CN105567736B - Construction and application of E3-deleted region replication complete type recombinant adenovirus type 4 vector system - Google Patents
Construction and application of E3-deleted region replication complete type recombinant adenovirus type 4 vector system Download PDFInfo
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Abstract
The invention relates to construction and application of a complete replication type recombinant adenovirus type 4 vector system with a deletion E3 region. The system provided by the invention comprises a skeleton plasmid, a shuttle plasmid and a packaging cell line, wherein the skeleton plasmid contains a fragment of 1-26569bp on the left side of an adenovirus AdV4 genome, the adenovirus AdV4 genome has a deletion gene, and the deletion gene is an E3 region gene; the shuttle plasmid contains a cloning site for inserting a target gene and an adenovirus AdV4 genome right inverted repeat sequence; the packaging cell line is HEK-293 cell line. The human adenovirus AdV4 genome is safe and reliable, so that it is expected to be used as gene therapy and recombinant vaccine in the infected host. Namely, the recombinant AdV4 adenovirus generated by the invention has safe and wide application.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to construction and application of a replication-complete recombinant adenovirus type 4 (AdV4) vector system with a deleted E3 region in the fields of gene therapy and recombinant vaccines.
Technical Field
Since the adenovirus type 5 recombination system has been successfully constructed in 1998, it has been widely used in various fields such as in vitro gene transfer, in vivo vaccination, and gene therapy, due to the advantages of adenovirus type 5 itself and the convenience of use of the system. However, adenovirus type 5 has certain harm to human body, can not be directly used as a live vector vaccine, and can only be prepared into a replication-defective vaccine (lacking E1 region), so that only transient and limited antigen expression can be generated in clinical use, and finally generated antibodies are correspondingly insufficient.
Research in recent years also finds that the application effect of adenovirus type 5 vectors is greatly reduced by pre-existing immunity against adenovirus type 5 in organisms, epidemiological investigation shows that the human body is infected with adenovirus type 5 quite commonly, and the seropositivity of adenovirus type 5 is about 60-70% in western developed countries; in Africa and southwest Asia, the adenovirus type 5 seropositive rate is as high as 98%; in China, the adenovirus type 5 seropositive rate also reaches 72 percent, and the situation makes the use of the adenovirus type 5 virus vector more obstructed.
Adenovirus type 4 (AdV4) belongs to the only serotype in the E subgroup of human adenovirus, and the virus has been widely used as a live vector vaccine by the military in the early 70 s for the prevention of ARD, so that respiratory diseases related to adenovirus are effectively restrained, and no adverse reaction is seen after immunization. In 1999, after the supply of the vaccine is stopped, the detection of a training center of a new soldier shows that the respiratory infection rate is obviously increased. New AdV4-AdV7 combination vaccines were developed in the united states and approved by the FDA in 2011, again effectively reducing the incidence of respiratory infectious diseases. Thus, adenovirus type 4 (AdV4) can be prepared as a replication-competent vaccine that can replicate in vivo, resulting in sustained and abundant antigen expression and ultimately higher immunopotency of the antibody.
In addition, studies show that adenovirus type 4 (AdV4) has higher infectivity than adenovirus type 5 virus, can induce more sufficient innate immunity, and when antibodies against adenovirus type 5 exist in a body, if the adenovirus type 4 is used for primary immunity and then adenovirus type 5 is used for boosting immunity, more specific cytokines and T lymphocytes against antigens are generated, and the result shows that the combined use of adenovirus type 4 and adenovirus type 5 vector vaccines can achieve better effect. Epidemiological investigations in china, argentina, the united states, egypt, etc. show that adenovirus type 4 has a lower infection rate in humans than adenovirus type 5, which means that there is less pre-existing immunity against adenovirus type 4 in the body than adenovirus type 5, which makes the use of adenovirus type 4 as a vector vaccine more advantageous.
Disclosure of Invention
The invention aims to provide a replication-complete recombinant adenovirus type 4 (AdV4 △ E3Genome) vector system with a deleted E3 region and application thereof.
