阅读说明：本技术 一种复制缺陷型重组人4型腺病毒及其制备方法和应用 (Replication-defective recombinant human adenovirus type 4, and preparation method and application thereof ) 是由 陈凌 冯立强 于 2019-08-19 设计创作，主要内容包括：本发明公开了一种复制缺陷型重组人4型腺病毒的制备方法和应用。所述复制缺陷型重组人4型腺病毒通过以下方法得到：将人4型腺病毒基因组质粒化,敲除其E1和E3基因,并将Ad4的E4基因的开放阅读框2、3、4、5、6替换为Ad5 E4基因的相对应开放阅读框。本发明所描述的复制缺陷型人4型腺病毒载体可潜在地应用于：抗人4型腺病毒疫苗研发；抗人4型腺病毒中和抗体及药物筛选；抗其他病原体疫苗的研发；生物学研究的报告示踪系统等。(the invention discloses a preparation method and application of replication-defective recombinant human adenovirus type 4. The replication-defective recombinant human adenovirus type 4 is obtained by the following method: human adenovirus type 4 genome was plasmid-granulated, knockout of its E1 and E3 genes, and replacement of open reading frame 2, 3, 4, 5, 6 of the E4 gene of Ad4 with the corresponding open reading frame of Ad5E4 gene. The replication-defective human adenovirus type 4 vectors described in the present invention can potentially be applied to: research and development of anti-human adenovirus 4 vaccine; screening anti-human adenovirus 4 neutralizing antibody and medicine; development of vaccines against other pathogens; biological studies report tracer systems, etc.)

1.A replication-defective human adenovirus type 4 vector, wherein the E1 and E3 genes are deleted and the E4 gene open reading frame 2, 3, 4, 6, 6/7 is replaced with the corresponding open reading frame of the Ad5E4 gene.

2. The replication-deficient human adenovirus type 4 according to claim 1, wherein the foreign gene expression cassette is integrated into the pro-E1 gene region of Ad 4.

3. The exogenous gene expression cassette of claim 2, comprising a eukaryotic gene promoter, any exogenous DNA sequence, and a transcription terminator.

4. Use of the replication deficient human adenovirus type 4 according to claims 1, 2 and 3 for the preparation of a vaccine against human adenovirus type 4or a medicament against human adenovirus type 4.

5. Use of the replication deficient human adenovirus type 4 according to claims 1, 2 and 3 for the preparation of vaccines, neutralizing antibodies against other pathogens.

6. the replication deficient human adenovirus type 4 according to claims 1, 2 and 3 in a biological reporter tracer system.

Technical Field

The invention relates to the technical field of biology, in particular to a replication-defective recombinant human adenovirus type 4 vector, and a preparation method and application thereof.

Background

Adenoviruses (Ad) are double-stranded DNA viruses, the genome of which is about 35-40kb in length. It is known that human adenoviruses are divided into 7 subgroups (A to G) including more than 50 serotypes and more than 90 genotypes, and cause mainly acute respiratory diseases (adenovirus subgroups B and C), conjunctivitis (adenovirus subgroups B and D), and gastroenteritis (adenovirus subgroups F41 and 42, subgroup G52) after infecting patients. Studies have reported that Ad4 is mainly concentrated in young people such as armies and schools and places where teenagers gather, and even causes death of patients. However, no specific medicine for treating Ad4 infection exists, and only supportive treatment can be adopted clinically.

Currently, vaccines against adenoviral infection are only available to the U.S. military. The vaccine is an enteric capsule type oral live virus vaccine prepared by passage of wild Ad4 and Ad7 on human embryonic kidney diploid fibroblasts, freeze dehydration, mixing with cellulose lactose and the like. The use of the vaccine effectively controls the outbreak of the adenovirus infection epidemic of the army. However, the Ad4 adenovirus vaccine used by the American military has great risk, the vaccine is mainly low-dose wild type adenovirus, the safety is poor, the risk of polluting the living environment after residual live virus is discharged from the intestinal tract exists, and the secondary pollution of the virus is easily caused, so that the vaccine cannot be widely applied to common people. Therefore, it is necessary to develop a replication-defective adenovirus vaccine which is highly safe and can prevent the strong Ad4 virus strain.

replication-defective adenovirus vectors have been widely used in the fields of vaccine development, gene therapy, etc., and not only have good safety, but also have strong immune response in organisms. It has been shown that adenovirus E1 gene is an essential gene for its replication and proliferation, and E3 gene plays a key role in the immune system against the host. After knocking out the E1 and E3 genes, adenovirus loses replication ability in normal human body, and has attenuation phenotype. Meanwhile, the main surface antigens of Ad4, such as Hexon and Fibre, are not affected, and the immunogenicity of the vaccine is not affected. Therefore, the replication-defective adenovirus is used as a vaccine, so that the universality and the application range of the vaccine can be effectively increased. Replication-defective adenoviruses can be produced in complementing cell strains, such as 293 cells, PerC6 cells expressing the Ad5E1 gene. However, many adenoviruses, especially non-C subgroup adenoviruses, have low yield in production cell lines after E1 and E3 genes are knocked out, and the main reasons are that Ad5E1B 55K cannot interact with subtype B adenovirus E4Orf6 protein, cannot effectively inhibit the enucleation of host cell mRNA, and cannot improve the expression of virus late protein. These adenoviruses require 293 cell lines or other cell lines expressing the corresponding E1 gene for production, however, these cell lines have not yet obtained permission for vaccine production, and therefore, replication-defective Ad4 with only E1, E3 gene knock-outs are difficult to produce in vaccine production cell lines 293 or PerC 6. Improving the production capacity of the cells in these cell lines is a bottleneck technical problem to be solved at present.

In order to improve the growth capacity of replication-defective adenovirus, the genes encoding Ad5E4Orf6 have been substituted for the corresponding genes such as Ad26 and Ad35, and these chimeric Ad26 and Ad35 can still replicate effectively in 293 cells and PerC6 cells after deletion of E1 gene. However, there has been no report of a similar strategy for engineering Ad4 into a replication-defective virus. In addition, because the adenovirus E4 gene contains reading frames of Orf1, Orf2, Orf 3, Orf 4, 34K (Orf6), Orf6/7 and the like, the encoded protein can interact with the protein encoded by the E1 gene, and plays an important role in the replication and packaging processes of the adenovirus; moreover, it is not clear whether the E4 gene of Ad4 itself has unknown cytotoxicity. Therefore, it is necessary to replace all or most of the coding cassettes of the Ad4E4 gene with the corresponding coding cassette of the Ad5E4 gene in order to improve the safety of replication-defective Ad4 and its replication capacity in producer cell lines.

in addition, replication-defective adenoviruses have been widely used as gene vectors in the fields of gene therapy, vaccines, and the like. Adenovirus vectors have a series of advantages in these fields, such as good safety, large gene load, availability of gene transduction, and easier mass production. Based on these advantages, there have been hundreds of clinical trials worldwide with adenovirus as the gene vector, the first of all (24.8%) of the vectors. Most of these studies used Ad5 or Ad2 as vectors. However, the use of conventional adenoviral vectors is limited by the anti-adenoviral immune response generated in most humans due to past adenoviral infection. Research shows that the neutralizing antibodies of Ad2 and Ad5 have extremely high positive rate in most of the population of developing countries and regions, and even exceed 90 percent in partial regions. These neutralizing antibodies inhibit the entry of adenovirus into cells of the body as a gene vector, making it difficult to perform the functions of immunogenicity and gene therapy. To overcome the pre-existing immune response against adenovirus, researchers have developed a series of techniques such as: (1) immunosuppressive agents such as cyclosporine, cyclophosphamide, FK506 and the like are adopted to inhibit partial anti-adenovirus immune response; (2) by modifying or transforming the surface protein of the adenovirus vector, the neutralization function of a pre-existing neutralizing antibody is avoided; (3) we have previously developed the AVIP technique of infecting PBMCs in vitro with adenovirus and then self-reinfusing; and so on. However, these strategies either have high toxic side effects (e.g., immunosuppressants), safety (through modification or engineering of adenoviral vector surface proteins), or are only once limiting (i.e., they cannot be reused because they generate an immune response against the new vector). However, Ad4 has not been popularized in a large scale in the population, and a neutralizing antibody against Ad4 is generally absent in the human body, so the human type 4-based adenovirus vector is a good substitute for vectors such as Ad2 and Ad 5.

Therefore, the replication-defective recombinant Ad4 with E1 and E3 gene deletion described in the invention can be applied to the following aspects: (1) development of vaccines against Ad 4; (2) screening tools for anti-Ad 4 neutralizing antibodies and drugs; (3) cell therapy and the development of genetic vaccines; (4) a biological tracer reporting system; and so on.

disclosure of Invention

The invention aims to overcome the defects of the prior art and provides replication-defective recombinant Ad4 capable of being amplified in a vaccine production cell strain in a large scale and a preparation method and application thereof.

The technical problem to be solved by the invention is realized by the following scheme:

A replication-defective recombinant human adenovirus type 4, which is obtained by the following method: human adenovirus type 4 genome was plasmid-granulated, knockout of its E1 and E3 genes, and replacement of open reading frame 2, 3, 4, 6, 6/7 of the E4 gene of Ad4 with the corresponding open reading frame of Ad5E4 gene.

Preferably, the replication-defective recombinant human adenovirus type 4 further integrates an expression cassette of a foreign gene into the E1 gene region of Ad 4.

A method for preparing replication-defective recombinant human adenovirus type 4, comprising the following steps:

And S1, amplifying the 5 'end and the 3' end of the Ad4 genome by PCR, connecting the 5 'end and the 3' end to an ampicillin resistance vector to obtain pT-Ad4(L + R), and carrying out homologous recombination with the Ad4 genome after linearization to obtain a genome plasmid pAd 4.

S2, amplifying the left and right homologous arms of the Ad4E3 region by PCR and connecting the homologous arms to a kanamycin-resistant vector to obtain a pVAX-Ad4-delE3 vector, and after linearization, carrying out homologous recombination with enzyme-digested and linearized pAd4 to obtain a genome plasmid pAd4 delta E3;

S3, carrying out PCR amplification on the left and right homologous arms of the Ad4E1 region and connecting the homologous arms to a kanamycin-resistant vector to obtain a pVAX-Ad4-delE1 vector, and carrying out homologous recombination with enzyme-digested and linearized pAd4 delta E3 to obtain a genome plasmid pAd4 delta E1 delta E3;

S4, amplifying Ad5E4Orf2-6 and Ad55E4 genes by PCR, replacing corresponding regions in the Ad4E4 gene with Ad5E4Orf2-6 to obtain pGK41-5E4, and after linearization, carrying out homologous recombination with enzyme-cut pAd4 delta E1 delta E3 to obtain a genome plasmid pAd4 delta E1 delta E3(5E 4);

Preferably, the specific method of step S1 is:

Using Ad4 genome as a template, performing PCR amplification to obtain Ad4-L and Ad4-R at the left and right ends of Ad4 genome as homologous arms, performing homologous recombination to a pSIMPLE-19(EcoRV) vector to obtain a pT-Ad4(L + R) vector, introducing enzyme digestion sites SpeI and BamHI between the left and right arms of the pT-Ad4(L + R) vector, performing linearization, performing homologous recombination with Ad4 genome DNA, and performing ampicillin resistance screening to obtain a circular plasmid pAd4 with all Ad4 genomes.

Most preferably, in one embodiment, the specific method of step S1 is:

Using Ad4 genome as a template, performing PCR amplification to obtain Ad4-L and Ad4-R at the left and right ends of Ad4 genome as homologous arms, performing homologous recombination to connect with a pSIMPLE-19(EcoRV) vector to obtain pT-Ad4(L + R) vector, introducing enzyme cutting sites SpeI and BamHI between the left and right arms of pT-Ad4(L + R) vector, performing double enzyme cutting linearization on pT-Ad4(L + R) by SpeI and BamHI, performing homologous recombination with Ad4 genome DNA, and screening ampicillin resistance to obtain the circular plasmid pAd4 with all Ad4 genome.

Preferably, the specific method of step S2 is:

using Ad4 genome as a template, carrying out PCR amplification to obtain homologous recombination arms delE3-4L and delE3-4R of an E3 region, carrying out homologous recombination on the homologous recombination arms delE3-4L and the delE3-4R to a pVax vector to obtain a pVAX-Ad4-delE3 vector, after linearization, carrying out homologous recombination on the vector and a pAd4 plasmid which is partially digested by EcoRI, and carrying out ampicillin resistance screening to obtain a genome plasmid pAd4 delta E3 which is knocked out an E3 gene and introduces a unique restriction site SwaI.

most preferably, in one embodiment, the specific method of step S2 is:

Taking Ad4 genome as a template, carrying out PCR amplification to obtain a homologous recombination arm delE3-4L delE3-4R of an E3 region and a pVax vector, carrying out three-fragment homologous recombination by Exnase enzyme to obtain a pVAX-Ad4-delE3 vector, after carrying out SpeI and XbaI double-enzyme digestion linearization on the pVAX-Ad4-delE3 vector, carrying out homologous recombination with a pAd4 plasmid partially digested by EcoRI, and carrying out ampicillin resistance screening to obtain a genome plasmid pAd4 delta E3 of which the E3 gene is knocked out and which introduces a unique digestion site SwaI.

Preferably, the specific method of step S3 is:

Taking Ad4 genome as a template, carrying out PCR amplification to obtain an E1 region homologous recombination arm L-delK (E1) and R-delK (E1), carrying out homologous recombination on a pVax vector to obtain a pVax-delE1 vector, carrying out homologous recombination on the linearized vector and PsiI linearized pAd4 delta E1 to obtain a genomic plasmid pAd4 delta E1 delta E3 with E1 and E3 removed and unique enzyme cutting site PacI introduced.

Most preferably, in one embodiment, the specific method of step S3 is:

Taking an Ad4 genome as a template, carrying out PCR amplification to obtain an E1 region homologous recombination arm L-delK (E1), carrying out three-fragment homologous recombination on an R-delK (E1) and a pVax vector by using an Exnase enzyme to obtain a pVAX-Ad4-delE1 vector, carrying out double digestion linearization on the pVAX-Ad4-delE1 vector by BstZ17I + SgrAI, carrying out homologous recombination with PsiI linearized pAd4 delta E1, and obtaining a genome plasmid pAd4 delta E1 delta E3 with E1 and E3 removed and a unique digestion site PacI introduced.

Preferably, the specific method of step S4 is:

using an Ad4 genome as a template, performing PCR amplification to obtain two gene fragments of a left arm and a right arm of an E4 gene, connecting the two gene fragments to a pGK143 vector to obtain a pGK143(L + R) vector, further performing PCR amplification to obtain an Ad5E4Orf2-6 gene fragment, performing homologous recombination with a linearized pGK143(L + R) vector to obtain pGK143-5E4(Orf2-6), and performing homologous recombination with a SwaI linearized pAd4 delta E1 delta E3 after linearization to obtain a pAd4 delta E1 delta E3(5E4) vector.

Most preferably, in one embodiment, the specific method of step S3 is:

Using an Ad4 genome as a template, carrying out PCR amplification to obtain two gene fragments of the left arm and the right arm of the E4 gene, carrying out homologous recombination with a vector framework recovered by BstZ17I + SgrAI double enzyme digestion pGK134-EGFP through an Exnase enzyme three-fragment to obtain a pGK143(L + R) vector, and introducing a unique BamHI enzyme digestion site between the left arm and the right arm of the E4 gene; further, using an Ad5 genome as a template, obtaining an Ad5E4Orf2-6 gene fragment through PCR amplification, carrying out homologous recombination with a BamHI single-enzyme digestion linearized pGK143(L + R) vector to obtain pGK143-5E4(Orf2-6), carrying out double-enzyme digestion linearization on a pGK143-5E4(Orf2-6) vector by BstZ17I + SgrAI, carrying out homologous recombination with a SwaI linearized pAd4 delta E1 delta E3 to obtain a pAd4 delta E1 delta E3(5E4) vector.

A method for preparing replication-defective recombinant human adenovirus type 4, further comprising the following steps:

S5, using Ad4 genome as a template, carrying out PCR amplification to obtain homologous recombination arms 4SE1L and 4SE1R of an E1 region, carrying out homologous recombination on a pVax vector to obtain a pGK41 vector, and introducing enzyme cutting sites PacI and EcoRV between the two arms; and (3) performing PCR amplification by taking pGA1-EGFP as a template to obtain an exogenous gene expression frame CMV-EGFP-BGH, connecting the exogenous gene expression frame CMV-EGFP-BGH with pGK41 linearized by EcoRV to obtain a pGK41-EGFP vector, and performing homologous recombination with pAd4 delta E1 delta E3(5E4) digested by PacI after linearization to obtain a genome plasmid pAd4 delta E1 delta E3(5E4) -EGFP.

Preferably, the specific method of step S5 is:

Using Ad4 genome as a template, carrying out PCR amplification to obtain homologous recombination arms 4SE1L and 4SE1R of an E1 region, carrying out homologous recombination on the homologous recombination arms to a pVax vector to obtain a pGK41 vector, and introducing enzyme cutting sites PacI and EcoRV between the two arms; and (3) performing PCR amplification by taking pGA1-EGFP as a template to obtain an exogenous gene expression frame CMV-EGFP-BGH, connecting the exogenous gene expression frame CMV-EGFP-BGH with pGK41 linearized by EcoRV to obtain a pGK41-EGFP vector, and performing homologous recombination with pAd4 delta E1 delta E3(5E4) digested by PacI after linearization to obtain a genome plasmid pAd4 delta E1 delta E3(5E4) -EGFP.

Most preferably, in one embodiment, the specific method of step S5 is:

Using Ad4 genome as a template, carrying out PCR amplification to obtain homologous recombination arms 4SE1L and 4SE1R of an E1 region and a pVax vector, carrying out homologous recombination through an Exnase enzyme to obtain a pGK41 vector, and introducing enzyme cutting sites PacI and EcoRV between the two arms; the method comprises the steps of carrying out PCR amplification by taking pGA1-EGFP as a template to obtain an exogenous gene expression frame CMV-EGFP-BGH, connecting the exogenous gene expression frame CMV-EGFP-BGH with pGK41 subjected to enzyme cutting linearization of EcoRV to obtain a pGK41-EGFP vector, carrying out double enzyme cutting linearization on the pGK41-EGFP vector through BstZ17I + SgrAI, and carrying out homologous recombination on the pGK41-EGFP vector and pAd4 delta E1 delta E3(5E4) subjected to enzyme cutting of PacI to obtain a genome plasmid pAd4 delta E1 delta E3(5E4) -EGFP.

pAd4 delta E1 delta E3(5E4) and pAd4 delta E1 delta E3(5E4) -EGFP were linearized by Asisi digestion, recovered by ethanol precipitation, transfected 293 cells were virus rescued, expanded and purified by CsCl2 density gradient centrifugation for Ad4 delta E1 delta E3(5E4) and Ad4 delta E1 delta E3(5E4) -EGFP.

The replication-defective recombinant human adenovirus type 4 is applied to the preparation of anti-human adenovirus type 4 vaccines.

the replication-defective recombinant human adenovirus type 4 is applied to the research and development of anti-human adenovirus type 4 drugs and neutralizing antibodies.

The replication-defective recombinant human adenovirus type 4 is applied to the preparation of other pathogen vaccines or gene therapy.

The replication-defective recombinant human adenovirus type 4 is applied to a biological report tracing system.

Has the advantages that: (1) the invention provides a novel replication-defective recombinant human adenovirus type 4, which can be produced in large quantities in vaccine production cell strains such as 293 and PerC6, and has an attenuated phenotype because normal human cells do not have replication capacity. (2) The recombinant vector can also express exogenous genes in target cells with high efficiency. (3) The recombinant vector can be used as a vaccine or a gene therapy vector, and can also be applied to the research and development of medicaments and neutralizing antibodies, a report tracing system and the like. (4) Example experimental data indicate that the replication-defective human type 4 adenovirus can induce a strong immune response in mice, resulting in specific neutralizing antibodies against Ad 4.

Drawings

FIG. 1 is a schematic diagram of the Ad4 genome cyclization principle.

FIG. 2 is a schematic diagram of the adenovirus E3 gene knock-out of the pAd4 vector.

FIG. 3 is a schematic diagram of the knock-out of adenovirus E1 gene in pAd 4. delta.E 3 vector.

FIG. 4 is a schematic diagram of the principle of E4 gene replacement in pAd4 Δ E1 Δ E3 vector by E4 gene in Ad5 genome.

FIG. 5 is a schematic diagram of the construction principle of pAd4 Δ E1 Δ E3(5E4) -EGFP plasmid carrying an expression cassette of a foreign gene.

FIG. 6 shows the results of double cleavage of vectors pAd4, pAd 4. delta. E3, pAd 4. delta. E1. delta. E3, pAd 4. delta. E1. delta. E3(5E4) and pAd 4. delta. E1. delta. E3(5E4) -EGFP.

FIG. 7 shows the production and purification of Ad4 replication-defective adenovirus.

FIG. 8 shows the results of plaque formation experiments with replication-deficient Ad4 vectors in HEK293 and A549 cells.

FIG. 9 shows the experimental protocol for Ad4 replication-deficient adenovirus immunized mice and the results of the detection of anti-Ad 4 neutralizing antibodies in post-immunization sera.

Detailed Description

The invention discloses a preparation method of a replication-defective human type 4 adenovirus vector. The preparation method and the construction idea of the adenovirus vector are suitable for research and development of vaccines aiming at adenovirus and other pathogens, screening of anti-adenovirus viruses and neutralizing antibodies, and reporting and tracing systems in biological research.

The term "human adenovirus type 4" according to the present invention refers to adenovirus type 4 known to those of ordinary skill in the art, and the Ad4 genome used in the examples is also derived from these known adenovirus type 4. The replication-defective human type 4 adenovirus vectors of the present invention are not limited to the specific clinical isolates employed in the examples.

The term "foreign sequence" according to the present invention refers to any DNA sequence not of adenovirus type 4 origin. It will be understood by those skilled in the art that the exogenous sequence may be an exogenous gene expression cassette, or may be an expression cassette for shRNA or miRNA, etc.

In the following examples, the exogenous gene expression cassette may comprise a eukaryotic promoter, an exogenous gene coding sequence, a transcription terminator, as understood by those skilled in the art. The exogenous gene coding sequence can be, but is not limited to, the coding sequence of green fluorescent protein, other virus antigens, shRNA and the like.

In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.