CN109295096B - Adenovirus vector system and recombinant adenovirus construction method - Google Patents

Abstract

Provides a set of adenovirus vector system and recombinant virus construction method. The vector system comprises adenovirus plasmids pKAD5f11p-EF1aP, pKAD5f11pES-PmeI and shuttle plasmid pUC 19-PM. The adenovirus plasmid contains E1/E3 deletion human adenovirus type 5 HAdV-5 genome, and the original HAdV-5fiber is replaced by HAdV-5 and HAdV-11p fusion gene F5-11 p; the PmeI site is an exogenous gene insertion site. pKAD5f11p-EF1aP contains the human EF1a promoter. The shuttle plasmid pUC19-PM can be used with pKAD5f11 pES-PmeI. The construction method comprises the following steps: carrying out PCR amplification on target genes containing homologous overlapping regions on two sides, and carrying out DNA assembly on the target genes and adenovirus plasmids linearized by PmeI to obtain adenovirus plasmids containing the target genes; or cloning the exogenous gene fragment into a shuttle plasmid, cutting the shuttle plasmid by endonuclease, and then carrying out DNA assembly with PmeI linearized pKAD5f11 pES-PmeI. The invention combines restriction enzyme cutting and DNA assembling technology, and fully simplifies the adenovirus vector construction process.

Description

Adenovirus vector system and recombinant adenovirus construction method

Technical Field

The invention belongs to the field of gene therapy, and particularly relates to an adenovirus vector system and a recombinant adenovirus construction method.

Background

Adenoviruses are non-enveloped linear double-stranded DNA viruses, currently divided into 5 genera in the family adenoviridae^[1]Various types of adenovirus have been developed as gene transfer vectors and have been widely used in basic research, gene therapy and vector vaccines^[2-4]。

The genome of adenovirus is about 26-46kb in length, which makes the construction of adenovirus vectors different from common plasmid vectors (generally less than 10kb in length). Too long a genome makes it very difficult to enrich one multiple cloning site on an adenovirus vector. Various methods have been developed for the construction of recombinant adenoviruses^[5-7]。

The adenovirus vector is constructed through 4 development stages of direct in-vitro connection, recombination in packaging cells, recombination in bacteria and site-specific recombination, and an improved in-vitro connection method is applied along with the development of molecular biology technology^[5-7]. (1) In vitro ligation method. This method requires a full-length adenoviral genome and a plasmid containing the left-terminal sequence of the adenoviral genome, including Inverted left-terminal repeat (ITR), packaging signal and enhancer sequence of E1A. Cloning a target gene to the downstream of a virus sequence of a plasmid, carrying out enzyme digestion by ClaI to obtain the left end of a genome containing the target gene, connecting the left end to a ClaI-digested wild adenovirus genome (ClaI only has one enzyme digestion site in the virus genome and is positioned in an E1A gene), and directly transfecting the obtained genomic DNA containing the target gene into a packaging cell 293 to generate recombinant virus particles. The method needs to culture wild adenovirus to extract viral genome, can use only one enzyme cutting site, has low connection efficiency, only deletes part of E1A gene, has limited foreign gene carrying capacity, and is eliminated without use at present. (2) Eukaryotic cell homologous recombination. In 1994, Graham F established a method for producing recombinant adenovirus by homologous recombination in 293 cells. This method requires 2 plasmids: the backbone plasmid contains most of the genome sequence of the adenovirus, but does not contain a packaging signal; the shuttle plasmid contains ITR, packaging signal and target gene cloning site, and two sequences overlapping with the skeleton plasmid. The target gene is cloned to a shuttle plasmid, and the shuttle plasmid and a skeleton plasmid are cotransfected to 293 cells, and because partial sequences of the two plasmids are overlapped, homologous recombination can occur in the cells, and finally, a complete vector genome is generated and the recombinant virus particles are packaged. This approach has been widely used and has prompted the development of adenoviral vectors. Due to low efficiency of intracellular recombinationThese recombination events may also produce replication-competent viruses (RCV), which must be plaque purified to obtain the correct cloned recombinant virus, are time-consuming and laborious, and are gradually being replaced by other methods. (3) Homologous recombination in bacteria. The skeleton plasmid and shuttle plasmid used in the method are similar to those used in the intracellular recombination method, after the target gene is cloned to the shuttle plasmid, the shuttle plasmid and the skeleton plasmid are co-transformed into bacterial cells (such as Escherichia coli BJ5183 strain), the recombinant enzyme in the bacterial cells is positive, the two plasmids are recombined in the bacteria, recombinant plasmids (containing adenovirus vector genome) are obtained through antibiotic screening, and the recombinant virus particles are rescued by transfecting packaging cells after amplification and purification. This method is represented by the AdEasy system (Stratagene, USA) which is widely used at present. Because the cloned adenovirus plasmid is obtained by the recombination in the bacterial cell, the uniform seed virus is obtained by rescuing in a packaging cell, and the method can be directly used for the amplification of the recombinant virus without time-consuming plaque purification. However, the recombination efficiency in bacteria is low, some false positive clones exist, recombinase positive bacteria generally cannot be used for massive amplification of adenovirus plasmids, and the obtained positive adenovirus plasmid clones need to transform new bacterial cells to prepare sufficient plasmids for virus rescue; in addition, when the plasmid with the repetitive sequence or the complex high-order structure is replicated by recombinase positive bacteria, the genome of the plasmid is unstable, partial sequence loss can occur, variant adenovirus plasmids are generated, and the recombinant virus which can not be rescued or can not be rescued is caused. (4) Site-specific recombination. Such methods use phage-derived recombinases that are capable of recognizing specific sequence sites (tens to hundreds of base pairs in length) and initiating recombination between sequences containing such sites; adding specific sites which can be identified by recombinase into an adenovirus skeleton and a shuttle plasmid, expressing corresponding phage recombinase in cells or using the recombinase in vitro to mediate site-specific recombination between the skeleton plasmid and the shuttle plasmid, introducing a target gene into the skeleton plasmid, and forming adenovirus plasmid with a recombinant adenovirus genome; transfecting the packaging cell to rescue the virus vector^[6,8]. The method further simplifies the preparation process of adenovirus plasmid, and has the limitation thatThere are certain sites in the adenovirus genome where recombinase-specific recognition sites inevitably remain. (5) Direct ligation using Homing Endonuclease (Homing Endonuclease). The length of the sequence recognized by the homing endonuclease is more than 20bp, wherein the specific sequence is at least 9-11 bp, which means that the sequence recognized and cut by the endonuclease is very rare, and the cutting site of the endonuclease is not existed in the genome of the wild-type adenovirus with the length of 36 kb. The method also uses 2 plasmids similar to the skeleton plasmid and the shuttle plasmid, but no sequence overlapping is needed among the 2 plasmids, the target gene is cloned to the shuttle plasmid, and is connected to the skeleton plasmid after being cut by using homing endonuclease to form a complete vector genome, and the recombinant virus is rescued by transfecting a packaging cell. The Adeno-X system from Clontech, USA, adopts the method. In addition, Clontech developed recently Adeno-X system 3, which directly inserted the PCR product with the gene of interest into the already linearized pAdenoX vector supplied by Clontech using In-Fusion technology, i.e.obtained the adenovirus plasmid. The method needs to purchase a linearized pAdenoX vector, and the insertion site of the foreign gene and other parts of the recombinant virus genome are determined and cannot be used for manual modification of the recombinant adenovirus.

The existing adenovirus vector systems have defects, some adenovirus vector systems have complicated construction steps and long time consumption, some adenovirus vector systems introduce unnecessary exogenous sequences into recombinant viruses, and some adenovirus vector systems cannot be used for reverse genetic modification of the recombinant adenoviruses. In recent years, new adenoviruses with different biological characteristics are continuously discovered, so that the need exists in the field for an adenovirus vector system and a recombinant virus construction method which have a quicker, simpler and more convenient construction process and are more convenient and flexible to artificially modify.

Disclosure of Invention

In order to meet the needs of the art, it is an object of the present invention to provide novel adenoviral vector systems and recombinant virus construction methods.

The invention establishes a vector system for directly and seamlessly inserting an exogenous target gene into an adenovirus vector genome and a recombinant adenovirus construction method by introducing a single enzyme cutting site into the adenovirus vector genome and by means of a DNA assembly technology.

In a first aspect, the present invention provides a set of adenoviral vector systems comprising two adenoviral plasmids and a shuttle plasmid.

Among them, adenovirus plasmid pKAD5f11p-EF1aP was designed and constructed by the present inventors (examples 1 to 4). The present inventors used the previously constructed pAdeassyf 11p adenovirus backbone plasmid (containing the modified fiber gene)^[9]pAd5GXP adenovirus plasmid (eliminating original PmeI restriction endonuclease site in the HAdV-5 genome)^[10]The adenovirus plasmid pKAD5f11p-EF1aP was constructed. The pKAD5F11p-EF1aP plasmid contains the HAdV-5 genome with the deletion of E1/E3 region, the original HAdV-5fiber gene is replaced by the fusion fiber gene (F5-11p) of HAdV-5 and HAdV-11p, and the original PmeI restriction endonuclease site in the genome is eliminated; adding a human EF1a promoter and an SV40 polyA tailing signal at the position of the original E1, and adding a single PmeI enzyme cutting site (a plasmid-wide unique PmeI site) between an EF1a promoter and an SV40 polyA tailing signal; it also contains pBR322 replication origin and kanamycin resistance gene for plasmid replication in prokaryotic cell. The fusion gene F5-11p consists of the tail domain of HAdV-5fiber and the shaft and knob domain coding regions of HAdV-11p fiber.