The vector system provided by the invention comprises 1 skeleton plasmid, 1 shuttle plasmid and 1 packaging cell line; the backbone plasmid contains a fragment of 1-26569bp on the left side of the adenovirus AdV4 genome; the shuttle plasmid contains a cloning site for inserting a target gene and an adenovirus AdV4 genome right inverted repeat sequence; the adenovirus AdV4 genome has deletion genes, and the deletion genes are E3 region genes; the packaging cell line is a HEK-293 cell line (ATCC, TMCRL-1573)。
the replication complete recombinant adenovirus AdV4 vector system of the deletion E3 area, the skeleton plasmid is pAd4FAST, and the skeleton plasmid pAd4FAST sequentially contains a replication origin of a pBR322 plasmid, an ampicillin resistance gene and an AdV4 genome 1-26569 bp.
The replication-complete recombinant adenovirus AdV4 vector system with the deletion of the E3 area comprises a Shuttle plasmid pAd4FAST-Shuttle, wherein the Shuttle plasmid sequentially comprises a replication origin of a PBR322 plasmid, a resistance gene of kanamycin, a right inverted repeat sequence of an AdV4 genome, and a right arm and a left arm (Rarm and Larm) for homologous recombination with a framework plasmid pAd4 FAST.
The replication-complete recombinant adenovirus AdV4 vector system with the deletion of the E3 region comprises a framework plasmid pAd4FAST, wherein a restriction enzyme PacI site is added at the left end of an AdV4 genome, and a restriction enzyme SpeI site is added at the right end.
The replication-complete recombinant adenovirus AdV4 vector system with the deletion of the E3 area comprises a recognition site ATTTAAAT of a restriction enzyme Swal between right arm (Rarm, Larm) sequences and left arm (Rarm, Larm) sequences which can be subjected to homologous recombination with framework plasmid pAd4FAST, and the right end of a right inverted repeat sequence of an AdV4 gene is added with the recognition site TTAATTAA of the restriction enzyme Pacl.
The vector system is used for preparing recombinant adenovirus.
The recombinant AdV4 adenovirus carrying target genes can be prepared by using the vector system. The target gene is cloned to the cloning site of a Shuttle plasmid pAd4FAST-Shuttle, the Shuttle plasmid carrying the target gene is converted into an escherichia coli BJ5183 strain (Stratagene company in America) containing a framework plasmid pAd4FAST after being cut and linearized by restriction enzyme SwaI, and the Shuttle plasmid pAd4FAST-Shuttle linearized in escherichia coli BJ5183 cells and the framework plasmid pAd4FAST are subjected to homologous recombination to obtain an adenovirus plasmid carrying the target gene. The plasmid is cut by restriction endonuclease PacI, a recombinant adenovirus genome is released, the recombinant adenovirus genome is transfected into a packaging cell line HEK-293 by a DNA transfection method, and the recombinant AdV4 adenovirus carrying a target gene is rescued in the packaging cell line. The small amount of adenovirus obtained initially can be further amplified by packaging cells to prepare a large amount of recombinant virus.
In the prepared recombinant adenovirus, except that the genome contains a part of exogenous sequence (target gene expression cassette), the rest part of the genome and all structural proteins of the virus are derived from the human adenovirus type 4 vaccine strain (GenBank accession number: AY 594254).
The human adenovirus AdV4 genome is safe and reliable, so that the human adenovirus AdV4 genome is expected to be used as gene therapy and recombinant vaccine in an infected host.
Drawings
FIG. 1 is the restriction enzyme identification diagram of the backbone plasmid pAd4 FAST; in the figure, 1 represents λ -EcoT 14I digest DNAmarker; 2 represents pAd4FAST plasmid after PacI and SpeI enzyme digestion; 3 represents pAd4FAST plasmid;
FIG. 2 is the sequencing identification diagram of backbone plasmid pAd4 FAST; in the figure, the horizontal line in PacI site shows the cloning marker site PacI; the middle horizontal line of the SpeI site shows the cloning marker site SpeI; the horizontal line in the AseI site shows the cloning marker site AseI;
FIG. 3 is a schematic diagram of the construction of backbone plasmid pAd4 FAST;
