阅读说明：本技术 一种新型腺病毒载体的构建和生产方法 (Construction and production method of novel adenovirus vector ) 是由 邹灵龙 饶亮明 于 2019-08-21 设计创作，主要内容包括：本发明属于分子生物学、载体规模化生产和基因治疗技术领域,尤其涉及一种新型腺病毒载体的构建和生产方法。本发明通过使用次黄嘌呤磷酸核糖基转移酶基因内含子序列作为填充基因片段,以及采用改进型Cre/LoxP体系的病毒载体生产方法的方式,达到有效生产高滴度第三代腺病毒载体的效果。本发明具有构建和生产方法中污染的辅助病毒少,生产所得的第三代腺病毒载体滴度高,目标基因表达稳定性高、表达时间长,以及可用于受损脑组织基因治疗的优点。(The invention belongs to the technical fields of molecular biology, vector large-scale production and gene therapy, and particularly relates to a method for constructing and producing a novel adenovirus vector. The invention achieves the effect of effectively producing the third-generation adenovirus vector with high titer by using the intron sequence of the hypoxanthine phosphoribosyltransferase gene as a filling gene fragment and adopting a mode of a virus vector production method of an improved Cre/LoxP system. The invention has the advantages of less pollution of helper virus in the construction and production method, high titer of the produced third generation adenovirus vector, high expression stability of target genes, long expression time and capability of being used for gene therapy of damaged brain tissues.)

1. A method for constructing and producing a novel adenovirus vector is characterized in that: the method comprises two steps of constructing helper virus which provides virus replication and packaging functions in trans, and supplementing genes required by adenovirus vector capsid packaging by virtue of filling fragment DNA, wherein packaging signals of the helper virus are specifically removed by Cre recombinase, loxP sites are respectively inserted at two sides of the packaging signals of the helper virus, amplification is carried out in Cre recombinase expression cell strains so as to reduce the mutual competition of the helper virus and third generation adenovirus, an intron sequence of hypoxanthine phosphoribosyltransferase gene is used as the filling fragment DNA so as to improve the stability of target genes on a vector, and adenovirus genomes can stably enter cell nuclei so as to prolong the expression time of the genes.

2. The method for constructing and producing a novel adenovirus vector according to claim 1, wherein: the titer of the third generation adenovirus vector reaches 10 per ml¹²A viral particle.

3. The method for constructing and producing a novel adenovirus vector according to claim 1, wherein: and (3) reserving all coding sequences of proteins required by the helper virus to produce the third generation adenovirus vector, and controlling the polluted helper virus to be below 0.1%.

4. The method for constructing and producing a novel adenovirus vector according to claim 1, wherein: the third generation adenovirus vector is hdAd delta and is used for gene therapy of damaged brain tissue after carrying beta geo gene.

5. The method for constructing and producing a novel adenovirus vector according to claim 1, wherein: the third generation adenovirus vector is hdAd delta, the expression product in the brain of the Fischer-344 rat after carrying the beta geo gene is the fusion protein of beta-galactosidase and neomycin, and the transgene expression time mediated by the third generation adenovirus vector is at least 66 days.

6. The method for constructing and producing a novel adenovirus vector according to claim 1, wherein: the third generation adenovirus vector is hdAd delta, which is transferred into aged rat brain after carrying beta geo gene, and the hdAd delta mediated gene transfer is 70% of that of the high peak at 183 days.

7. The method for constructing and producing a novel adenovirus vector according to claim 6, wherein: the first generation adenovirus vector fgAdv expresses more microglial cells, astrocytes and macrophages than the third generation adenovirus vector hdAd delta.

8. The method for constructing and producing a novel adenovirus vector according to claim 1, wherein: in the step of constructing the helper virus for providing the virus replication and packaging function in a trans-form manner, an FLP-frt recombination system is adopted, FLP recombinase is adopted to cut packaging proteins, the steps are carried out in FLP expression cell strains (239FLP and 239CreFLP), and frt sites are added on two sides of a helper virus packaging signal to form the FLP-frt recombination system.

9. The method for constructing and producing a novel adenovirus vector according to claim 8, wherein: in the step of constructing the helper virus for providing the virus replication and packaging function in trans, a phi C31-attB/attP recombination system is adopted, a phi C31 recombinase is used for reducing the contamination rate of the helper virus, and a phi C31 recombinase is used for identifying homologous chromosome sequences of attB/attP and has the same shearing capacity as the 293 cell strain Cre, so that when an attB/attP sequence inserted at two sides of a packaging signal is cut off by a phi C31, attR/attL can be obtained to prevent reverse recombination.

10. The method for constructing and producing a novel adenovirus vector according to claim 1, wherein: in the step of constructing the helper virus for providing the virus replication and packaging function in a trans-form manner, a non-adenovirus vector is used as the helper virus, the baculovirus containing most adenovirus genes is used as the helper virus to infect 293Cre cells, a Cre recombinase acts on LoxP sites on the baculovirus, the baculovirus is divided into a framework part and an adenovirus part, the framework part is kept at a transcription level and cannot be replicated, the adenovirus part can provide all functions of the helper virus of the third generation adenovirus, the third generation adenovirus vector is constructed together with a plasmid carrying a target gene, and finally pollution of the helper virus is avoided.

