CN103923943B - A kind of expression vector based on adenovirus AdC7 and construction method thereof - Google Patents
A kind of expression vector based on adenovirus AdC7 and construction method thereof Download PDFInfo
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Abstract
本发明涉及一种基于腺病毒AdC7的表达载体及其构建方法。本发明人设计了一种有效的策略，成功地基于黑猩猩型腺病毒AdC7构建成一种新型表达载体。所述的疫苗载体可应用于制备可高效表达且具有良好免疫原性的病毒疫苗。The present invention relates to an expression vector based on adenovirus AdC7 and a construction method thereof. The inventors designed an effective strategy and successfully constructed a novel expression vector based on the chimpanzee adenovirus AdC7. The vaccine vector can be used to prepare a virus vaccine that can be expressed efficiently and has good immunogenicity.
Description
Technical field
The invention belongs to biotechnologys and field of virology；More particularly it relates to which a kind of be based on adenovirus AdC7
Expression vector and its construction method.
Background technique
Recombinant adenoviral vector has the characteristics that high transduction efficiency, expression are high, the duration is long, is easy to purify, and
And it can induce the cellular immunity and humoral immune reaction of foreign gene specificity after being immunized, therefore be a kind of ideal vaccine load
Body.Expression vector based on 2 type of adenovirus human serotype (AdHu2) and 5 types (AdHu5) is in vaccine research and gene therapy
Once it was widely used, but since the individual of 40-60% in crowd has infected corresponding adenovirus, exists and prestore neutralization accordingly
Antibody, to limit the clinical application of AdHu2 and AdHu5 carrier.To overcome the influence for prestoring neutralizing antibody, a kind of method is to change
Make the capsid protein of AdHu2 and AdHu5 carrier.Some studies have shown thats can realize that this is thought by exchange fiber protein gene
Method.But after this embedded virus can still be neutralized by the antibody for hexon epitope, and some hexon albumen are engineered
Embedded virus it is unstable；Another method be select people's rare serotypes or from other animal reservoirs adenovirus as
Expression vector.Due to generally will be free from the neutralizing antibody of anti-vaccine carrier for chimpanzee type adenovirus in crowd, it is based on vaccine carrier for chimpanzee type gland
The vaccine carrier of virus, immune effect are significantly better than with the vaccine carrier of human serotype's adenovirus construction.Currently, being based on black orangutan
The novel vaccine carrier of orangutan type adenovirus becomes important selection in vaccine research and development.In May, 2013, Bill cover hereby foundation (Bill&
Melinda Gates Foundation) 29,000,000 dollars of research unit (company) subsidizing the U.S., Britain and Switzerland, it is used for
The research and development of novel chimpanzee adenoviral vector.
There are three ways to constructing recombinant adenoviral vector.Traditional method is that foreign gene is carried out in package cell line
It is time-consuming and laborious to obtain recombined adhenovirus, but this method is complicated for operation with the homologous recombination of wild-type virus, it is not easy to
Obtain single recombined adhenovirus；Second method is based on the homologous recombination in Escherichia coli, the disadvantage is that relatively time consuming, appearance
Easily mutate.The third method is by adenoviral gene group Direct Cloning to plasmid vector, and then linearisation transfection is packed
Cell line saves out adenovirus.Since adenoviral gene group is relatively large, size is about 36kb, is carried out to its genome direct
There are certain difficulty by clone.
Summary of the invention
The purpose of the present invention is to provide a kind of expression vector and its construction method based on adenovirus AdC7.
In the first aspect of the present invention, a kind of method for preparing adenovirus expression carrier is provided, which comprises with open country
Based on raw type adenovirus AdC7 genome (preferably, sequence such as GenBank accession number AY530878.1), deletes part E1 and compile
Code area and the whole code area E3 delete area in E1 and increase I-Ceu I and PI-Sce I restriction enzyme site, delete area in E3 and increase Rsr
II restriction enzyme site obtains the recombinant adenoviral expressing vector of replication defect type.
In a preferred embodiment, the deletion part code area E1, which refers to, deletes 458- in wild type AdC7 genome
The sequence in 3026 (the sequence sequence of calculation site based on GenBank accession number AY530878.1).
In another preferred example, the code area deletion whole E3, which refers to, deletes the in wild type AdC7 genome
The sequence in 27094-31799 (the sequence sequence of calculation site based on GenBank accession number AY530878.1).
In another preferred example, the method includes:
(1) origin sequence, the LITR segment from AdC7 adenoviral gene group, source of pNEB193 will be derived from
Segment OLN is fused by fusion DNA vaccine in the Nde I-Age I segment of AdC7 adenoviral gene group, with Spe I and Age I enzyme
It is sliced section OLN；With Nde I, Age I and Spe I digestion AdC7 adenoviral gene group, segment Age (the 4028)-SpeI that will be cut
(10610) (the sequence sequence of calculation site based on GenBank accession number AY530878.1) is connected with the segment OLN of digestion
It connects to form plasmid pOIN；
(2) Hind III and Avr II digestion AdC7 genome, the target fragment Hind III (7153)-that will be cut are used
Avr II (23363) is inserted into the site Hind III and Avr II of plasmid pOIN, and the plasmid of acquisition is named as pOINH (base
In the sequence sequence of calculation site of GenBank accession number AY530878.1)；
(3) it is located at the segment in AdC7 adenoviral gene between 31800-36535 from amplification in AdC7 adenoviral gene group,
Avr II and Rsr II restriction enzyme site are introduced at 5 ' ends, Pac I and Asis I site is introduced at 3 ' ends, the segment of amplification is inserted into
Into the Avr II and Asis I site of pOINH, the plasmid of acquisition is named as pOINHR；
(4) it is located at the segment in AdC7 adenoviral gene between 23165-27093 from amplification in AdC7 adenoviral gene group,
The site Rsr II is introduced at 3 ' ends, the segment of amplification is inserted into the site Avr II and Rsr II of pOINHR, is replicated
The recombinant adenoviral expressing vector of deficiency.
In another preferred example, the origin sequence is the SEQ ID NO:1 (nucleosides of i.e. appended pAdC7 carrier
Acid sequence) in sequence shown in 1-1882.
In another preferred example, the LITR segment from AdC7 adenoviral gene group is in SEQID NO:1
Sequence shown in 1883-2382.
In another preferred example, the NdeI-AgeI segment from AdC7 adenoviral gene group is SEQ ID
Sequence shown in 2383-3422 in NO:1.
In another preferred example, the segment between the Rsr II-Asis I is 26483- in SEQ ID NO:1
Sequence shown in 31241.
In another preferred example, the segment between the Avr II-Rsr II is 22751- in SEQ ID NO:1
Sequence shown in 26482.
In another aspect of this invention, a kind of adenovirus expression carrier is provided, the expression vector is in wild-type adenovirus
Delete the code area part E1 and the whole code area E3 on the basis of AdC7 genome, also, E1 delete area increase I-Ceu I and
PI-Sce I restriction enzyme site deletes area in E3 and increases Rsr II restriction enzyme site.
It in a preferred embodiment, further include external source between restriction enzyme site PI-Sce I and the I-Ceu I of the expression vector
Antigen encoding gene.
In another preferred example, the exogenous antigen be the haemagglutinin antigen of (but being not limited to) influenza virus (such as
H5N1HA)。
In another aspect of this invention, a kind of method preparing vaccine is provided, which comprises
(1) adenovirus expression carrier is provided；
(2) by the antigen encoding gene of external source be inserted into (1) expression vector restriction enzyme site PI-Sce I and I-Ceu I it
Between；
(3) the recombinant expression carrier virus infection of (2) is produced into cell, virus is packed in the cell, to be had
The vaccine of immunogenicity.
In another aspect of this invention, a kind of kit for being used to prepare vaccine is provided, the kit includes: that front is appointed
Adenovirus expression carrier described in one.
In a preferred embodiment, the kit further include: virus production cell；And/or antigen encoding gene.
In another preferred example, the virus production cell is HEK293 cell.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
The digestion of Fig. 1, AdC7 genomic DNA is identified.
1,1kb ladder(NEB)；2, Bgl II digestion.
The digestion identification of Fig. 2, recombinant adenoviral vector pAdC7 (i.e. pAdC7- Δ E1 Δ E3).
1,1kb ladder(NEB)；2,Bgl II；3.Xho I；4.Mfe I.
Fig. 3, building recombined adhenovirus AdC7-eGFP.
A: recombinant adenoviral vector pAdC7-eGFP digestion identification；
B: the plaque formation of recombined adhenovirus AdC7-eGFP
Fig. 4, building recombined adhenovirus AdC7-H5N1HA.
A:pAdC7-H5N1HA digestion identification；
B:AdC7-H5N1 digestion identification；
C: the expression of detection HA albumen.
Fig. 5, the strategy for constructing replication-defective adenoviral vector pAdC7.
Specific embodiment
The present inventor passes through in-depth study, devises a kind of effective strategy, is successfully based on vaccine carrier for chimpanzee type adenovirus
AdC7 is built into a kind of new expression vector.The vaccine carrier, which can be applied to preparation, high efficient expression and to be had good immune
The viral vaccine of originality.
The present invention provides a kind of adenovirus expression carrier, the expression vector includes: the expression vector in wild type
The code area part E1 and the whole code area E3 are deleted on the basis of adenovirus AdC7 genome, also, are deleted area in E1 and increased I-Ceu
I and PI-Sce I restriction enzyme site delete area in E3 and increase Rsr II restriction enzyme site.
For adenovirus AdC7 genome, the present inventor passes through careful sequence alignment, has finally determined using digestion position
Insertion point of point I-Ceu I and the PI-Sce I as foreign gene, to will not cause in the other of adenovirus expression carrier
It causes to shear in position.
Vaccine carrier for chimpanzee type adenovirus AdC7, i.e. SAdV24, and it is named as Pan7, it is isolated from chimpanzee lymph nodes.
The present invention, by genome Direct Cloning method, deletes part E1 and the whole code area E3, in E1 based on wild type AdC7
It deletes area and increases I-Ceu I and PI-Sce I restriction enzyme site, delete area in E3 and increase Rsr II enzyme site, be finally obtained duplication
The AdC7 recombinant adenoviral expressing vector of deficiency.
The expression vector also contains replication orgin and/or marker gene etc..Side well-known to those having ordinary skill in the art
Method can be used to construct the required expression vector of the present invention.These methods include recombinant DNA technology in vi, DNA synthetic technology, in vivo
Recombinant technique etc..The DNA sequence dna can be effectively connected in the appropriate promoter (such as CMV) in expression vector, with guidance
MRNA synthesis.Expression vector further includes the ribosome bind site and transcription terminator of translation initiation.In addition, expression vector is excellent
Selection of land includes one or more selected markers, to provide the phenotypic character for selecting the host cell of conversion.
The present invention also provides a kind of methods for preparing adenovirus expression carrier, which comprises with wild type adenopathy
Based on malicious AdC7 genome, the code area part E1 and the whole code area E3 are deleted, area is deleted in E1 and increases I-Ceu I and PI-
Sce I restriction enzyme site deletes area in E3 and increases Rsr II restriction enzyme site, and the recombined adhenovirus expression for obtaining replication defect type carries
Body.
As preferred embodiment of the invention, cloning process includes: the segment that 1. Overlap PCR is obtained, through Age I with
After Spe I digestion, the segment obtained with NdeI, AgeI and SpeI digestion AdC7 postgenome connects into plasmid pOIN；②Hind
IIII and Avr II digested plasmid pOIN and AdC7 genome, connect into plasmid pOINH；3. Rsr II and Asis I digestion PCR
Obtained segment and plasmid pOINH, connects into plasmid pOINHR；4. segment and matter that Avr II and Rsr II digestion PCR are obtained
Grain pOINHR, connects into plasmid pAdC7.
After obtaining the adenovirus expression carrier, by transfected virus produce cell, carry out virus breeding.After transfection
After a period of time, virus can be harvested.As preferred embodiment of the invention, the virus of harvest can repeated infection virus production it is thin
Born of the same parents, it is lasting to pass on.Virus titer (TCID50) measurement can be carried out according to conventional method in that art.
Adenovirus expression carrier of the invention is suitable for expressing a variety of antigens, to make as an expression vector platform
Standby viral vaccine.The antigen is not particularly limited.
As preferred embodiment of the invention, for the validity for verifying AdC7, the present inventor is by the HA base of influenza virus H 5 N 1
Because being cloned into AdC7 carrier, researches show that AdC7 carrier energy overexpression protection protogenes (such as HA gene), it was demonstrated that the present inventor's success
A kind of novel adenovirus vector is obtained, can be used for vaccine research and development and other biomedical basic research, be new generation vaccine
Research and development provide a kind of novel carrier platform.
The method that the present invention passes through adenoviral gene group Direct Cloning, and STBL2 Escherichia coli stable conversion system is utilized,
Avoid it is traditional by methods of homologous recombination construct adenovirus vector, largely reduce adenovirus mutation can
Can, the recombinant adenoviral vector genetic stability of acquisition is good.Therefore, external source base is expressed using the adenovirus vector that the present invention constructs
Because will be more stable, efficient.The present invention constructs adenovirus vector using external direct enzyme cutting connection method, screens sun in bacteria levels
Property clone be easier, be quick, highly shortened the building time of adenovirus vector.In addition, the present invention is suitable for all adenopathies
The building of poisonous carrier, it is thus only necessary to adenoviral gene group restriction enzyme site is analyzed and is rationally applied, it can be general
All over being applied to all biomedical laboratories, and transformation can be optimized to adenovirus internal sequence, construct various spies
Anisotropic adenovirus vector.Therefore, the present invention provides not only a kind of new expression vector, and provides a kind of novel gland
The construction method of viral vectors is more advantageous to and adenovirus vector is promoted to develop in biomedical basic research field and clinical application
Aspect plays its advantageous feature, to ensure that human health, improving the quality of living lays the foundation.
Based on new improvement of the invention, the present invention also provides a kind of kit for being used to prepare vaccine, the kits
Including the adenovirus expression carrier.
The kit may also include virus production cell, the encoding gene of the antigen to be expressed.In addition, described
It may also include the operation instructions for illustrating vaccine preparation method in kit.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
1, material
1.1 Strain and cell
Wild-type adenovirus AdC7 is purchased from ATCC (Manassas, VA)；HEK293 cell strain is purchased from Chinese Academy of Sciences Shanghai
Biochemistry and cell biological research institute, culture medium are the DMEM containing 10% fetal calf serum (FCS).
1.2 restriction enzymes, strain and plasmid
Restriction enzyme is purchased from New England Biolabs；DH5 α and Stbl2 are purchased from Invitrogen；pNEB193
Purchased from New England Biolabs.
Pshuttle-CMV carrier is purchased from CLONTECH Laboratories, Inc.
PUC57-EGFP plasmid: EGFP sequence (SEQ ID NO:2) by Nanjing Jin Sirui company at and be cloned into pUC57 carry
Body obtains pUC57-EGFP.
PUC57-H5N1HA plasmid: H5N1HA sequence (SEQ ID NO:3) is synthesized and is cloned by Nanjing Jin Sirui company
PUC57 carrier, obtains pUC57-H5N1HA.
2, method
The amplification and purifying of 2.1 adenovirus
HEK293 cell is cultivated in the DMEM containing 10%FCS, and AdC7 adenovirus cultivates amplification in HEK293 cell.
Adenovirus infection HEK293 is carried out in the DMEM of no FCS, after two hours, adds FCS that FCS final concentration is made to reach 5%.When cell whole
After showing Virus plaque (CPE), infected cell is collected, is centrifuged.With DMEM be resuspended cell, multigelation cell three times,
Make cell cracking.Virus is purified through CsCl density gradient centrifugation, and glycerol is then added and sets -80 DEG C of refrigerators preservations.
2.2 extract adenovirus genomic dna
After AdC7 Adenovirus Purification, genomic DNA is extracted with standard method.It is performed an analysis mirror with BglII digested genomic dna
It is fixed.
The adenovirus vector of 2.3 building replication defect types
2.3.1 constructing plasmid pOIN
Using pNEB193 as template, with primer outside primer of the origin and antisense primer
Of the origin (table 1) obtains segment the origin by PCR amplification.
Using AdC7 adenoviral gene group as template, with primer sense primer of LITR and antisense primer
Of LITR.I-Cue obtains segment LITR by PCR amplification.
Using AdC7 adenoviral gene group as template, with primer sense primer of NdeI-AgeI.PI-Sce with
Outside primer of NdeI-AgeI, obtains segment NdeI-AgeI by PCR amplification.
By fusion DNA vaccine, segment the origin, segment LITR and segment NdeI-AgeI are fused into segment OLN, melted
When closing PCR, primer inside primer of origin and inside primer of NdeI-AgeI are used.By PCR with
The 458-3026 bit slice section of E1 in AdC7 genome is deleted the (sequence based on GenBank accession number AY530878.1 by fusion DNA vaccine
Column count sequence site), while introducing some restriction enzyme sites, the 5 ' ends of segment the origin introduce spe I, Avr II with
Asis I restriction enzyme site introduces Pac I restriction enzyme site, in segment LITR and NdeI- between segment the origin and LITR
I-Ceu I and PI-Sce I restriction enzyme site are introduced between AgeI.With Spe I and Age I endonuclease bamhi OLN, with DNA common products
Purification kit recycling；With Nde, Spe I and Age I (Spe I and Age I digestion AdC7 is used, two sizes almost phase can be obtained
Same segment, i.e. segment Age I (4028)-Spe I (10601) and AgeI (25823)-Spe I (32770), in low melting point fine jade
It is inseparable in lipolysaccharide electrophoresis, therefore with three kinds of enzyme digestion AdC7 genomes) digestion AdC7 genome, low melting-point agarose electrophoresis,
6.5kb target fragment AgeI (4028)-Spe I (10610) (sequence based on GenBank accession number AY530878.1 can be obtained
Sequence of calculation site).Then, make connection reaction, conversion in low melting-point agarose, selected clone performs an analysis identification；It obtains
POIN plasmid.
Table 1, the primer
2.3.2 constructing plasmid pOINH
With Hind III and Avr II digestion pOIN, agarose electrophoresis recycles target fragment (segment of 6.5kb)；Use Hind
III and Avr II digestion AdC7 genome, low melting-point agarose electrophoresis obtain target fragment: from Hind III (7153) to Avr
The segment (the sequence sequence of calculation site based on GenBank accession number AY530878.1) of II (23363).Then, in low melting point
Make connection reaction in agarose, converts, identification；Obtain pOINH.
2.3.3 constructing plasmid pOINHR
Using AdC7 adenoviral gene group as template, with primer sense primer of RsrII.AsisI.PacI with
Antisense primer of RsrII.AsisI.PacI obtains segment RsrII-AsisI by PCR amplification, and purifying PCR is produced
Object, Rsr II and Asis I endonuclease bamhi RsrII-AsisI.With Rsr II and Asis I digestion pOINHR, low melting-point agarose
Electrophoresis obtains target fragment.Then, make connection reaction in low melting-point agarose, convert, identification.
2.3.4 constructing plasmid pAdC7
Using AdC7 adenovirus genomic dna as template, with primer sense primer of AvrII-RsrII with
Antisense primer of AvrII-RsrII obtains segment Avr II-Rsr II, purified pcr product by PCR amplification；
With Avr II and Rsr II endonuclease bamhi AvrII-RsrII.With Avr II and Rsr II digestion pOINHR, low melting-point agarose electricity
Swimming obtains target fragment.Then, make connection reaction in low melting-point agarose, convert, identification.Plasmid pAdC7 is obtained, is passed through
2.3.2 27094-31799 bit sequence in AdC7 genome is deleted into (i.e. deletion E3) with 2.3.3 similar step.
2.4 building recombined adhenovirus AdC7-eGFP
2.4.1 eGFP is cloned into pAdC7
With Nhe I and Not I digestion pUC57-EGFP plasmid, Ago-Gel recycles target fragment EGFP, connects
To the pShuttle-CMV carrier through identical digestion, plasmid pShuttle-CMV-EGFP is obtained after digestion identification.With PI-Sce I
With I-Ceu I digestion pshuttle-eGFP, runs glue and recycle target fragment；With PI-SceI and I-Ceu I digestion pAdC7, eutectic
Point agarose electrophoresis, obtains target fragment.Then, make connection reaction in low melting-point agarose, convert, identification.It obtains
pAdC7-eGFP。
2.4.2 saving AdC7-eGFP recombined adhenovirus
Pac I digestion pAdC7-eGFP, makes its linearisation.6 orifice plates are spread with HEK293 cell, when cell degree of collecting reaches
When 70-80%, the pAdC7-eGFPDNA that 1.5 μ g have been linearized is transfected with X-tremeGENE.The feelings of observation cell fluorescence daily
Condition.
2.5 building recombined adhenovirus AdC7-H5N1HA
2.5.1 H5N1HA is cloned into pAdC7
With Apa I and Not I digestion pUC57-H5N1HA plasmid, Ago-Gel recycles target fragment HA, connects
To the pShuttle-CMV carrier through identical digestion, digestion is identified and is sequenced to obtain plasmid pShuttle-H5N1HA.Use PI-Sce
I and I-Ceu I digestion pshuttle-H5N1HA runs glue and recycles target fragment；With PI-Sce I and I-Ceu I digestion pAdC7,
Low melting-point agarose electrophoresis obtains target fragment.Then, make connection reaction in low melting-point agarose, convert, identification；It obtains
pAdC7-H5N1HA。
2.5.2 saving AdC7-H5N1HA recombined adhenovirus
Pac I digestion pAdC7-H5N1HA, makes its linearisation.6 orifice plates are spread with HEK293 cell, when cell degree of collecting reaches
When 70-80%, the pAdC7-H5N1HA that 1.5 μ g have been linearized is transfected with X-tremeGENE.
2.5.3 amplification and purifying group adenovirus AdC7-H5N1HA
HEK293 cell is cultivated in the DMEM containing 10%FCS, and AdC7-H5N1HA adenovirus is trained in HEK293 cell
Support amplification.Adenovirus infection HEK293 is carried out in the DMEM of no FCS, after two hours, adds FCS that FCS final concentration is made to reach 5%.When thin
After born of the same parents all show Virus plaque (CPE), infected cell is collected, is centrifuged.Cell, multigelation cell is resuspended with DMEM
Three times, make cell cracking.Virus is purified through CsCl density gradient centrifugation, and glycerol is then added and sets -80 DEG C of refrigerators preservations.
2.5.4 AdC7-H5N1HA is identified in digestion
After AdC7-H5N1HA Adenovirus Purification, genomic DNA is extracted with standard method.Base is digested with Bgl II and Xho I
Because a group DNA performs an analysis identification.
2.5.5Western blot detects the expression of H5N1HA
6 orifice plates are spread with HEK293 cell.When cell degree of collecting reaches 80-90%, 1 × 109Vp, 3 × 109Vp and 6 ×
109Vp AdC7-H5N1HA virus removes infection cell, while with 6 × 109Vp AdC7 virus removes infection cell, receives after 24 hours thin
Born of the same parents, Western blot detect the expression of H5N1.
3, embodiment
Embodiment 1, the amplification of adenovirus, purifying and adenoviral gene group extraction
It after wild type AdC7 is expanded in HEK293 cell, is purified through CsCl density gradient centrifugation, extracts disease later
Virus gene group DNA carries out agarose electrophoresis with Bgl II digestion genome.The results show that expanding, the virus that purifies is
AdC7 adenovirus.See Fig. 1.
Embodiment 2, the adenovirus vector for constructing replication defect type
According to flow chart (Fig. 5), segment the ori, LITR, NdeI-AgeI are obtained by PCR；By fusion DNA vaccine by this
Three segments are fused into segment OLN；With Spe I and Age I digestion OLN, with Hind III, Age I, Spe I digestion AdC7 gene
Group, connection forms plasmid pLITR later.
With Hind III and Avr II digestion AdC7 genome, digestion is then obtained into large dna fragment cloning to pLITR, is formed
Plasmid pOINH.
Segment RsrII-AsisI will be obtained by PCR, is cloned into pOINH, form pOINHR.
Finally, segment AvrII-RsrII will be obtained by PCR, it is cloned into plasmid pOINHR, obtains pAdC7- Δ E1 Δ
E3。
With Bgl II, Xho I, Mfe I digestion pAdC7- Δ E1 Δ E3, agarose electrophoresis is carried out, the results showed that pAdC7-
Δ E1 Δ E3 is correct purpose carrier.See Fig. 2.
Embodiment 3, building recombined adhenovirus AdC7-eGFP
After eGFP is cloned into pAdC7, the digestion identification such as Fig. 3 A of recombinant adenoviral vector pAdC7-eGFP.
, there is Virus plaque on the tenth day, subsequent plaque gradually expands in transfected HEK 293 after recombinant plasmid dna linearisation
Greatly.Such as Fig. 3 B.
Embodiment 4, building recombined adhenovirus AdC7-H5N1HA
After H5N1HA is cloned into pAdC7, transfected HEK 293 packs out virus.After amplification, purified virus, gene is mentioned
Group digestion identification, such as Fig. 4 A-B.
HEK293 cell is infected with the AdC7-H5N1HA of different titers.After 24 hours, cell, Western blot are collected
Detect the expression of HA.Such as Fig. 4 C.
Conclusion
By the method for the above viral genome Direct Cloning, wild-type adenovirus AdC7 is built by the present inventor can table
Up to the carrier of foreign gene.Therefore the present inventor researches and develops for new generation vaccine and the basic research of various biomedicines provides one
New expression vector of the kind based on AdC7, while providing a kind of method of novel building adenovirus vector.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (7)
1. a kind of method for preparing adenovirus expression carrier, which is characterized in that the described method includes:
Based on the wild-type adenovirus AdC7 genome shown in the GenBank accession number AY530878.1, deletes part E1 and compile
Code area and the whole code area E3 delete area in E1 and increase I-Ceu I and PI-Sce I restriction enzyme site, delete area in E3 and increase Rsr
II restriction enzyme site obtains the recombinant adenoviral expressing vector of replication defect type；
Wherein, the deletion part code area E1 refers to 458-3026 in deletion wild type AdC7 genome sequences；
The deletion whole code area E3 refers to 27094-31799 in deletion wild type AdC7 genome sequences；
Wherein, the method includes:
(1) by origin sequence shown in SEQ ID NO:1 1-1882, from the LITR of AdC7 adenoviral gene group
Segment is fused into segment OLN by fusion DNA vaccine from the Nde I-Age I segment of AdC7 adenoviral gene group, with Spe I
With Age I endonuclease bamhi OLN；With Nde I, Age I and Spe I digestion AdC7 adenoviral gene group, the segment Age- that will be cut
SpeI is connected to form plasmid pOIN with the segment OLN of digestion；
(2) Hind III and Avr II digestion AdC7 genome are used, the target fragment Hind III-Avr II cut is inserted into
In the site Hind III and Avr II of plasmid pOIN, the plasmid of acquisition is named as pOINH；
(3) it is located at the segment in AdC7 adenoviral gene between 31800-36535 from amplification in AdC7 adenoviral gene group, 5 '
End introduces Avr II and Rsr II restriction enzyme site, introduces Pac I and Asis I site at 3 ' ends, the segment of amplification is inserted into
In the Avr II and Asis I site of pOINH, the plasmid of acquisition is named as pOINHR；With
(4) it is located at the segment in AdC7 adenoviral gene between 23165-27093 from amplification in AdC7 adenoviral gene group, 3 '
End introduces the site Rsr II, and the segment of amplification is inserted into the site Avr II and Rsr II of pOINHR, obtains replication defective
The recombinant adenoviral expressing vector of type.
2. a kind of adenovirus expression carrier, which is characterized in that the expression vector is shown in the GenBank accession number AY530878.1
Wild-type adenovirus AdC7 genome on the basis of delete the code area part E1 and the whole code area E3, also, in E1 deletion area
Increase I-Ceu I and PI-Sce I restriction enzyme site, deletes area in E3 and increase Rsr II restriction enzyme site；
Wherein, the deletion part code area E1 refers to 458-3026 in deletion wild type AdC7 genome sequences；
The deletion whole code area E3 refers to 27094-31799 in deletion wild type AdC7 genome sequences；
The adenovirus expression carrier is prepared by the following method:
(1) by origin sequence shown in SEQ ID NO:1 1-1882, from the LITR of AdC7 adenoviral gene group
Segment is fused into segment OLN by fusion DNA vaccine from the Nde I-Age I segment of AdC7 adenoviral gene group, with Spe I
With Age I endonuclease bamhi OLN；With Nde I, Age I and Spe I digestion AdC7 adenoviral gene group, the segment Age- that will be cut
SpeI is connected to form plasmid pOIN with the segment OLN of digestion；
(2) Hind III and Avr II digestion AdC7 genome are used, the target fragment Hind III-Avr II cut is inserted into
In the site Hind III and Avr II of plasmid pOIN, the plasmid of acquisition is named as pOINH；
(3) it is located at the segment in AdC7 adenoviral gene between 31800-36535 from amplification in AdC7 adenoviral gene group, 5 '
End introduces Avr II and Rsr II restriction enzyme site, introduces Pac I and Asis I site at 3 ' ends, the segment of amplification is inserted into
In the Avr II and Asis I site of pOINH, the plasmid of acquisition is named as pOINHR；With
(4) it is located at the segment in AdC7 adenoviral gene between 23165-27093 from amplification in AdC7 adenoviral gene group, 3 '
End introduces the site Rsr II, and the segment of amplification is inserted into the site Avr II and Rsr II of pOINHR, obtains replication defective
The recombinant adenoviral expressing vector of type.
3. adenovirus expression carrier as claimed in claim 2, which is characterized in that the restriction enzyme site PI-Sce of the expression vector
It further include the antigen encoding gene of external source between I and I-Ceu I.
4. adenovirus expression carrier as claimed in claim 3, which is characterized in that the exogenous antigen is the blood of influenza virus
Solidifying element antigen.
5. a kind of method for preparing vaccine, which is characterized in that the described method includes:
(1) adenovirus expression carrier as claimed in claim 3 is provided；
(2) antigen encoding gene of external source is inserted between restriction enzyme site PI-Sce I and the I-Ceu I of (1) expression vector；
(3) the recombinant expression carrier virus infection of (2) is produced into cell, virus is packed in the cell, is immunized to obtain and have
The vaccine of originality.
6. a kind of kit for being used to prepare vaccine, which is characterized in that the kit includes:
Any adenovirus expression carrier of claim 2-4.
7. kit as claimed in claim 6, which is characterized in that the kit further include:
Virus production cell；And/or
Antigen encoding gene.
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| SG11201900808SA (en) * | 2016-08-01 | 2019-02-27 | Wistar Inst | Compositions and methods of replication deficient adenoviral vectors for vaccine applications |
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| CN107574175A (en) * | 2017-09-11 | 2018-01-12 | 南方医科大学 | A kind of expression vector and its construction method based on recombined adhenovirus |
| CN107686843A (en) * | 2017-09-11 | 2018-02-13 | 南方医科大学 | A kind of expression vector and its construction method based on adenovirus AdC6 |
| CN107988258B (en) * | 2017-12-12 | 2020-08-04 | 中国科学院微生物研究所 | A kind of Zika virus vaccine based on chimpanzee adenovirus vector and preparation method thereof |
| CN112760341B (en) * | 2020-07-13 | 2022-12-09 | 中国科学院微生物研究所 | Application of recombinant vector and chimpanzee adenovirus packaged with the recombinant vector in the preparation of 2019 novel coronavirus vaccine |
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