FIG. 4 is a restriction enzyme identification map of the vector pShuttle-1; in the figure, 1 represents DL15000 DNA Marker; 2 represents a pShuttle-1 vector; 3-5 sequentially represent pShuttle-1 vectors after enzyme digestion by PacI, SwaI and SpeI; 6 represents 250bp ladder DNA Marker;
FIG. 5 is a schematic diagram of the construction of Shuttle plasmid pAd4 FAST-Shuttle;
FIG. 6 is the restriction enzyme identification map of Shuttle plasmid pAd4 FAST-Shuttle; in the figure, 1 denotes DL15000 DNAmarker; 2 denotes pAd4FAST-Shuttle vector; 3-5 represent pAd4FAST-Shuttle vector after enzyme digestion by PacI, SwaI and SpeI in sequence;
FIG. 7 is the restriction enzyme identification diagram of plasmid pAd4FAST-Shuttle-EGFP carrying the target gene; in the figure, 1 represents DL15000 DNA Marker; 2 represents pAd4FAST-Shuttle-EGFP vector; 3 represents pAd4FAST-Shuttle-EGFP (counterclockwise cloning of EGFP) vector after SwaI and SpeI enzyme digestion; 4 represents pAd4FAST-Shuttle-EGFP (EGFP clockwise cloning) vector after SwaI and SpeI enzyme digestion;
FIG. 8 is the restriction enzyme identification map of recombinant plasmid pAd4 FAST-EGFP;
FIG. 9 is a diagram showing the recombination of the backbone plasmid pAd4FAST and Shuttle plasmid pAd4FAST-Shuttle-EGFP carrying the target gene in E.coli BJ 5183;
FIG. 10 is a graph showing the cytopathic effect (CPE) and fluorescence expression of the rescued P3 generation AdV4-EGFP virus; in the figure, the row corresponding to a shows the pathological conditions of the P3 generation AdV4-EGFP virus at different times after infection of HEK-293 cells; b, the horizontal lines correspond to the expression of the fluorescent protein at different times;
FIG. 11 is a graph of the growth of a rescued AdV4-EGFP virus (■) versus the parental virus AdV4 △ E3(◆).
Detailed Description
In one embodiment of the invention, the backbone plasmid of the invention comprises a fragment of 1-26569bp to the left of the adenovirus AdV4 genome.
In a specific embodiment, the backbone plasmid is pAd4FAST, and contains the replication origin of the pBR322 plasmid, the ampicillin resistance gene, and a fragment of 1-26569bp to the left of the AdV4 adenovirus genome.
In a specific embodiment, the backbone plasmid, pAd4FAST, adds a restriction enzyme, PacI, site at the left end and a SpeI site at the right end of the AdV4 genome.
In one embodiment of the present invention, the Shuttle plasmid of the present invention is pAd4FAST-Shuttle, which comprises the replication origin (Ori) of the pBR322 plasmid, the resistance gene for kanamycin (Kan), the right inverted repeat (ITR) of the AdV4 genome, the right and left arms (R arm, L arm) for homologous recombination with the backbone plasmid pAd4FAST (the fragment position of L arm on the genome of AdV4(GenBank accession: AY 594254): Int-1000nt, and the fragment position of R arm on the genome of AdV4(GenBank accession: AY 594254): 25833nt-27476nt) in this order.
In a specific embodiment of the present invention, the Shuttle plasmid, pAd4FAST-Shuttle, contains a recognition site for the restriction enzyme SwaI (ATTTAAAT) between the left and right arms capable of undergoing homologous recombination with the backbone plasmid, pAd4FAST, to which a recognition site for the restriction enzyme PacI (TTAATTAA) is added at the right end of the right inverted repeat of the AdV4 genome.
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
1. Construction of backbone plasmid pAd4FAST and preparation of electrotransformation competent cells
(1) Construction of backbone plasmid pAd4 FAST:
① using pAdeasy-1 vector (Stratagene, USA, GenBank accession number AY370909) as template, using primers P1: CCTTAATTAA(PacI) CATGC (SEQ ID NO:1) and P2: GGACTAGA (SpeI) TCTAGTTTCG (SEQ ID NO:2) to amplify the fragment (fragment size is 3878bp) with position 27246-31123bp, the fragment contains pBR322 replication origin, ampicillin resistance gene and other vector basic components, the product is digested by PacI and SpeI, purified and recovered by DNA gel recovery kit, obtaining product 1;
amplification was performed in a standard Polymerase Chain Reaction (PCR) system, 50. mu.l: 5 XPCR buffer 10uL, template 10ng, 10mM deoxyribonucleoside triphosphate (dNTP)1 uL, upstream and downstream primers (25 uM) 1 uL each, high fidelity thermostable DNA polymerase 1 unit, ddH2O is complemented; the reaction conditions are as follows: pre-denaturation at 98 ℃ for 2min, amplification for 1 cycle, denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 15s, extension at 68 ℃ for 10min, filling the tail end, and amplification for 30 cycles;
② using the virus genome (human adenovirus type 4 vaccine strain, GenBank accession number: AY594254) as the template, amplifying the 1-13666bp fragment with primers P3: CCTTAATTAA(PacI) CATCATCAATAATATAC (SEQ ID NO:3) and P4: GGACTAGTAACACGCTGCTGT (SEQ ID NO:4), using PacI and AseI to carry out enzyme digestion, purifying and recovering the fragments by a DNA gel recovery kit to obtain a product 2, using primers P5: GGACTAGTCTTCACGGACAGCGGT (SEQ ID NO:5) and P6: GGACTAGT (SpeI) GAATTTCCTGGTACAC (SEQ ID NO:6) to amplify the 12686-26569bp fragment of the virus genome, using AseI and SpeI to carry out enzyme digestion, purifying and recovering the fragments by the DNA gel recovery kit to obtain a product 3, wherein the AseI site used by the product 2 and the product 3 is located between 12686bp and 13666bp of the virus genome;
③ product 1 from step ① was ligated together with products 2 and 3 from step ② in 10. mu.l ligation system of 10 Xligation buffer (1. mu.l), 2. mu.l each of 3 fragments, 1. mu. l T4 DNA ligase (NEB, USA, 400U/. mu.l), ddH2Reacting O2 mu l at 16 ℃ for 12 hours to obtain a connecting product A;
④ the ligation product A obtained in step ③ is transformed into DH5 α competent cells, the concrete operation steps of the transformation are that 5 mul ligation product A is added into 100 mul DH5 α competent cells, the cells are mixed gently, ice bath is carried out for 30min, heat shock is carried out for 90s at 42 ℃, 1ml LB liquid culture medium is added, shaking is carried out at the rotating speed of 200rpm at the temperature of 37 ℃ for 1h, 200 mul is spread on an LB agar plate containing ampicillin after the shaking is finished, the cells are cultured at the temperature of 37 ℃ for 18h, a single colony is picked after the culture is finished, plasmids are extracted by shaking, enzyme digestion (figure 1) and sequencing identification (figure 2) are carried out, the framework plasmid pAd4FAST is obtained, and the construction process of the whole framework plasmid pAd4FAST is shown in figure 3.
FIG. 1 is a restriction enzyme map, wherein 1 is a lambda-EcoT 14I digest DNA Marker; FIG. 2 shows the identification result of pAd4FAST plasmid after PacI and SpeI enzyme digestion, and the construction process of the backbone plasmid pAd4FAST is shown in FIG. 3: PacI and SpeI carry out enzyme digestion on the framework plasmid to obtain two fragments, and two bands are shown in an identification picture; 3 is the identification result of pAd4FAST plasmid; in the identification process, the position has a certain deviation during electrophoresis due to the overlarge gene;
FIG. 2 is the sequencing identification diagram of the backbone plasmid pAd4 FAST.
(2) Preparation of electrotransformation competent cells:
transferring the skeleton plasmid pAd4FAST obtained in the step (1) into BJ5183 Escherichia coli, wherein the transformation method is the same as the step ④, and preparing the BJ5183 Escherichia coli containing the skeleton plasmid pAd4FAST into an electric transformation competent cell by streaking the BJ5183 Escherichia coli thallus containing the skeleton plasmid pAd4FAST, selecting a single colony, placing the single colony into 5mL of LB culture medium, shaking the colony at 37 ℃ and 200rpm overnight to obtain a bacterial liquid, inoculating the bacterial liquid into 500mL of LB liquid culture medium according to the volume ratio of the bacterial liquid to the culture liquid of 1:500, shaking the bacterial liquid at 200rpm until the OD600 is 0.8, immediately placing the bacterial liquid into an ice-water bath for 30min, then centrifuging the bacterial liquid in a 250mL precooled centrifuge cup at 4 ℃ and 4000rpm for 30min, discarding supernatant, using precooled 10% glycerol to perform isometric centrifugation at 4 ℃ and 20 rpm, repeating the previous step, collecting precipitates, suspending the cells in a glycerol suspension tube with 1mL 10% of the glycerol, and then preserving the suspended cells in an EP tube with 40. mu.l per tube at 5 ℃ and re-20 mL EP tubes to re-suspension tubes.
2. Construction of Shuttle plasmid pAd4FAST-Shuttle
(1) Construction of vector pShuttle-1:
① using virus genome (human adenovirus type 4 vaccine strain) as template, using primer P7: CCTTAATTAA(PacI) CATCATCAATAATATAC (SEQ ID NO:7) and primer P8: GGACTAGT (SpeI) GATTTAAAT(SwaI) ACGGCTTGCACTCCA (SEQ ID NO:8) to amplify the 1-1000bp segment of virus genome, using PacI and SpeI to cut enzyme, purifying and recycling through DNA gel recycling kit to obtain product 4, using primer P9: GGACTAGT (SpeI) GGGCTCGGAGCAGCGTCT (SEQ ID NO:9) and primer P10: CCTTAATTAA(PacI) CATCATCAATAATATACCTT (SEQ ID NO:10) to amplify the 27813 and 32766bp segment of virus genome, using PacI and SpeI to cut enzyme, purifying and recycling through DNA gel recycling kit to obtain product 5;
② cutting pShuttle-CMV vector (Stratagene, USA) with PacI, recovering 2922bp fragment (refer to pShuttle-CMV sequence, version 017005, fragment position: 3692-6613bp) and dephosphorizing, the fragment contains pBR322 replication origin and kanamycin resistance gene, purifying and recovering by DNA gel recovery kit to obtain product 6;
③, connecting the product 4 and the product 5 in the step ① with the product 6 in the step ② together under the same connecting conditions as in the step 1 (1) or ③ to obtain a connecting product B;
④, taking 5 μ l of the ligation product B in the step ③, transforming the ligation product B into DH5 α competent cells, specifically transforming the DH5 α competent cells in the same steps as the step 1 (1) ④ to obtain the competent cells containing the ligation product B, taking 200 μ l of DH5 α competent cells which are transformed, coating the competent cells on an LB agar plate containing kanamycin, culturing for 18 hours at 37 ℃, picking out a single colony after the culture is finished, extracting plasmids by shaking bacteria, and carrying out restriction enzyme digestion identification (figure 4) to obtain a vector pShuttle-1, wherein the construction process of the plasmid pShuttle-1 is shown in figure 5;
in FIG. 4, 2 represents the result of the identification of the vector pShuttle-1, which is about 8875bp in size; 3, the identification result of the pShuttle-1 vector after the PacI enzyme digestion is shown, the identification result can be obtained by a construction process of the pShuttle-1 shown in a figure 5, and two fragments which form the pShuttle-1 vector are obtained after the PacI enzyme digestion, wherein the size of one fragment is about 5954bp, and the size of the other fragment is about 2922 bp; 4 represents the result of identifying the pShuttle-1 vector after digestion with SwaI, 5 represents the result of identifying the pShuttle-1 vector after digestion with SpeI, as shown in FIG. 5, and the linearized vector pShuttle-1 obtained after digestion with SwaI or SpeI has a size of about 8876 bp; the results of the identification in FIG. 4 show that the corresponding product sizes all correspond to the standard size, i.e., the desired vector pShuttle-1 was obtained by ligation.
(2) Construction of Shuttle plasmid pAd4 FAST-Shuttle:
① using a virus genome (human adenovirus type 4 vaccine strain) as a template, amplifying a fragment of 25833-27476bp of the virus genome by using a primer P11: CCAAGCATTTAAAT(SwaI) GCGGGGGCACA (SEQ ID NO:11) and a primer P12: GGACTAGT (SpeI) GAACAACGGCGATTG (SEQ ID NO:12), carrying out enzyme digestion by using SwaI and SpeI, and carrying out purification and recovery by using a DNA gel recovery kit to obtain a product 7;
② ligation of product 7 and product 8 from step ① was performed under conditions (10. mu.l system) of 10 Xligation buffer 1. mu.l, 2. mu.l each of 2 fragments, 1. mu. L T4 DNA ligase (NEB, USA, 400U/. mu.l), 4. mu.l ddH2O, reacting for 12 hours at 16 ℃ to obtain a connecting product C;
③ mu.l of the ligation product C obtained in step ② is taken and transformed into DH5 α competent cells, the transformation is carried out in the same manner as in step 1, (1) ④, after the transformation is finished, 200. mu.l of DH5 α competent cells are taken and spread on LB agar plate containing kanamycin, the cells are cultured for 18h at 37 ℃, after the culture is finished, single colonies are picked, after the plasmids are extracted by shaking bacteria, the plasmids are identified by enzyme digestion (figure 6), a vector pAd4FAST-Shuttle is obtained, the construction process of the whole Shuttle plasmid vector pAd4FAST-Shuttle is shown in figure 5, the SpeI restriction enzyme cutting site in the vector pAd4FAST-Shuttle is positioned in the deletion region of E3 and is used for loading of foreign genes, and the SwaI restriction enzyme cutting site is positioned between the left and right homologous arms and is used for linearization of vectors before recombination.
In FIG. 6, 2 represents the result of the identification of the vector pAd4FAST-Shuttle, which is about 10520bp in size; 3, the identification result of the pAd4FAST-Shuttle vector after PacI enzyme digestion is used, the construction process of the pAd4FAST-Shuttle is shown in figure 5, and two fragments forming the pAd4FAST-Shuttle vector are obtained after PacI enzyme digestion, wherein the size of one fragment is about 7598bp, and the size of the other fragment is about 2922 bp; 4 represents the result of the identification of the product digested with SwaI and 5 represents the result of the identification of the product digested with SpeI, as can be seen from FIG. 5, and the linearized vector pAd4FAST-Shuttle, which is approximately 10520bp, is obtained after both SwaI and SpeI; the results of the identification in FIG. 6 show that the corresponding product sizes correspond to the standard sizes, i.e.the desired vector pAd4FAST-Shuttle is obtained by ligation.
Example 2
The embodiment is a preparation process of a recombinant AdV4 virus carrying a target gene, and concretely relates to a preparation example of a recombinant adenovirus AdV4-EGFP carrying an EGFP reporter gene, which is described as follows:
the vector system can be used for preparing recombinant AdV4 adenovirus carrying a target gene, and the target gene is cloned to a cloning site of a Shuttle plasmid pAd4 FAST-Shuttle. After being cut and linearized by restriction enzyme SwaI, shuttle plasmids carrying target genes are transformed into escherichia coli BJ5183 strains (Stratagene company, USA) carrying framework plasmids pAd4FAST, adenovirus plasmids carrying the target genes are obtained through homologous recombination in BJ5183 cells, the plasmids are cut by restriction enzyme PacI to release recombinant adenovirus genomes, the recombinant adenovirus genomes are transfected into a packaging cell line HEK-293 by utilizing a DNA transfection method, recombinant AdV4 adenoviruses carrying the target genes are obtained through rescue in the packaging cells, a small amount of adenoviruses obtained at the beginning can be further amplified by the packaging cells, and a large amount of recombinant adenoviruses are prepared.
1. Cloning of the EGFP Gene and preparation of adenovirus plasmid pAd4FAST-EGFP
(1) ① was digested with NheI and SpeI from pZJ-1-EGFP vector (SEQ ID NO:13), and a 1769bp fragment (the position of the fragment in pZJ-1-EGFP vector was 402-2170bp) was recovered, which contained the CMV-EGFP-SV40pA framework;
the synthesis process of the pZJ-1-EGFP vector comprises the following steps: firstly, artificially synthesizing a CMV-EGFP-SV40pA sequence (SEQ ID NO:14) (the upstream of the sequence sequentially contains EcoRI and NheI sites, and the downstream thereof isContaining SpeI and HindIII sites) and then is cloned on a pUC19 vector which is cut by EcoRI and HindIII after being cut by EcoRI and HindIII; specific ligation conditions were (10 μ l system): 10 Xligation buffer 1. mu.L, 2. mu.L of each of 2 fragments, 1. mu. L T4 DNA ligase (NEB Corp., USA, 400U/. mu.L), 4. mu.L ddH2And reacting at 16 ℃ for 12 hours to obtain the vector pZJ-1-EGFP.
② enzyme digestion pAd4FAST-Shuttle carrier by using SpeI and dephosphorylation to obtain a linearized pAd4FAST-Shuttle carrier;
③ connecting the fragment recovered in step ① with the linearized pAd4FAST-Shuttle vector in step ② under the same conditions as in step 2 (2) ② of example 1 to obtain a connection product D, transforming the connection product D into DH5 α competent cells under the same conditions as in step 1 (1) ④ of example 1, spreading 200. mu.l of DH5 α competent cells on an LB agar plate, culturing at 37 ℃ for 18h, picking out a single colony after the completion of the culture, and performing SpeI and SwaI enzyme digestion identification on shake bacteria extracted plasmids (FIG. 7) to obtain the pAd4FAST-Shuttle-EGFP vector;
FIG. 7 is a restriction enzyme identification diagram of the vector pAd4FAST-Shuttle-EGFP, and FIG. 2 shows pAd4FAST-Shuttle-EGFP with a size of 12289 bp; 3 is a pAd4FAST-Shuttle-EGFP vector which is cut by SwaI and SpeI under the condition of anticlockwise cloning, and the recombination process of the pAd4FAST-Shuttle in the figure 5 shows that the pAd4FAST-Shuttle-EGFP is cut by SwaI and SpeI to form two fragments, the EGFP is connected with the small fragment, the size of the EGFP is 3413bp, and the size of the other large fragment is 8876 bp; under the condition of clockwise cloning, the pAd4FAST-Shuttle-EGFP vector is obtained by the recombination process of the pAd4FAST-Shuttle in the figure 5 after enzyme digestion by SwaI and SpeI, the pAd4FAST-Shuttle-EGFP is subjected to enzyme digestion by SwaI and SpeI to form two fragments, the EGFP is connected with the large fragment, the size of the EGFP is 10645bp, and the size of the other small fragment is 1644 bp; the identification results in fig. 7 show that: the sizes of the corresponding fragments are the same as the standard sizes of the corresponding fragments, and the vector pAd4FAST-Shuttle-EGFP is obtained. It can also be derived that: EGFP clockwise cloning and EGFP anticlockwise cloning are connected with different fragment ends in two different cloning directions.
(2) Carrying out enzyme digestion linearization on the pAd4FAST-Shuttle-EGFP carrier obtained in the step (1) by using SwaI, extracting the linearized pAd4FAST-Shuttle-EGFP carrier by phenol chloroform, and dissolving the vector in deionized water to obtain a solution containing the pAd4FAST-Shuttle-EGFP carrier; 500ng of the solution was then electroporated into the electroporation competent cells prepared and stored in step 1 (2) of example 1 under the following conditions: electric shock is carried out for 5ms under the condition of 2.5 KV/cm; 200. mu.l of the electrically shocked competent cells were spread on an LB plate (containing the resistance gene Kan of 50. mu.g/ml kanamycin), and cultured at 37 ℃ for 20 hours.
(3) After the culture in the step (2) is finished, picking small colony shake bacteria to extract plasmid electrophoresis detection (figure 8A), wherein the plasmid with higher position in the figure 8A is suspicious positive plasmid, and the detection results with the labels 1, 4, 10, 11, 12 and 15 are obtained from the figure and have higher positions and are suspicious positive plasmid; selecting the 6 suspicious positive plasmids, and detecting the existence of the EGFP gene by using PCR (fig. 8B), wherein the 6 suspicious positive plasmids all contain the EGFP gene and are positive plasmids as can be seen from fig. 8B; then, the positive plasmids marked as 1, 2, 3 and 4 are further subjected to PacI enzyme digestion identification (figure 8C), and finally, an adenovirus plasmid pAd4FAST-EGFP is obtained;
the recombination process of the framework plasmid pAd4FAST and the Shuttle plasmid pAd4FAST-Shuttle-EGFP carrying the target gene in Escherichia coli BJ5183 is shown in FIG. 9.
2. Rescue of recombinant adenovirus AdV4-EGFP
(1) After the adenovirus plasmid pAd4FAST-EGFP is linearized by PacI enzyme digestion, phenol chloroform is extracted once, the plasmid is precipitated with absolute ethyl alcohol at the temperature of-20 ℃ overnight, the precipitate is centrifuged at 10000rpm for 30min, the supernatant is discarded, the precipitate is collected and washed by 75% glacial ethyl alcohol once, the precipitate is centrifuged at 10000rpm for 10min, the supernatant is discarded, the precipitate is collected and naturally dried at room temperature for 5min, 1 × TE is dissolved, and the content of the adenovirus plasmid pAd4FAST-EGFP is determined;
(2) transfection: the transfection procedure was performed in 6-well plates, seeded at 1X 10 per well6HEK-293 cells, HEK-293 cells using 10% fetal bovine serum in 1 × DMEM medium, 37 degrees 5% CO2Culturing for 18-24 hours in an incubator, and discarding the supernatant after the culture is finished; then, 2. mu.g of the adenovirus plasmid pAd4FAST-EGFP and 10ul liposome obtained by dissolving 1 XTE in step (1) were addedLipofectamine2000 and 1ml opti-MEM medium, culturing for 6 hours, and then discarding the supernatant; adding 2ml of 1 × DMEM maintenance solution containing 2% fetal calf serum, 5% CO at 37 deg.C2Culturing in an incubator, supplementing each cell well with 500 mu l of maintenance solution every 3 days, harvesting cells after 7 days, carrying out freeze-thaw lysis for 3 times, infecting HEK-293 cells again with supernatant (P0 generation virus supernatant) after centrifugation, increasing EGFP cells with the increase of the number of passage, and enabling the cultured HEK-293 cells to generate complete cytopathic effect (CPE) with the virus supernatant of P3 generation (figure 10a), and increasing the expression of fluorescent protein with the increase of time (figure 10b), which indicates that the virus is amplified.
3. Determination of growth characteristics of recombinant viruses
Firstly, recombinant virus AdV4-EGFP and parental virus AdV4 △ E3 are respectively taken to measure Plaque Forming Unit (PFU), the two viruses infect cells (HEK-293 cells) according to the MOI of 0.05, and the test is carried out in a T25 cell bottle, each bottle is inoculated with 2 multiplied by 106Inoculating the cells with the virus within 18-24 h; after inoculation is completed, three bottles are respectively taken at each time point of 12h, 24h, 36h, 48h, 60h, 72h, 84h and 96h for freezing and storing at-70 ℃, and finally TCID50 of samples at each time point is measured, and a growth curve is drawn, wherein the result is shown in figure 11; as can be seen from the growth curve of FIG. 11, the growth trends of the recombinant adenovirus and the parental virus are not very different, i.e., the growth of the adenovirus is not greatly affected after the foreign gene is cloned into the human adenovirus, so that the human adenovirus AdV4 genome is expected to be used as gene therapy and recombinant vaccine in the host which can be infected by the human adenovirus AdV4, and the safety and other properties of the human adenovirus are not affected.
Claims (4)
1. A replication-competent recombinant adenovirus AdV4 vector system lacking E3 region, comprising a backbone plasmid, a shuttle plasmid and a packaging cell line, wherein:
the backbone plasmid contains a fragment of 1-26569bp on the left side of the genome GenBank accession number AY594254 of the human adenovirus AdV 4;
the shuttle plasmid contains a cloning site for inserting a target gene and a right inverted repeat sequence of a genome GenBank accession number AY594254 of a human adenovirus AdV 4;
the genome GenBank accession number of the human adenovirus AdV4 is AY594254, and the deletion gene is an E3 region gene;
the packaging cell line is HEK-293 cell line, the cell line is from ATCC cell bank and is numbered as CRL-1573TM；
The skeleton plasmid is pAd4FAST, and the skeleton plasmid pAd4FAST sequentially contains a replication origin of a pBR322 plasmid, a resistance gene of ampicillin and a fragment of 1-26569bp on the left side of an AdV4 adenovirus genome;
the Shuttle plasmid is pAd4FAST-Shuttle, the Shuttle plasmid sequentially contains a replication origin of a PBR322 plasmid, a resistance gene of kanamycin, an inverted repeat sequence on the right side of an AdV4 genome, a cloning site for inserting a target gene, a right arm and a left arm for carrying out homologous recombination with the framework plasmid pAd4FAST, a recognition site ATTTAAAT of a restriction enzyme Swal is contained between the sequences of the right arm and the left arm which can carry out homologous recombination with the framework plasmid pAd4FAST, the target gene is cloned to the cloning site of the Shuttle plasmid pAd4FAST-Shuttle, the Shuttle plasmid carrying the target gene is linearized by restriction enzyme SwaI and then carries out homologous recombination with the framework plasmid pAd4FAST to obtain an adenovirus plasmid carrying the target gene.
2. The replication-competent recombinant adenovirus AdV4 vector system of claim 1 lacking the E3 region, wherein: in the backbone plasmid pAd4FAST, a restriction enzyme PacI site is added at the left end of the genome of AdV4, and a restriction enzyme SpeI site is added at the right end.
3. The replication-competent recombinant adenovirus AdV4 vector system of claim 2 lacking the E3 region, wherein: in the Shuttle plasmid pAd4FAST-Shuttle, a recognition site ATTTAAAT of a restriction enzyme Swal is contained between the sequences of the right arm and the left arm which can be subjected to homologous recombination with the framework plasmid pAd4FAST, and a recognition site TTAATTAA of a restriction enzyme Pacl is added at the right end of the right inverted repeat sequence of the AdV4 gene.
4. Use of a vector system according to any one of claims 1 to 3 for the preparation of a recombinant adenovirus.
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