The recombinant virus can be rescued by introducing the exogenous target gene directly into the PmeI site of pKAD5f11p-EF1aP by the DNA assembly method described later. Adenovirus recognizes and binds to a cell receptor through fiber expressed in the penton of the viral particle, and then infects a target cell, wherein the receptor of the fiber protein of HAdV-5 binding cell is CAR molecule, and the cell receptor of HAdV-11p binding recognition by fiber is DSG2 or CD46 molecule^[11,12]. The recombinant virus constructed by the adenovirus plasmid pKAd5f11p-EF1aP carries fused fiber protein, thereby having tropism of HAdV-11p virus, being capable of infecting cells by combining with DSG2 or CD46 molecules, and the DSG2 or CD46 molecules have higher expression abundance in tumor cells or hematopoietic cells than CAR, so the recombinant virus can infect the tumor cells or hematopoietic cells more efficiently^[9,13]. EF1a is a constitutive high-efficiency promoter, and the target gene controlled by it can be expressed efficiently in the target cell^[14]。

The adenovirus plasmid pKAD5f11pES-PmeI is obtained by further deleting EF1a promoter on the basis of constructing pKAD5f11p-EF1aP (example 7), which meets the requirement of user to select target gene regulatory element (mainly referring to promoter) or carry multiple target gene expression cassettes at the same time. The pKAD5f11pES-PmeI plasmid does not contain the EF1a promoter, and the other components are identical to those of pKAD5f11p-EF1aP, and the single enzyme cutting site PmeI site is positioned after the adenovirus packaging signal (ES). pKAd5f11pES-PmeI can be used alone, for example, by introducing a plurality of gene elements directly into the PmeI site (position of E1) of pKAd5f11pES-PmeI plasmid by the DNA assembly method described later, recombinant viruses can be rescued (examples 9 and 10); it can also be used in combination with the shuttle plasmid pUC 19-PM. When too many genetic elements are desired to be introduced or the PCR method is not convenient to use, a strategy of cloning the genetic elements into a shuttle plasmid may be employed (example 12).

The shuttle plasmid pUC19-PM is formed by cloning a synthetic sequence into the pUC19 plasmid at the multiple cloning site by DNA assembly. The kit contains a multiple cloning site, and the multiple cloning site consists of restriction enzymes EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, EcoRV, SalI, PstI, SphI, HindIII, ClaI and EcoRV in sequence; the upstream and the downstream of the multiple cloning site respectively contain a homologous overlapping region which is consistent with the sequences on both sides of the PmeI site of the pKAD5f11pES-PmeI plasmid; the outer sides of the homologous overlapping regions respectively contain a PacI enzyme cutting site; also, the plasmid contained pBR322 origin of replication derived from pUC19, ampicillin resistance gene, which was required for plasmid replication in prokaryotic cells (FIG. 18). When it is necessary to introduce multiple gene elements, multiple gene expression cassettes into an adenovirus vector or the PCR method is not convenient (for example, the secondary structure of the fragment or the amplification is difficult due to too long product, or there is a fear that the PCR reaction may cause mutation), these fragments can be sequentially cloned into the multiple cloning site of the shuttle plasmid by the enzyme digestion-ligation method, and then these fragments are excised together with homologous overlapping regions at both ends using PacI, and cloned into the PmeI site of the pKAD5f11pES-PmeI plasmid by the DNA assembly method described later, to obtain the adenovirus plasmid carrying the target gene (example 12).

In a second aspect, the present invention provides a method for preparing a recombinant adenovirus carrying a foreign gene of interest using the adenoviral vector system of the first aspect.

The inventor provides a method for constructing the recombinant adenovirus vector which is simpler and more convenient than the prior method. The method comprises the following steps: cutting the adenovirus plasmid from the foreign gene insertion site by using restriction endonuclease, and recovering linearized adenovirus plasmid DNA; carrying out PCR or enzyme digestion to obtain linear DNA containing a target gene; mixing the two sections of linear DNA, carrying out DNA assembly reaction, transforming the reaction product into escherichia coli competent cells, and screening to obtain the adenovirus plasmid containing the target gene. We will refer to this method as restriction-assembly. The subsequent method for generating recombinant viruses from adenovirus plasmids is a general method that has been established by the predecessor, and comprises: using selected restriction endonuclease to cut the adenovirus plasmid containing target gene and release complete recombinant virus genome, i.e. cutting plasmid skeleton (generally including antibiotic resistance gene and plasmid replication origin), recovering DNA, transfecting packaging cell and saving recombinant virus; the packaging cells can be used for continuously amplifying and rescuing the obtained recombinant viruses. The method provided by the inventor can realize the introduction of the exogenous target gene into the adenovirus plasmid through DNA assembly reaction and bacterial transformation experiments, and is more convenient and faster than the existing method. The use of this method requires 2 preconditions: (1) it is necessary to introduce a single cleavage site(s) at the site of insertion of the foreign gene into the adenovirus plasmid. When adenovirus plasmid is cut by using corresponding restriction enzyme, the plasmid can be cut at the cloning site of the exogenous target gene to form a linearized DNA chain, so that DNA assembly can be carried out, and the directional cloning of the exogenous gene is ensured. The natural adenovirus genome does not have such a single enzyme cutting site generally, so the adenovirus genome to be used must be modified through complicated molecular cloning operation in the early stage, and the usable single enzyme cutting site is introduced to construct adenovirus plasmid for cloning exogenous genes. Once the adenovirus plasmid containing the single enzyme cutting site is successfully constructed, the subsequent target gene insertion is simplified due to the use of the method. (2) Homologous overlapping regions required for DNA assembly must be introduced at both ends of the gene of interest (or sequence of interest). The homologous overlapping region is generally 15-60bp long, and can be added to the 5' end of a primer by designing a PCR primer and directly added to both sides of the exogenous gene by utilizing PCR amplification (examples 5 and 9); alternatively, the foreign gene may be flanked by other methods, for example, a shuttle plasmid is constructed in advance, the shuttle plasmid contains homologous overlapping regions on both sides of the multiple cloning site, and after cloning the target gene into the multiple cloning site, the homologous overlapping regions can be excised together with the target gene by restriction enzyme cleavage to form a linear DNA strand (example 12). DNA assembly reactions refer to techniques that fuse linear DNA fragments together by homologous overlap regions, commonly known as Gibson assembly techniques. In the case where the first precondition described above is satisfied (i.e., an adenovirus plasmid for cloning a foreign gene containing a single cleavage site has been prepared), the foreign gene can be directly cloned into the site by a conventional cleavage-ligation method. However, the DNA assembly method has the advantages that: the ligation efficiency is higher, and more positive colony clones can be generated after competent bacteria are transformed; when a single restriction enzyme site is used, the restriction enzyme-ligation method will produce two positive colonies inserted in the forward and reverse directions, whereas the DNA assembly method only produces directional clones; when the enzyme digestion-connection method is used, the inserted target fragment is limited by the enzyme digestion site, namely the target fragment cannot contain the restriction enzyme digestion site to be used, otherwise the target fragment is cut into two or more fragments, the connection efficiency is seriously influenced or the target fragment cannot be successfully connected, and the target fragment is not limited by the enzyme digestion site when the DNA assembly method is used.

In a third aspect, the present invention provides the use of an adenoviral vector system according to the first aspect or an adenoviral vector construction method according to the second aspect for the preparation of an adenoviral vector construction kit, a gene therapy kit or a recombinant vaccine. The foreign target gene may be a target protein gene whose expression level is to be increased, such as, but not limited to, an immune regulator, a virus structural protein gene, or the like. The recombinant virus prepared by the adenovirus vector system is a replication-defective virus with deletion of an E1/E3 region. Unlike common recombinant viruses based on the HAdV-5 genome, this class of viruses contains a fusion fiberThe gene (F5-11p) is formed by fusing the tail coding region of HAdV-5fiber with the shaft and knob coding regions of HAdV-11p, and the expressed fusion protein F5-11p endows the recombinant virus with the capacity of recognizing and combining cell receptors CD46 and DSG-2, so that the gene has stronger exogenous gene transduction capacity on cells with high expression abundance of the receptors. The existing research shows that the expression level of CD46 and DSG-2 is higher in tumor cells or hematopoietic cells, so that the constructed recombinant virus has higher infection efficiency on the tumor cells or the hematopoietic cells^[9,11,13]. The restriction enzyme cutting-assembling method provided by the invention greatly simplifies the construction process of adenovirus plasmid. The traditional bacterial internal homologous recombination method needs to undergo 3 times of bacterial transformation, while the restriction enzyme cutting-assembling method only needs 1 time of bacterial transformation, so that the adenovirus vector construction becomes convenient, fast, time-saving and labor-saving, and is expected to become the mainstream adenovirus vector construction method.

Therefore, the adenovirus vector system and the recombinant virus construction method provided by the invention can save and obtain the corresponding recombinant adenovirus. The adenovirus vector system and the recombinant virus construction method can be used as a good tool for constructing recombinant vaccines and gene therapy kits, foreign target gene expression frames are introduced into adenovirus plasmids, and then recombinant adenoviruses carrying foreign target genes are obtained through rescue, and the obtained recombinant adenoviruses carrying foreign target genes can be used for preparing recombinant vaccines or gene therapy reagents with corresponding functions. Obviously, the adenovirus vector system and the recombinant virus construction method have the advantages that other adenovirus vector systems in the prior art do not have, and are widely applied.

In summary, the present invention provides the following technical solutions:

1. a set of adenovirus vector system, which comprises two adenovirus plasmids pKAD5f11p-EF1aP and pKAD5f11pES-PmeI and a shuttle plasmid pUC 19-PM; adenovirus plasmids pKAD5F11p-EF1aP and pKAD5F11pES-PmeI both contain E1/E3 deleted human adenovirus type 5 (HAdV-5) genome, the HAdV-5 genome original HAdV-5fiber gene is replaced by fusion gene (F5-11p) of HAdV-5 and HAdV-11p fiber, the original PmeI site of HAdV-5 genome is removed by mutation, and the PmeI site also contains pBR322 replication origin and kanamycin resistance gene which are needed for plasmid replication in prokaryotic cells; the adenovirus plasmids pKAD5F11p-EF1aP and pKAD5F11pES-PmeI contain fusion fiber genes (F5-11p) obtained by fusing tail domain of HAdV-5fiber with the shaft and knob domain coding regions of HAdV-11p fiber; the adenovirus plasmid pKAD5f11p-EF1aP contains the following characteristics: adding a human EF1a promoter and an SV40 polyA tailing signal at the position of an original HAdV-5E1 zone, and containing a single restriction enzyme PmeI restriction site (GTTTAAAC) between an EF1a promoter and an SV40 polyA tailing signal; the adenovirus plasmid pKAD5f11pES-PmeI contains the following characteristics: a single restriction enzyme PmeI restriction site (GTTTAAAC) is added at the position of the original HAdV-5E1 zone; the shuttle plasmid pUC19-PM contained the following characteristics: contains a multiple cloning site, which consists of restriction enzyme cutting sites of EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, EcoRV, SalI, PstI, SphI, HindIII, ClaI and EcoRV in sequence, wherein the upstream and the downstream of the multiple cloning site respectively contain a homologous overlapping region which is consistent with the sequences at two sides of the PmeI site of the pKAD5f11pES-PmeI plasmid, the outside of the homologous overlapping region respectively contains a PacI cutting site, and also contains a pBR322 replication origin required for plasmid replication in prokaryotic cells and an ampicillin resistance gene.

2. A method of constructing a recombinant adenovirus, the method comprising: cutting the adenovirus plasmid from the foreign gene insertion site by using restriction endonuclease, and recovering linearized adenovirus plasmid DNA; carrying out PCR or enzyme digestion to obtain linear DNA containing a target gene; mixing the two sections of linear DNA, carrying out DNA assembly reaction, transforming the reaction product into escherichia coli competent cells, and screening to obtain the adenovirus plasmid containing the target gene.

3. The method according to item 2, wherein said adenovirus plasmid contains an artificially introduced restriction enzyme cleavage site at the foreign gene insertion site, which is present only at the foreign gene insertion site and is not present at other sites of said adenovirus plasmid.

4. The method according to item 2, wherein the DNA containing the gene of interest is linearized, characterized in that: both ends of the DNA respectively contain a homologous overlapping region consistent with sequences on both sides of the insertion part of the foreign gene of the adenovirus plasmid; the homologous overlapping region can meet the requirement of Gibson assembly reaction, and the length is 15-60 bp.

5. An adenovirus vector construction kit comprising the adenovirus vector system described in item 1.

6. Use of the adenoviral vector system or the recombinant adenoviral construction method according to any one of items 1 to 4 in the preparation of an adenoviral vector construction kit, a gene therapy kit or a recombinant vaccine.

Drawings

The above features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic diagram of the construction of E1/E3 deleted adenovirus plasmid pAd5f11p-EF1a containing the human EF1a promoter. Firstly, using overlap extension PCR to amplify to obtain a DNA fragment (ES-EF1ap-MCS) containing HAdV-5 packaging signal, human EF1a promoter and multiple cloning sites; cloning the ES-EF1ap-MCS fragment to a BsrGI/EcoRV site of a shuttle plasmid pShuttle-CMV by means of an enzyme digestion-ligation method to obtain a shuttle plasmid pSh5-EF1a with an exogenous gene promoter replaced by EF1 ap; then, the linearized shuttle plasmid was digested with PmeI, the E.coli BJ5183 strain was transformed electrically, and homologous recombination was performed with the backbone plasmid pAdEasyf11p (the previously constructed adenovirus backbone plasmid pAdEasyf11p contains fusion gene F5-11p of HAdV-5 and HAdV-11p fiber), and adenovirus plasmid pAd5F11p-EF1a was obtained. In the pAd5F11p-EF1a plasmid, the E1/E3 region of HAdV-5 was deleted, the E1 region was replaced with the EF1a promoter and the SV40 polyA tailing signal, and the fiber gene of HAdV-5 was replaced with F5-11 p. In the figure, Amp: an ampicillin resistance open reading frame (ampicillin resistance ORF); CMVp: the human cytomegalovirus promoter; EF1 ap: the human EF1a promoter; ES: a packaging signal (encapsidation signal); f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; ITR: inverted terminal repeat (inverted terminal repeat); kan: kanamycin resistance open reading frame (kanamycin resistance ORF); ori: the pBR322 origin of replication; pA: SV40 polyA tailed signal; 1411Sh5EF1aF1, 1411Sh5EF1aR1, 1411Sh5EF1aF2, 1411Sh5EF1aR2, 1411Sh5EF1aF3 and 1411Sh5EF1aR3 are 6 primers used for overlap extension PCR.

FIG. 2 is a schematic diagram of the construction of adenovirus plasmid pAd5f11p-CGXP with PmeI cleavage sites removed by mutation. Firstly, carrying out BamHI digestion on pAd5F11p-EF1a to separate a 10999bp fragment containing the F5-11p gene; and replacing the corresponding part in the pAd5GXP plasmid with the modified fragment by an enzyme digestion-ligation method to obtain the pAd5f11p-CGXP plasmid (the original PmeI site in the genome of the previously constructed adenovirus plasmid pAd5GXP is mutated and removed). The pAd5F11p-CGXP adenovirus plasmid contains F5-11p genes, and the original single PmeI restriction site is mutated (namely, the PmeI site is not contained). In the figure, CMVp: the human cytomegalovirus promoter; EF1 ap: the human EF1a promoter; f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; GFP: a green fluorescent protein coding frame; kan: kanamycin resistance open reading frame (kanamycin resistance ORF); ori: the pBR322 origin of replication.

FIG. 3 is a schematic diagram of the construction of a small plasmid pKAD5-EF1aBP with a reformed multiple cloning site in E1 region. Firstly, designing and synthesizing a pair of primers with 3' ends capable of being complementarily paired, annealing the primers by self, and extending by using DNA polymerase to obtain a 109bp double-stranded DNA fragment (EcoRI-PacI-BamHI); BstZ17I enzyme-cleaves pAd5f11p-EF1a plasmid, recovers 7210bp fragment containing EF1a promoter and multiple cloning site region after electrophoresis, and carries out DNA assembly with the 109bp fragment to obtain plasmid pKAD5-EF1aBstZ 17I; the KpnI/EcoRV double enzyme digestion pKAD5-EF1aBstZ17I plasmid removes multiple cloning sites, recovers 7233bp fragments after electrophoresis, and carries out DNA assembly with the synthesized 58nt single-stranded oligonucleotide fragment to obtain the pKAD5-EF1aBP plasmid. In the pKAD5-EF1aBP plasmid, the original multiple cloning site is replaced by a PmeI site, and two homologous overlapping regions which are convenient for DNA assembly operation are added at the original BstZ17I site. In the figure, EF1 ap: the human EF1a promoter; f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; kan: kanamycin resistance open reading frame (kanamycin resistance ORF); ori: the pBR322 origin of replication; 1805F11p-EF1PmeA1-1805F11p-EF1PmeA3 is a chemically synthesized single-stranded oligonucleotide.

FIG. 4 is a schematic diagram of the construction of adenovirus plasmid pKAD5f11p-EF1aP for cloning foreign genes. EcoRI is used for carrying out enzyme digestion on pKAD5-EF1aBP, and 7242bp linearized fragment is recovered by electrophoresis; BstZ17I/PacI double enzyme digestion pAd5f11p-CGXP plasmid, electrophoresis recovery of 26781bp fragment; the above 2 fragments were combined and DNA assembly was carried out to obtain pKAD5f11p-EF1 aP. In pKAD5F11p-EF1aP plasmid, the E1/E3 region of HAdV-5 is deleted, the E1 region is replaced by EF1a promoter and SV40 polyA tailing signal, the fiber gene of HAdV-5 is replaced by fusion gene of HAdV-5 and HAdV-11p fiber (F5-11p), and the foreign gene coding region can be directly cloned to the PmeI site by DNA assembly. The details of the PmeI unique cleavage site and 40bp flanking sequences of pKAD5f11p-EF1aP plasmid are shown below, and homologous overlapping regions designed for DNA assembly can be selected from the PmeI flanking sequences. In the figure, CMVp: the human cytomegalovirus promoter; EF1 ap: the human EF1a promoter; f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; GFP: a green fluorescent protein coding frame; kan: kanamycin resistance open reading frame (kanamycin resistance ORF); ori: the pBR322 origin of replication; pA: SV40 polyA tailed signal.

FIG. 5 is an electrophoretogram of the adenovirus plasmid pKAD5f11p-EF1aP for cloning of foreign genes, which was identified by digestion with EcoRV. M1 Lambda/HindIII DNA marker; m2 DL2000 DNA molecular weight marker; EcoRV enzyme pKAD5f11P-EF1aP plasmid (33964bp), the predicted fragment molecular weight is: 1238,2052,2617,3075,4545,7637,12800 bp.

FIG. 6 is a schematic diagram of the construction of cloning of the exogenous green fluorescent protein gene into an adenovirus plasmid. Performing PCR amplification to obtain GFP fragments with overlapping areas at two ends; the PCR product is directly assembled with PmeI linearized pKAD5f11p-EF1aP to obtain the adenovirus plasmid pKAD5f11p-EPG carrying GFP gene. The overlapping region of the two segments of the PCR product (about 25bp) was added by synthesis at the 5' end of the primer. In the figure, CMVp: the human cytomegalovirus promoter; EF1 ap: the human EF1a promoter; f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; GFP: a green fluorescent protein coding frame; kan: kanamycin resistance open reading frame (kanamycin resistance ORF); OL1 and OL 2: upstream and downstream overlap regions (overlap 1, overlap 2); ori: the pBR322 origin of replication; pA: SV40 polyA tailed signal; 1805F11p-EF1PmeGFP1/1805F11p-EF1PmeGFP2 is a pair of primers for PCR amplification.

FIG. 7 shows the results of restriction enzyme analysis screening and identification of the adenovirus plasmid pKAD5f11p-EPG clone. The restriction result of HindIII plasmid pKAD5f11p-EF1aP was used as a control. M is Lambda/HindIII DNA marker; HindIII digestion of pKAD5f11p-EPG predicted fragment molecular weight: 75,2081,2993,4597,5209,5322,6421,8009 bp; HindIII digestion of pKAD5f11p-EF1aP plasmid predicted fragment molecular weight: 75,2081,4597,5209,5322,8009,8671 bp. The electrophorogram showed that the actual molecular weight of the digested fragment was consistent with that predicted, indicating that all of the randomly selected 5 clones were pKAD5f11p-EPG plasmid.

FIG. 8 shows the rescue of the HAdV5f11p-EPG recombinant virus in 293 cells. After the linearized pKAD5f11p-EPG adenovirus plasmid is digested by PacI, 293 cells are transfected, and after 5 days of culture, fluorescence foci are formed on GFP positive cells on monolayer cells, which indicates that the recombinant virus HAdV5f11p-EPG is successfully rescued by a fluorescence microscope.

FIG. 9 shows the results of restriction enzyme analysis and identification of recombinant adenovirus HAdV5f11p-EPG genomic DNA. M1 Lambda/HindIII DNA marker; m2 DL2000 DNA molecular weight marker; HindIII cleavage predicted fragment molecular weight: 75,1008,2081,2429,2993,4597,5209,5322,8009 bp; the molecular weight of the fragment predicted by EcoRV cleavage is: 1238,2052,2181,2617,3075,4545,7637,8378 bp. The electrophorogram shows that the actual molecular weight of the enzyme fragment is consistent with that predicted, and the rescued virus is indeed HAdV5f11 p-EPG.

FIG. 10 shows the expression of GFP by fluorescence microscopy after infection of A549 cells with recombinant adenovirus HAdV5f11 p-EPG. The recombinant virus HAdV5f11p-EPG infects A549 cells with the infection intensity of 200 vp/cell; fluorescence microscopy 2 days after infection showed that more than 90% of the cells expressed GFP.

FIG. 11 is a diagram showing the construction of adenovirus plasmid pKAD5f11pES-PmeI for cloning foreign gene expression cassette. The EF1a promoter and multiple cloning site were first deleted from the pKAd5-EF1 acbstz 17I plasmid: AatII/EcoRV double enzyme digestion pKAD5-EF1aBstZ17I plasmid, recovering 5892BP fragment, and carrying out DNA assembly with synthetic single-stranded oligonucleotide to obtain pKAD5ES-BP plasmid (5904 BP); and carrying out EcoRI digestion linearization on the pKAD5ES-BP plasmid, carrying out DNA assembly on the plasmid and a 26781BP fragment recovered by BstZ17I/PacI double digestion pAd5f11p-CGXP plasmid electrophoresis, and obtaining the pKAD5f11pES-PmeI plasmid. In pKAD5F11pES-PmeI, the E1/E3 region of HAdV-5 is deleted, the PmeI site is added to the original E1 region, and the fiber gene of HAdV-5 is replaced by a fusion gene of HAdV-5 and HAdV-11p fiber (F5-11 p). The details of the PmeI unique cleavage site and the 40bp flanking sequence of the pKAD5f11pES-PmeI plasmid are also shown in the figure, and homologous overlapping regions designed for DNA assembly can be selected in the PmeI flanking sequence. In the figure, CMVp: the human cytomegalovirus promoter; EF1 ap: the human EF1a promoter; ES: a packaging signal (encapsidation signal); f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; GFP: a green fluorescent protein coding frame; ITR: inverted terminal repeat (inverted terminal repeat); kan: kanamycin resistance open reading frame (kanamycin resistance ORF); ori: the pBR322 origin of replication; pA: SV40 polyA tailed signal.

FIG. 12 is an electrophoretogram of cloning of foreign gene expression cassette identified by digestion with adenovirus plasmid pKAD5f11pES-PmeI using EcoRV. M1 Lambda/HindIII DNA marker; EcoRV digested pKAD5f11pES-PmeI plasmid (32626bp), predicted fragment molecular weight: 1238,2052,2617,3075,4545,5526,5936,7637 bp.

FIG. 13 is a schematic diagram of the construction of inducible expression lentiviral vector plasmid pLVX-TRE 3G-TetGFP. Firstly, fusing a Tet-on3G coding region and WPRE into a fragment by using overlap extension PCR, inserting the fragment between two PstI sites of a pLVX-TRE3G plasmid by using an enzyme digestion-connection method, and obtaining the plasmid which is named as pLVX-TRE 3G-Teton; then, the GFP coding region obtained by PCR amplification is cloned between BamHI/EcoRI sites of pLVX-TRE3G-Teton by an enzyme digestion-ligation method to obtain pLVX-TRE3G-TetGFP plasmid. In the figure, Amp: an ampicillin resistance open reading frame (ampicillin resistance ORF); GFP: a green fluorescent protein coding frame; LTR: a long terminal repeat; MCS: a multiple cloning site; ori: the pBR322 origin of replication; pPGK: the murine phosphoglycerate kinase promoter (Mouse phosphoglycerate kinase 1 promoter); pTR: the TRE3GV promoter; puro: a puromycin resistance gene; Tet-On 3G: Tet-On3G transcriptional activator protein; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element (woodchuck hepatitis virus posttranscriptional regulatory element); 1306TetonP1-1306TetonP4 are 4 primers used for overlap extension PCR; 1306TET-GFPF1 and 1306TET-GFPR1 are a pair of primers used to amplify the GFP coding region.

FIG. 14 is a schematic diagram showing the construction of cloning of a GFP expression regulatory element amplified by PCR into an adenovirus plasmid. Carrying out PCR amplification by using pLVX-TRE3G-TetGFP plasmid as a template to obtain a GFP expression regulatory element (TG-teton) with two ends provided with overlapping regions; the PCR product is directly assembled with PmeI linearized pKAD5f11pES-PmeI plasmid to obtain adenovirus plasmid pKAD5f11p-TGFPT carrying GFP expression regulatory element. The overlapping region of the two segments of the PCR product (about 25bp) was added by synthesis at the 5' end of the primer. In the figure, GFP: a green fluorescent protein coding frame; ES: a packaging signal (encapsidation signal); f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; kan: kanamycin resistance open reading frame (kanamycin resistance ORF); ori: the pBR322 origin of replication; pPGK: the murine phosphoglycerate kinase promoter (Mouse phosphoglycerate kinase 1 promoter); pTR: the TRE3GV promoter; Tet-On 3G: Tet-On3G transcriptional activator protein; 1805F11p-TGteton1 and 1805F11p-TGteton2 are a pair of primers used for PCR amplification.

FIG. 15 shows the results of restriction enzyme analysis screening and identification of adenovirus plasmid pKAD5f11p-TGFPT clone. M1 Lambda/HindIII DNA marker; m2 DL2000 DNA molecular weight marker; EcoRV enzyme pKAD5f11p-TGFPT predicted fragment molecular weight: 1238,2052,2617,3075,4545,5936,7637,7942 bp. The electrophorogram showed that the actual molecular weight of the digested fragment was consistent with that predicted, indicating that all 5 randomly selected clones were pKAD5f11p-TGFPT plasmid.

FIG. 16 is the result of restriction enzyme analysis identification of recombinant adenovirus HAdV5f11p-TGFPT genomic DNA. M is Lambda/HindIII DNA marker; BglII cleavage predicted fragment molecular weight: 271,1268,1497,1551,2151,5177,5533,6180,8430 bp; the molecular weight of the fragment predicted by EcoRV cleavage is: 1238,2052,2181,2617,2777,3075,4545,5936,7637 bp; HindIII cleavage predicted fragment molecular weight: 75,1008,1125,2081,4597,4632,5209,5322,8009 bp; NdeI enzyme digestion predicted fragment molecular weight: 539,4095,8901,18523 bp. The electrophorogram showed that the actual molecular weight of the cleaved fragment was consistent with that predicted, indicating that the rescued virus was indeed HAdV5f11 p-TGFPT.

FIG. 17 shows the induced expression of GFP by fluorescence microscopy after infection of A549 cells with recombinant adenovirus HAdV5f11 p-TGFPT. The purified recombinant virus infected A549 cells at an infection intensity of 1000vp/cell, with no or a final concentration of 1. mu.g/ml doxycycline (Dox) added to the medium; fluorescence microscopy was performed 2 days after infection. No Dox was added, no GFP + cells were found; with addition of Dox, more than 50% of the cells expressed GFP. Indicating that Dox is able to regulate GFP expression in cells following viral infection.

FIG. 18 is a schematic diagram of the construction of shuttle plasmid pUC19-PM containing homologous overlapping regions. Designing and synthesizing 6 primers, synthesizing a 190bp DNA fragment by overlap extension PCR, wherein the middle part of the fragment is a multiple cloning site, two homologous overlapping regions (about 30bp, used for assembling DNA with adenovirus plasmid pKAD5f11 pES-PmeI) which are consistent with sequences on two sides of a PmeI site of the adenovirus plasmid pKAD5f11pES-PmeI are arranged on the outer sides of the multiple cloning sites, each homologous overlapping region is arranged on the outer sides of the overlapping regions, and the homologous overlapping regions are consistent with sequences on two sides of the multiple cloning site of the pUC19 plasmid (used for assembling DNA with linearized pUC19 plasmid at this time); the PCR product was directly DNA-assembled with EcoRI/HindIII double digested linearized pUC19 plasmid to obtain pUC19-PM plasmid. Details of the two PacI sites of the pUC19-PM plasmid and their internal sequences are also shown in the lower part of the figure, with homologous overlapping regions indicated in lower case letters adjacent to the PacI site and a multiple cloning site in the middle. In the figure, Amp: an ampicillin resistance open reading frame (ampicillin resistance ORF); MCS: a multiple cloning site; ori: the pBR322 origin of replication; 1800PacI-MCS1-1800PacI-MCS6 are 6 primers used for overlap extension PCR.

FIG. 19 is a schematic diagram of the construction of an adenovirus plasmid by means of the shuttle plasmid pUC 19-PM. Firstly, ClaI/KpnI double enzyme digestion pLVX-TRE3G-TetGFP plasmid is carried out, 3140bp fragment is recovered through electrophoresis, the fragment (containing GFP expression regulation and control elements) is cloned between ClaI/KpnI of pUC19-PM multiple cloning site by using an enzyme digestion-connection method, and shuttle plasmid pUC19-PTGFPW containing a target gene expression frame is obtained; PacI cuts pUC19-PTGFPW plasmid, recovers 3217bp segment (containing homologous overlapping area on two sides) by electrophoresis, and directly carries out DNA assembly with PmeI linearized pKAD5f11pES-PmeI plasmid to obtain pKAD5f11p-TGFPW adenovirus plasmid. In the figure, Amp: an ampicillin resistance open reading frame (ampicillin resistance ORF); ES: a packaging signal (encapsidation signal); f5-11 p: fusion gene of HAdV-5 and HAdV-11p fiber; GFP: a green fluorescent protein coding frame; kan: kanamycin resistance open reading frame (kanamycin resistance ORF); LTR: a long terminal repeat; ori: the pBR322 origin of replication; pPGK: the murine phosphoglycerate kinase promoter (Mouse phosphoglycerate kinase 1 promoter); pTR: the TRE3GV promoter; Tet-On 3G: Tet-On3G transcriptional activator protein; WPRE: woodchuck hepatitis virus posttranscriptional regulatory element (woodchuck hepatitis virus posttranscriptional regulatory element).

FIG. 20 shows the results of restriction enzyme analysis screening and identification of the adenovirus plasmid pKAD5f11p-TGFPW clone. M is Lambda/HindIII DNA marker; HindIII digestion of pKAD5f11p-TGFPW the predicted fragment molecular weight: 75,2081,4597,4631,5209,5322,5857,8009 bp. The electrophorogram showed that the actual molecular weight of the digested fragment was consistent with that predicted, indicating that all of the 5 randomly selected clones were pKAD5f11p-TGFPW plasmids.

FIG. 21 shows the results of restriction enzyme analysis and identification of recombinant adenovirus HAdV5f11p-TGFPW genomic DNA. M is Lambda/HindIII DNA marker; BglII cleavage predicted fragment molecular weight: 271,1268,1497,1551,2151,5177,5533,6180,9169 bp; the molecular weight of the fragment predicted by EcoRV cleavage is: 1238,2052,2181,2617,3075,3516,4545,5936,7637 bp; HindIII cleavage predicted fragment molecular weight: 75,1008,1865,2081,4597,4631,5209,5322,8009 bp; NdeI enzyme digestion predicted fragment molecular weight: 1279,4095,8901,18522 bp. The electrophorogram shows that the actual molecular weight of the enzyme cutting fragment is consistent with that predicted, and the rescued virus is really HAdV5f11p-TGFPW recombinant virus.

FIG. 22 shows the induced expression of GFP by fluorescence microscopy after infection of A549 cells with recombinant adenovirus HAdV5f11 p-TGFPW. The purified recombinant virus HAdV5f11p-TGFPW infected A549 cells at an infection intensity of 1000vp/cell, with no or a final concentration of 1. mu.g/ml doxycycline (Dox) added to the medium; fluorescence microscopy was performed 2 days after infection. Dox was not added, and GFP expression was not observed; with Dox added, most cells expressed GFP. Indicating that Dox is able to regulate GFP expression in target cells following viral infection.

Detailed Description

The invention is further described below with reference to specific examples, but it will be understood by those skilled in the art that the invention is not limited to these specific examples.

The construction of plasmids pAdEasyf11p and pAd5GXP used in the following examples has been published. pAdEasyf11p is obtained by modifying the fiber gene of adenovirus backbone plasmid pAdEasy-1(Genbank accession No. AY370909)^[9](ii) a pAd5GXP is an HAdV-5 adenovirus plasmid with original PmeI sites deleted^[10]. pShuttle-CMV (https:// www.addgene.org/16403/), pShuttle (Genbank access No. AF334399) plasmids and E.coli BJ5183 strain were purchased from Stratagene, USA. The pLVX-EF1a-Tet3G and pLVX-TRE3G plasmids were purchased from Clontech, USA. PCR thermostable DNA polymerase (Q5 high fidelity DNA polymerase), DNA Assembly reagents (NEBuilder HiFi DNA Assembly Master Mix, Cat. E2621), calf alkaline phosphatase (CIP) were used and purchased from NEB corporation, USA; DNA ligation kit (cat.6022q) was purchased from Takara Bio corporation; agarose gel DNA recovery kit (cat. d4045) was purchased from ZYMO RESEARCH; various restriction enzymes were purchased from NEB or Takara Bio; coli TOP10 competent cells and pUC19 plasmid (Genbank accession No. M77789) were purchased from Tiangen Biochemical technology, Inc.; the PCR primers were synthesized by Beijing Liuhe Hua Dagen science and technology Co., Ltd; human cell lines 293(Cat. CRL-1573) and A549(Cat. CCL-185) were purchased from American Type Culture Collection (ATCC); plasmid transfection was performed using Lipofectamine 3000 from ThermoFisher Scientific.

Example 1 construction of adenovirus plasmid pAd5F11p-EF1a carrying fusion of fiber Gene (F5-11p) and human EF1a promoter

The construction process of adenovirus plasmid containing human EF1a promoter in E1 region using the backbone plasmid pAdEasyf11p carrying F5-11p fusion gene is shown in FIG. 1.