Technical Field

The invention belongs to the technical fields of molecular biology, vector large-scale production and gene therapy, and particularly relates to a method for constructing and producing a novel adenovirus vector.

Background

Adenovirus is a DNA double-stranded virus, which enters cells through receptor-mediated endocytosis to accomplish high-efficiency gene expression. Vectors derived from adenovirus are widely used in gene transfer and human gene therapy studies. The third generation adenovirus vector removes all virus gene components and is the most perfect adenovirus vector at present.

The carrier derived from adenovirus has the obvious advantages of high target gene transduction efficiency, low pathogenicity, high titer, no integration into host cell chromosome in vivo and the like. Adenovirus vectors are therefore widely used in gene transfer and human gene therapy research.

Adenoviruses are DNA viruses whose genome is linear double-stranded DNA, approximately 36kb in length, surrounded by an icosahedral protein coat, but without an envelope. The adenovirus genome can be divided into two parts, coding region and non-coding region. The coding region may be divided into an early gene region and a late gene region according to the replication cycle of DNA. Early gene regions have regions E1-E4, which encode primarily viral regulatory proteins. The late gene region is divided into five regions L1-L5, and encodes virus structural proteins, and the expression of the five regions is regulated by the expression product of the early gene region. The proteins expressed by the early genes (E1 and E2) are essential for adenoviral genome replication, viral packaging, and translation of other protein expression, but the products of these early genes are significantly cytotoxic and are the regions most in need of removal or inactivation. The noncoding region of adenovirus is very short, and the adenovirus has a small Inverted Terminal Repeat (ITR) at each of its two ends, each of about 100bp, and this ITR is not long but contains cis-acting elements necessary for replication and packaging of virus, and it is also a DNA replication origin necessary for replication of viral DNA, and inside the ITR is a viral packaging signal (. psi.), which together with the ITR constitutes the noncoding region of adenovirus.

Early adenoviral vector modifications focused on the removal of early genes, the first generation adenoviral vectors removed the E1 or E3 region of the adenoviral genome, viral toxicity was much reduced, but since some competent cells may exist in humans, they all contained E1-like proteins that allow the expression of the E2 gene, even though their expression levels were low, they could still cause viral DNA replication and synthesis of late structural proteins, even form replication-competent adenovirus (RCA reaction), with great potential hazards, while the second generation adenoviral vectors removed more early genes, but still did not completely avoid the trace expression of residual viral gene expression in vivo and the resulting immune response, and the production of the second generation viral vectors was rather difficult.

Therefore, the latest third generation adenovirus vector removes all virus-encoding genes, and only retains ITR of non-coding region and packaging signal (. psi.) of adenovirus, so it is called naked adenovirus (Gutless adenovirus). Third generation adenoviral vectors require helper-dependent adenoviral vectors to replicate, and are therefore also known as helper-dependent adenoviral vectors. Because the coding region of the virus gene is completely removed, the third generation adenovirus vector has the super-large capacity of 36kbDNA, and can accommodate a plurality of exogenous genes and mediate the transfer of polygenes. Meanwhile, due to the removal of all viral genes, the cells transduced by the vector have no expression of viral proteins, and the monitoring and the elimination of the transduced cells by an immune system are avoided. Thus, long-term survival of the transduced cells is warranted. This is critical for applications requiring long-term expression of the transgene. However, third generation adenoviral vector production has presented significant challenges, and the use of such vectors is greatly limited if high titers are not available.

The existing third generation adenovirus vector construction method generally has the following two problems:

First, the helper virus and third generation adenovirus required for the construction method have the same capsid, and compete with each other to affect the packaging efficiency of the viral vector, resulting in the production of adenovirus vectors with less than the desired titer.

Secondly, the stuffer DNA necessary for the construction method may affect the stability of the viral vector or change the expression efficiency of the target, and a suitable stuffer DNA selection optimization method is not available at present to solve the problem that the stuffer DNA affects the stability of the vector.

The Chinese patent with patent publication No. CN 106868047A and publication No. 2017.06.20 discloses a recombinant adenovirus vector and a construction method thereof, which is realized by replacing the corresponding part of Ad5 with an antigenic determinant gene of human rare serotype adenovirus Ad 35.

However, the construction method in the patent of the invention has the problem that the titer of the adenovirus vector obtained by construction and production is low.

Disclosure of Invention

The invention aims to provide a method for constructing and producing a novel adenovirus vector, which can achieve the effect of effectively producing a high-titer third-generation adenovirus vector by using an intron sequence of an inosine phosphate transferase gene as a filling gene segment and adopting a mode of a virus vector production method of an improved Cre/LoxP system. The invention has the advantages of less pollution of helper virus in the construction and production method, high titer of the produced third generation adenovirus vector, high expression stability of target genes, long expression time and capability of being used for gene therapy of damaged brain tissues.

