b is the viability assay of MHCC97, all expressed as mean ± standard deviation.

2. In vivo anticancer effects of recombinant Newcastle disease Virus

The experimental animals of the immune reconstitution SCID/beige mouse (Hu-PBMC-SCID) and human liver cancer HepG2 model are randomly divided into 5 groups, and when the average tumor volume of the mouse reaches 150mm³200ul PBS, 10 ul was applied every other day⁷pfu NDV、10⁷pfu NDV-MMP8 were injected intratumorally for 5 treatments. Observing the growth condition of the tumor of the mouse, and exploring the treatment effect of the recombinant virus on the tumor, wherein the result is shown in figure 6, and figure 6a is the tumor volume change and Kaplan-Meier survival curve of a mouse HepG2 transplanted tumor model; FIG. 6b is a Kaplan-Meier survival curve of a mouse transplanted tumor model, tumor size measured using a caliper. Data are presented as mean ± standard deviation, tumor volumes on day 30 were compared using one-way anova. (. P < 0.05,. P < 0.001). As can be seen from FIG. 6, the tumor volume of the PBS group is increased with time after the mice are respectively treated by PBS, NDV and NDV-MMP8, the tumor volume of the PBS group is not treated or inhibited, and the tumor volume of the recombinant virus treatment group is increased slowly.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

SEQUENCE LISTING

<110> Guangxi university of medical science

<120> recombinant Newcastle disease oncolytic virus, preparation method and application

<130> 2021.05.18

<160> 5

<170> PatentIn version 3.3

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atgcaacaaa tacctcaaga gaagtcaatt aatgactacc tggaaaagtt ctaccaatta 60

ccaagcaacc agtatcagtc tacaaggaag aatggcacta atgtgatcgt tgaaaagctt 120

aaagaaatgc agcgattttt tgggttgaat gtgacgggga agccaaatga ggaaactctg 180

gacatgatga aaaagcctcg ctgtggagtg cctgacagtg gtggttttat gttaacccca 240

ggaaacccca agtgggaacg cactaacttg acctacagga ttcgaaacta taccccacag 300

ctgtcagagg ctgaggtaga aagagctatc aaggatgcct ttgaactctg gagtgttgca 360

tcacctctca tcttcaccag gatctcacag ggagaggcag atatcaacat tgctttttac 420

caaagagatc acggtgacaa ttctccattt gatggaccca atggaatcct tgctcatgcc 480

tttcagccag gccaaggtat tggaggagat gctcattttg atgccgaaga aacatggacc 540

aacacctccg caaattacaa cttgtttctt gttgctgctc atgaatttgg ccattctttg 600

gggctcgctc actcctctga ccctggtgcc ttgatgtatc ccaactatgc tttcagggaa 660

accagcaact actcactccc tcaagatgac atcgatggca ttcaggccat ctatggactt 720

tcaagcaacc ctatccaacc tactggacca agcacaccca aaccctgtga ccccagtttg 780

acatttgatg ctatcaccac actccgtgga gaaatacttt tctttaaaga caggtacttc 840

tggagaaggc atcctcagct acaaagagtc gaaatgaatt ttatttctct attctggcca 900

tcccttccaa ctggtataca ggctgcttat gaagattttg acagagacct cattttccta 960

tttaaaggca accaatactg ggctctgagt ggctatgata ttctgcaagg ttatcccaag 1020

gatatatcaa actatggctt ccccagcagc gtccaagcaa ttgacgcagc tgttttctac 1080

agaagtaaaa catacttctt tgtaaatgac caattctgga gatatgataa ccaaagacaa 1140

ttcatggagc caggttatcc caaaagcata tcaggtgcct ttccaggaat agagagtaaa 1200

gttgatgcag ttttccagca agaacatttc ttccatgtct tcagtggacc aagatattac 1260

gcatttgatc ttattgctca gagagttacc agagttgcaa gaggcaataa atggcttaac 1320

tgtagatatg gctaa 1335

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ccaaggtcca actctgttta aacttagaaa aaatacgggt agaagtgcca ccgacccccg 60

ggtccg 66

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attgccgctt gggtttaaac ttattacttg tacagctc 38

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gttagatgca gccgggtcg 19

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<213> Artificial sequence

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aatgggcaga atcaaagta 19

Claims

1. A recombinant Newcastle disease virus, which is characterized by comprising a nucleotide sequence shown as SEQ ID NO. 1.

2. The recombinant newcastle disease virus according to claim 1, wherein said recombinant newcastle disease virus is constructed based on the oncolytic newcastle disease virus Lasota strain.

3. The recombinant newcastle disease virus of claim 1, further comprising an hTERT promoter.

4. The recombinant newcastle disease virus according to any of claims 1-3, further comprising N, P and NP genes of newcastle disease virus.

5. Use of the recombinant newcastle disease virus according to any of claims 1-3 for the preparation of an anti-tumor medicament.

6. The use of claim 5, wherein the tumor is a hepatoma tumor cell.

7. The use of claim 6, wherein said individual liver cancer tumor cells are HepG2 or MHCC 97.