The CMV promoter of the pShuttle-CMV plasmid was first replaced with the human EF1a promoter. Firstly, taking pShuttle plasmid as a template, and taking 1411Sh5EF1aF 1: ccggtgtaca caggaagtga caat and 1411Sh5EF1aR 1: cttttgtatg aattactcga cgtcagtatt acgcgctatg agtaacacaa is used as a primer, the PCR amplification is carried out to obtain a 181bp product which starts from an upstream BsrGI site and contains a HAdV-5 packaging signal (ES); and taking pLVX-EF1a-Tet3G plasmid as a template, 1411Sh5EF1aF 2: cgcgtaatac tgacgtcgag taattcatac aaaaggactc gc and 1411Sh5EF1aR 2: acggtacctc acgacacctg aaatggaaga a is used as a primer, a 1360bp product is obtained by PCR amplification, and the product contains a human EF1a promoter; then 1411Sh5EF1aF 3: ttccatttca ggtgtcgtga ggtaccgtcg acgcggccgc acgcgttcta and 1411Sh5EF1aR 3: the ggccgatatc ttagctagca agcttaggtc tagaacgcgt gcggccgcgt oligonucleotide annealed and extended by itself to obtain 80bp double-stranded DNA product, which mainly contains multiple enzyme cutting sites (used as multiple cloning sites for constructing shuttle plasmid). The 181, 1360 and 80bp products are mixed to be used as a template, 1411Sh5EF1aF1 and 1411Sh5EF1aR3 are used as primers, and PCR is overlapped and extended to obtain a fused product 1558bp of the three products, and the product is subjected to electrophoretic recovery and named as ES-EF1 ap-MCS. The pShuttle-CMV plasmid (generating 6678bp and 792bp fragments) is double digested by BsrGI/EcoRV, and the 6678bp fragment is recovered by electrophoresis; carrying out double digestion on the ES-EF1ap-MCS fragment by using BsrGI/EcoRV, and carrying out electrophoresis to recover a 1546bp fragment; 2, transforming TOP10 competent cells of Escherichia coli, coating LB plate containing kanamycin, liquid culturing positive colony, extracting plasmid, and obtaining shuttle plasmid named pSh5-EF1 a. Coli BJ5183 strain was co-electroporated with the backbone plasmid pAdEasyf11p using PacI linearized pSh5-EF1a, and selected to obtain adenovirus plasmid pAd5f11p-EF1a, according to the AdEasy adenovirus vector systems instructions (Clontech). pAd5f11p-EF1a is an adenovirus plasmid without the gene of interest.

Example 2 removal of the original PmeI site of pAd5f11p-EF1a adenovirus plasmid

The process for constructing the F5-11p fusion gene and the PmeI site mutated from gtttaaac to gtttaaat integrated into the same adenovirus plasmid is shown in FIG. 2.

In the early constructed adenovirus plasmid pAd5GXP^[10]，HAThe original PmeI site (gtttaaac) of the dV-5 genome is artificially mutated into gtttaaat, and the mutation is synonymous mutation and does not influence the amino acid sequence coded by a virus gene containing the site. The original PmeI site of the HAdV-5 genome is removed by mutation, and the site is reserved for cloning of a foreign gene. BamHI enzyme digestion pAd5F11p-EF1a plasmid, recycling 10999bp fragment containing F5-11p gene; BamHI enzyme digestion pAd5GXP, calf alkaline phosphatase (CIP) treatment, 5' P removal, electrophoresis recovery of 24694 bp fragment; and connecting the 2 fragments, and identifying the forward and reverse directions of the product plasmid to obtain the forward connected plasmid pAd5f11 p-CGXP. In the adenovirus plasmid pAd5F11p-CGXP, the F5-11p fusion gene combines the two properties of the mutated PmeI site.

Example 3 introduction of PmeI site into cloning site of foreign Gene in adenovirus E1 region

Further modification of the cloning site for the foreign gene in the E1 region of adenovirus is shown in FIG. 3. To facilitate molecular biology procedures such as site-directed mutagenesis, a smaller plasmid was isolated from the pAd5f11p-EF1a adenovirus plasmid constructed in example 1.

The plasmid pAd5f11p-EF1a was digested with BstZ17I, and a 7210bp fragment containing the cloning site of the foreign gene was recovered by electrophoresis. To facilitate later recovery of the engineered fragment, 2 oligonucleotide primers were designed and synthesized, 1805F11p-EF1PmeA 1: gctgtccgtg tccccgtata cagacttgag aggcctgtcg aattcccaaa ataaggta and 1805F11p-EF1PmeA 2: gccgcatagt taagccagta tacggatcct taattaacat catcaataat ataccttatt ttgggaattc gac, annealing the pair of primers by themselves and extending to obtain a double-stranded DNA product of 109 bp; the 7210bp and 109bp fragments were mixed and DNA assembly was carried out to obtain pKAD5-EF1aBstZ17I plasmid (kanamycin resistance). Finally, a 7233bp fragment (original multiple cloning site is removed) is recovered after electrophoresis by using pKAD5-EF1aBstZ17I plasmid obtained by KpnI/EcoRV double enzyme; and the synthetic single-stranded oligonucleotide 1805F11p-EF1PmeA 3: cttccatttc aggtgtcgtg aggtaccgtt taaacatccg atccaccgga tctagata (58nt) were subjected to DNA assembly to obtain pKAD5-EF1aBP plasmid. pKAD5-EF1aBP contains a cloning site of an exogenous gene (the position of the original E1 region), and a PmeI site is introduced into the downstream of an EF1a promoter; and two homologous overlapping regions are added on two sides of the original pAd5f11p-EF1a plasmid BstZ17I enzyme cutting fragment so as to facilitate the subsequent recovery process to adopt DNA assembly operation.

Example 4 construction of adenovirus plasmid pKAD5f11p-EF1aP for cloning foreign Gene

The process for constructing adenovirus plasmid pKAD5f11p-EF1aP for cloning foreign gene is shown in FIG. 4.

BstZ17I/PacI double enzyme digestion example 2 constructed pAd5f11p-CGXP, electrophoresis after gel cutting recovery 26781bp fragment; EcoRI is used for carrying out enzyme digestion linearization on the pKAD5-EF1aBP plasmid constructed in the embodiment 3, and a 7242bp fragment is recovered; the two fragments were mixed and DNA assembly was carried out to obtain adenovirus plasmid pKAD5f11p-EF1aP (SEQ ID NO: 1). The sequence on both sides of the PmeI site is closely related to the construction method of the recombinant virus described later and is the main selection range of homologous overlapping regions, and the specific sequence conditions of 40bp on both sides are shown in FIG. 4. The restriction enzyme identification map of the pKAD5f11p-EF1aP plasmid is shown in FIG. 5, and the size of the fragment obtained after restriction enzyme is the same as the expected size, which indicates that the construction of the pKAD5f11p-EF1aP plasmid is successful.

Through the molecular biology operations of the embodiments 1 to 4, the obtained adenovirus plasmid pKAD5f11p-EF1aP has the following characteristics: (1) linearization of this with PacI gave 2 fragments, one containing the plasmid backbone, containing the kanamycin resistance gene and the origin of replication of the plasmid, and the other containing the HAdV-5 genome deleted in the E1/E3 region; (2) the part of the original E1 genome contains an EF1a promoter and an SV40 polyA tailing signal, and 1 PmeI single enzyme cutting site (GTTTAAAC) is added between the promoter and the SV40 polyA tailing signal; (3) the fiber gene of the original HAdV-5 was replaced by the F5-11p fusion gene.

Example 5 introduction of exogenous Gene of interest into PmeI site of pKAD5f11p-EF1aP adenovirus plasmid Using GFP as example

A method for cloning a foreign gene into pKAD5f11p-EF1aP adenovirus plasmid is exemplified. The construction of the cloning of the GFP gene into the adenovirus plasmid is schematically shown in FIG. 6.

2 primers were designed and synthesized, 1805F11p-EF1PmeGFP 1:aggtgtcgtg aggtaccgtt taaaccatgg tgagcaaggg cg and 1805F11p-EF1PmeGFP 2:atccggtgga tcggatgttt aaacaagctt tagagtccgg acttgtacag ct, 5' ends of the pair of primers (underlined)Marker part) is identical to the sequence at both ends of the PmeI plasmid pKAD5f11p-EF1aP (see FIG. 4), which is called the homologous overlap region, and the 3' end is capable of binding to the template to be amplified. Using pAd5GXP plasmid carrying GFP gene as template, and amplifying the primer to obtain GFP CDS fragment (length 784 bp); PmeI enzyme digestion pKED 5f11p-EF1aP plasmid, and recovery of linearized 33964bp fragment; mixing the 2 segments, and carrying out DNA assembly reaction to obtain the adenovirus plasmid pKAD5f11 p-EPG. Randomly selected 5 plasmid clones were identified using HindIII restriction (fig. 7), and the restriction pattern was consistent with expectations, indicating that these 5 plasmids were all positive plasmid clones with correct GFP insertion. In the pKAD5f11p-EPG plasmid, the GFP coding region was introduced between the EF1a promoter and SV40 polyA to become an expressible target gene. In this example, the PmeI sites flanking the GFP-encoding region were retained, and could be removed or altered to other sequences during the actual construction procedure. In the case of not reserving the PmeI site, in order to ensure the efficiency of DNA assembly reaction, the homologous overlapping region of the primer may be extended by several bases toward both sides when designing the synthetic primer, so that the melting temperature (Tm value) of the homologous overlapping region satisfies the requirement of DNA assembly.

Example 6 rescue and identification of recombinant Virus HAdV5f11p-EPG

PacI enzyme digestion of pKAD5f11p-EPG plasmid obtained in example 5, ethanol precipitation recovery of DNA, use of liposome transfection 293 cells, 37 ℃ culture for 3-7 days, day 5 visible GFP positive cells form fluorescence foci (figure 8), day 7 visible rescued virus in monolayer cells form plaque, fluorescence microscope visible GFP high efficiency expression. The above results indicate that the recombinant adenovirus (named HAdV5f11p-EPG) was successfully rescued. The cells with multiple plaques were frozen and thawed 3 times together with the culture medium, centrifuged at low speed, and the supernatant (about 5ml) was collected and frozen in a freezer at-80 ℃ as seed virus HAdV5f11 p-EPG. After the seed virus was grown in 293 cells, it was purified by the general cesium chloride ultracentrifugation method to obtain a purified virus. Viral genome DNA is extracted by using a Hirt method, then HindIII and EcoRV are selected to carry out enzyme digestion identification on the genome DNA (figure 9), the size of the enzyme digestion fragment is consistent with that predicted, and the result shows that the obtained recombinant virus is HAdV5f11 p-EPG. Purified HAdV5f11p-EPG was used to infect A549 cells at an infection intensity of 200vp/cell (200viral particle per cell); after 2 days of culture, fluorescence microscopy showed that more than 90% of the cells highly expressed GFP protein (FIG. 10), indicating that the HAdV5f11p-EPG recombinant virus had higher gene transfer efficiency.

Example 7 construction of adenovirus plasmid pKAD5f11pES-PmeI for cloning foreign Gene

The pKAD5f11p-EF1aP can be used for cloning a single target gene and preparing recombinant adenovirus of which the EF1a promoter controls the expression of the single target gene. When it is desired to select another promoter or to carry multiple gene expression cassettes simultaneously, the EF1a promoter of pKAD5f11p-EF1aP is an unnecessary element, and it is necessary to construct an adenovirus plasmid for cloning a foreign gene that does not contain a predetermined promoter. FIG. 11 is a schematic diagram of the construction of adenovirus plasmid pKAD5f11pES-PmeI meeting such requirements.

The plasmid pKAD5-EF1aBstZ17I constructed in example 3 was further modified. The AatII/EcoRV double-digested plasmid pKAD5-EF1aBstZ17I, recovered 5892bp fragment (EF 1a promoter and multiple cloning site removed), and synthetic single-stranded oligonucleotide 1805F11 p-ESPmeA: tgttactcat agcgcgtaat actgacgtcg tttaaacgat atccgatcca ccggatctag ata to obtain plasmid pKAD5 ES-BP. This plasmid does not contain the EF1a promoter and multiple cloning site, and adds a PmeI site (GTTTAAAC) downstream of the adenovirus packaging signal (ES). Cutting pKAD5ES-BP plasmid with EcoRI, and recovering 5904BP band; BstZ17I/PacI double enzyme digestion pAd5F11p-CGXP plasmid, electrophoresis recovery containing F5-11p gene 26781bp fragment; the above 2 fragments were mixed and DNA assembly was carried out to obtain pKAD5f11pES-PmeI (SEQ ID NO: 2). Since the sequences flanking the PmeI site are closely related to the recombinant virus construction method described below, they are the main selection region for the homologous overlapping region, and the specific sequence of 40bp flanking each of them is further shown in FIG. 11. The restriction enzyme identification map of the obtained pKAD5f11pES-PmeI plasmid EcoRV is shown in figure 12, and the restriction enzyme fragment is consistent with the expectation, which indicates the success of plasmid construction. The pKAD5f11pES-PmeI plasmid has the same characteristics as pKAD5f11p-EF1aP, except that it does not contain the EF1a promoter.

Example 8 construction of plasmid pLVX-TRE3G-TetGFP containing multiple Gene expression cassettes

To illustrate that pKAD5f11pES-PmeI plasmid can be used to carry multiple gene expression cassettes, the inventors first constructed a plasmid carrying 2 gene expression cassettes. The functional coupling of the two expression cassettes can ensure that the expression of the target protein GFP can be artificially regulated, which is convenient for verifying that the two expression cassettes can normally work. FIG. 13 is a schematic diagram of the construction of plasmid pLVX-TRE3G-TetGFP carrying the double gene expression cassette.

Tet-on3G is a commonly used induction regulation expression system, and needs to express an exogenous transcription factor Tet-on3G in a target cell, and meanwhile, the target cell contains a target gene expression frame controlled by a TRE3G promoter; when doxycycline (doxycycline, Dox) is added into a culture medium, Dox entering a target cell is combined with Tet-on3G protein, so that Tet-on3G is converted into a transcription activator of a TRE3G promoter, and transcription and expression of a target gene are started, and thus, the expression of the target protein can be regulated and controlled by adding or not adding Dox. The Tet-on3G and the protein of interest are often expressed separately using two retroviral vectors, which in the present example are carried in a single adenoviral vector in combination. For this purpose, a plasmid pLVX-TRE3G-TetGFP carrying a double gene expression cassette was constructed.

pLVX-EF1a-Tet3G plasmid is taken as a template, and 1306TetonP 1: ggccctgcag cccaagctta ccatgtctag actggacaag agc and 1306TetonP 2: gattgttcca gacgcgcccc gttatccagg gagcatgtca aggtc is used as a primer, and 790bp of fragments containing a Tet-on3G coding region are amplified by PCR; pLVX-EF1a-Tet3G plasmid is taken as a template, and 1306TetonP 3: gaccttgaca tgctccctgg ataacggggc gcgtctggaa caatc and 1306TetonP 4: ggccctgcag aattaattcc aggcgggg is used as a primer, and 652bp of a fragment containing WPRE element is amplified by PCR; the two products are mixed, 1306Teton P1 and 1306Teton P4 are used as primers, and 1397bp fragment TET-WRPE is obtained by overlap extension PCR amplification. Digesting a PCR product by PstI enzyme, and recovering a 1383bp fragment; carrying out enzyme digestion on the pLVX-TRE3G plasmid by PstI, carrying out CIP treatment, and carrying out electrophoresis to recover a 6583bp fragment; the above 2 fragments were ligated to obtain plasmid pLVX-TRE 3G-Teton. Using pAd5GXP plasmid as a template, and adopting 1306TET-GFPF 1: ggccggatcc gccaccatgg tgagcaaggg and 1306TET-GFPR 1: ggccgaattc ttagagtccg gacttgtaca gctc is used as a primer, and 755bp tet-GFP fragment containing a GFP coding frame is obtained by PCR amplification; the tet-GFP fragment was double digested with BamHI/EcoRI and cloned into the BamHI/EcoRI sites of pLVX-TRE3G-Teton, yielding pLVX-TRE3G-TetGFP plasmid. Both the GFP expression cassette under the control of the TRE3G promoter and the Tet-on3G expression cassette under the control of the PGK promoter were contained in the pLVX-TRE3G-TetGFP plasmid (FIG. 13).

Example 9 cloning of foreign genes into pKAD5f11pES-PmeI plasmid Using PCR method

Using a method similar to that of example 5, a plurality of foreign genes can be cloned into pKAD5f11pES-PmeI plasmid together. FIG. 14 is a schematic diagram showing the construction of cloning of GFP and its expression control elements into an adenovirus plasmid by PCR amplification.

2 primers were designed and synthesized, 1805F11p-TGteton 1:tcatagcgcg taatactgac gtcgtttaaa cttccagacg cgccccgtta t and 1805F11p-TGteton 2:tctagatccg gtggatcgga tatcgtttaa acgatgaggc cctttcgtct tcact, similar to example 5, the underlined parts are homologous overlapping regions (see FIG. 11). Firstly, taking pLVX-TRE3G-TetGFP plasmid as a template, and 1805F11p-TGteton1 and 1805F11p-TGteton2 as primers, and carrying out PCR amplification to obtain a 2471bp TG-teton fragment, wherein the fragment contains GFP and a Tet-on3G expression cassette at the same time. PmeI enzyme cutting pKAD5f11pES-PmeI adenovirus plasmid, recovering 32626bp fragment, mixing with PCR product, and carrying out DNA assembly to obtain adenovirus plasmid pKAD5f11p-TGFPT simultaneously containing GFP and Tet-on3G expression frame. Randomly selecting 5 plasmid clones for enzyme digestion identification, wherein the enzyme digestion fragments are consistent with the expected fragments, and the results show that the obtained plasmids are all TG-teton correctly inserted pKAD5f11p-TGFPT plasmids (FIG. 15).

Example 10 rescue and characterization of recombinant Virus HAdV5f11p-TGFPT

PacI digested pKAD5f11p-TGFPT plasmid obtained in example 9, ethanol precipitated to recover DNA, transfected 293 cells with liposome, cultured at 37 ℃ for 7 days, and the rescued virus was observed to form plaques in monolayer cells on day 7. The above results indicate that the recombinant adenovirus HAdV5f11p-TGFPT was successfully rescued. The cells with multiple plaques and the culture solution are frozen and thawed for 3 times, centrifuged at low speed, and the supernatant (about 5ml) is collected, subpackaged and frozen in a refrigerator at minus 80 ℃ to be used as seed viruses. After the seed virus was grown in 293 cells, it was purified by the general cesium chloride ultracentrifugation method to obtain a purified virus. Viral genomic DNA was extracted by Hirt method, and the genomic DNA was digested with BglII, EcoRV, HindIII and NdeI (FIG. 16), and the size of the fragments was consistent with that predicted, indicating that the recombinant virus was HAdV5f11 p-TGFPT. Purified HAdV5f11p-TGFPT was used to infect A549 cells at an infection intensity of 1000vp/cell, with or without addition of 1. mu.g/ml Dox to the culture medium; after 2 days of culture, more than 50% of the cells in the culture system with Dox added highly expressed GFP protein, and no GFP positive cells were observed in the culture system without Dox added (fig. 17). The results indicate that after HAdV5f11p-TGFPT infection, the expression of the target gene GFP can be regulated by adding Dox, namely the HAdV5f11p-TGFPT recombinant virus has a predetermined function.

Example 11 construction of shuttle plasmid pUC19-PM

The pKAD5f11pES-PmeI plasmid can be used for inserting a plurality of target genes and control sequences, and when the sequences are difficult to be introduced together by using PCR alone, it is very beneficial if a shuttle plasmid with intermediate transition function is available: thus, the fragments can be cloned into shuttle plasmid and then transferred into adenovirus plasmid once again. In order to meet such requirements, the inventors designed and constructed a shuttle plasmid pUC19-PM capable of being used with pKAD5f11pES-PmeI plasmid. FIG. 18 is a schematic diagram of the construction thereof, with the homologous overlap region and the multiple cloning site shown below the figure.

Firstly, 6 single-stranded oligonucleotides are designed and synthesized,

1800PacI-MCS1：cgttgtaaaa cgacggccag tgaattttaa ttaatcatag c，

1800PacI-MCS2：ctcgaattca aacgacgtca gtattacgcg ctatgattaa ttaaaattca ctggc，

1800PacI-MCS3：ctgacgtcgt ttgaattcga gctcggtacc cggggatcct ctagagatat cgtcg，

1800PacI-MCS4：atcgtttatc gataagcttg catgcctgca ggtcgacgat atctctagag gatcc，

1800PacI-MCS5：atgcaagctt atcgataaac gatatccgat ccaccggatc tagttaatta acttg，1800PacI-MCS6：cagctatgac catgattacg ccaagttaat taactagatc cggtgg；

mixing them together, splicing into DNA fragments with the length of 190bp by using an overlap extension PCR method; EcoRI/HindIII double digestion pUC19 plasmid, recovery of 2635bp fragment, and 190bp PCR product DNA assembly, pUC19-PM (SEQ ID NO:3) was obtained. The sequence between the 2 PacI sites of this plasmid is shown below in FIG. 18, including homologous overlapping regions and multiple cloning sites that match the pKAD5f11pES-PmeI plasmid at both ends.

Example 12 cloning of foreign genes into pKAD5f11pES-PmeI plasmid by means of shuttle plasmid

Firstly, a plurality of exogenous gene segments are cloned to a multiple cloning site of a shuttle plasmid, and then a plurality of exogenous genes are cloned to an adenovirus plasmid together by using the existing homologous overlapping regions on two sides of the multiple cloning site of the shuttle plasmid and a DNA assembly method. FIG. 19 is a schematic diagram of the construction of pKAD5f11pES-PmeI plasmid in which two foreign gene expression cassettes were introduced together with the aid of pUC19-PM shuttle plasmid.

Firstly, ClaI/KpnI is used for double enzyme digestion of the pLVX-TRE3G-TetGFP plasmid constructed in the example 8, GFP and Tet-on3G expression frames (3140bp) are cut off, and fragments are recovered through electrophoresis; the fragment was cloned to ClaI/KpnI site of the shuttle plasmid by the enzyme digestion-ligation method to obtain a shuttle plasmid pUC19-PTGFPW carrying the target gene. In this example, the inventors cloned 2 expression cassettes into a shuttle plasmid by one-time enzyme digestion-ligation; however, it will be appreciated by those skilled in the art that multiple genes or regulatory elements of interest may alternatively be cloned sequentially into the multiple cloning site of the shuttle plasmid. PacI enzyme cuts pUC19-PTGFPW plasmid, and after electrophoresis, a fragment (3217bp) containing the exogenous gene is recovered; the PmeI enzyme cuts the linearized pKAD5f11pES-PmeI plasmid, recovers 32626bp segment, and carries out DNA assembly with 3217bp segment to obtain adenovirus plasmid pKAD5f11p-TGFPW carrying exogenous gene. The randomly selected 5 plasmid clones were subjected to enzyme digestion identification, and the obtained fragments were consistent with those expected, and the results indicated that the obtained plasmids were all pKAD5f11p-TGFPW plasmids with the correct insertion of foreign genes (FIG. 20). pKAD5f11p-TGFPW differs from the adenovirus plasmid pKAD5f11p-TGFPT constructed in example 9 only in that: pKAD5f11p-TGFPW adds a WPRE element downstream of the Tet-on3G coding region.

Example 13 rescue and characterization of recombinant Virus HAdV5f11p-TGFPW

PacI digested pKAD5f11p-TGFPW plasmid obtained in example 12, ethanol precipitated to recover DNA, transfected 293 cells with liposome, cultured at 37 ℃ for 7 days, and the rescued virus was observed to form plaques in monolayer cells on day 7. The above results demonstrate that the recombinant adenovirus (named HAdV5f11p-TGFPW) was successfully rescued. The cells with multiple plaques and the culture solution are frozen and thawed for 3 times, centrifuged at low speed, and the supernatant (about 5ml) is collected, subpackaged and frozen in a refrigerator at minus 80 ℃ to be used as seed viruses. After the seed virus was grown in 293 cells, it was purified by the general cesium chloride ultracentrifugation method to obtain a purified virus. Viral genomic DNA was extracted by Hirt method, and the genomic DNA was digested with BglII, EcoRV, HindIII and NdeI (FIG. 21), and the size of the fragments was consistent with that predicted, indicating that the recombinant virus was HAdV5f11 p-TGFPW. Purified HAdV5f11p-TGFPW was used to infect A549 cells at an infection intensity of 1000vp/cell, with or without addition of 1. mu.g/ml Dox to the culture medium; after 2 days of culture, fluorescence microscopy showed that more than 50% of the cells in the culture system with Dox added highly expressed GFP protein, and no GFP positive cells were observed in the culture system without Dox added (fig. 22). The results indicate that after HAdV5f11p-TGFPW infection, the expression of the target gene GFP can be regulated by adding Dox, namely the HAdV5f11p-TGFPW recombinant virus has a predetermined function.

It should be understood that while the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein, and any combination of the various embodiments may be made therein without departing from the spirit and scope of the present invention as defined by the following claims.

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Claims (5)

1. A set of adenovirus vector system, which comprises two adenovirus plasmids pKAD5f11p-EF1aP and pKAD5f11pES-PmeI and a shuttle plasmid pUC 19-PM; adenovirus plasmid pKAD5F11p-EF1aP contains E1/E3 deleted human adenovirus type 5 HAdV-5 genome, HAdV-5 genome original HAdV-5fiber gene is replaced by fusion gene F5-11p of HAdV-5, HAdV-11p fiber, F5-11p is composed of tail domain of HAdV-5fiber and shaft and knob domain coding regions of HAdV-11p fiber, original PmeI site of HAdV-5 genome is removed by mutation, human EF1a promoter and SV 40A tailing signal are added to original HAdV-5E1 position, and a single restriction endonuclease PmeI site is contained between human EF1a promoter and SV40 polyA tailing signal, and the plasmid replication origin, kanamycin resistance gene required for plasmid replication in cells are also contained; the adenovirus plasmid pKAD5f11pES-PmeI does not contain a human EF1a promoter, the other components are completely the same as pKAD5f11p-EF1aP, and the single restriction enzyme PmeI restriction site is positioned behind the adenovirus packaging signal ES; the shuttle plasmid pUC19-PM contained the following characteristics: contains a multiple cloning site, which consists of restriction endonuclease EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, EcoRV, SalI, PstI, SphI, HindIII, ClaI and EcoRV cutting sites in sequence, wherein the upstream and downstream of the multiple cloning site respectively contain a homologous overlapping region which is consistent with the sequences at both sides of the PmeI site of the pKAD5f11pES-PmeI plasmid, the outside of the homologous overlapping region respectively contains a PacI cutting site, and the replication origin of pBR322 required for plasmid replication in prokaryotic cells and an ampicillin resistance gene.

2. A method of constructing a recombinant adenovirus, the method comprising: cutting the adenovirus plasmid pKAD5f11p-EF1aP or pKAD5f11pES-PmeI described in claim 1 from the insertion site of the foreign gene by using a restriction enzyme PmeI, and recovering linearized adenovirus plasmid DNA; carrying out PCR or enzyme digestion to obtain linear DNA containing a target gene; mixing the two sections of linear DNA, carrying out Gibson assembly reaction, transforming escherichia coli competent cells by reaction products, and screening to obtain the adenovirus plasmid containing the target gene.

3. The method of claim 2, wherein the linear DNA comprising the gene of interest is characterized by: the two ends of the DNA respectively contain a homologous overlapping region which is consistent with the sequences at the two sides of the insertion part of the exogenous gene of the adenovirus plasmid pKAD5f11p-EF1aP or pKAD5f11 pES-PmeI; the homologous overlapping region can meet the requirement of Gibson assembly reaction, and the length is 15-60 bp.

4. An adenovirus vector construction kit comprising the adenovirus vector system of claim 1.

5. Use of an adenoviral vector system or a recombinant adenoviral construction method according to any one of claims 1-3 in the preparation of an adenoviral vector construction kit, a gene therapy kit or a recombinant vaccine.

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