The technical scheme adopted by the invention for solving the problems is as follows: a method for constructing and producing a novel adenovirus vector comprises two steps of constructing a helper virus for providing a virus replication and packaging function in trans, and supplementing genes required by adenovirus vector capsid packaging by means of filling fragment DNA, wherein a packaging signal of the helper virus is specifically removed by Cre recombinase, loxP sites are respectively inserted at two sides of the packaging signal of the helper virus, amplification is carried out in a Cre recombinase expression cell strain to reduce the mutual competition between the helper virus and third generation adenovirus, an intron sequence of an inosine phosphate group transferase gene is used as the filling fragment DNA to improve the stability of a target gene on the vector, and an adenovirus genome can stably enter a cell nucleus so as to prolong the expression time of the genes.

The further preferred technical scheme is as follows: the titer of the third generation adenovirus vector reaches 10 per ml¹²a viral particle.

The further preferred technical scheme is as follows: and (3) reserving all coding sequences of proteins required by the helper virus to produce the third generation adenovirus vector, and controlling the polluted helper virus to be below 0.1%.

The further preferred technical scheme is as follows: the third generation adenovirus vector is hdAd delta and is used for gene therapy of damaged brain tissue after carrying beta geo gene.

The further preferred technical scheme is as follows: the third generation adenovirus vector is hdAd delta, the expression product in the brain of the Fischer-344 rat after carrying the beta geo gene is the fusion protein of beta-galactosidase and neomycin, and the transgene expression time mediated by the third generation adenovirus vector is at least 66 days.

The further preferred technical scheme is as follows: the third generation adenovirus vector is hdAd delta, which is transferred into aged rat brain after carrying beta geo gene, and the hdAd delta mediated gene transfer is 70% of that of the high peak at 183 days.

The further preferred technical scheme is as follows: the first generation adenovirus vector fgAdv expresses more microglial cells, astrocytes and macrophages than the third generation adenovirus vector hdAd delta.

The further preferred technical scheme is as follows: in the step of constructing the helper virus for providing the virus replication and packaging function in a trans-form manner, an FLP-frt recombination system is adopted, FLP recombinase is adopted to cut packaging proteins, the steps are carried out in FLP expression cell strains (239FLP and 239CreFLP), and frt sites are added on two sides of a helper virus packaging signal to form the FLP-frt recombination system.

The further preferred technical scheme is as follows: in the step of constructing the helper virus for providing the virus replication and packaging function in trans, a phi C31-attB/attP recombination system is adopted, a phi C31 recombinase is used for reducing the contamination rate of the helper virus, and a phi C31 recombinase is used for identifying homologous chromosome sequences of attB/attP and has the same shearing capacity as the 293 cell strain Cre, so that when an attB/attP sequence inserted at two sides of a packaging signal is cut off by a phi C31, attR/attL can be obtained to prevent reverse recombination.

The further preferred technical scheme is as follows: in the step of constructing the helper virus for providing the virus replication and packaging function in a trans-form manner, a non-adenovirus vector is used as the helper virus, the baculovirus containing most adenovirus genes is used as the helper virus to infect 293Cre cells, a Cre recombinase acts on LoxP sites on the baculovirus, the baculovirus is divided into a framework part and an adenovirus part, the framework part is kept at a transcription level and cannot be replicated, the adenovirus part can provide all functions of the helper virus of the third generation adenovirus, the third generation adenovirus vector is constructed together with a plasmid carrying a target gene, and finally pollution of the helper virus is avoided.

The invention achieves the effect of effectively producing the third-generation adenovirus vector with high titer by using the intron sequence of the hypoxanthine phosphoribosyltransferase gene as a filling gene fragment and adopting a mode of a virus vector production method of an improved Cre/LoxP system. The invention has the advantages of less pollution of helper virus in the construction and production method, high titer of the produced third generation adenovirus vector, high expression stability of target genes, long expression time and capability of being used for gene therapy of damaged brain tissues.

Drawings

FIG. 1 is a schematic diagram of the design of Ad Δ E1E 3/SRa-. beta.geo) and hdAd Δ/SRa-. beta.geo vectors of example 1 of the present invention.

FIG. 2 is a graph showing comparative observation data of the expression time (A) and the gene persistence (B) of the β geo gene mediated by two vectors in example 1 of the present invention.

FIG. 3 is a graph comparing the immune responses after transduction with two vectors in example 1 of the present invention.

FIG. 4 is a comparative observation of the expression time of the β geo gene mediated by two vectors in aged brain tissue (hippocampus, A; cerebrospinal fluid, B) in example 2 of the present invention.

FIG. 5 is a graph comparing the immune responses of two vectors in example 2 of the present invention after transduction in aged brain tissue.

FIG. 6 is a graph showing the comparison of the expression time of β geo gene mediated by two vectors in injured brain tissue according to example 3 of the present invention.

FIG. 7 is a graph comparing the immune responses of two vectors of example 3 of the present invention after transduction in damaged brain tissue.

FIG. 8 is a graph comparing cytokine responses after transduction of damaged brain tissue by two vectors in example 3 of the present invention.

Detailed Description

The following description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention.