CN104651404A - Human type 3 adenovirus display vector and construction method thereof - Google Patents
Human type 3 adenovirus display vector and construction method thereof Download PDFInfo
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- CN104651404A CN104651404A CN201510125605.8A CN201510125605A CN104651404A CN 104651404 A CN104651404 A CN 104651404A CN 201510125605 A CN201510125605 A CN 201510125605A CN 104651404 A CN104651404 A CN 104651404A
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Abstract
The invention discloses a human type 3 adenovirus display vector and a construction method thereof and aims at providing a human type 3 adenovirus display vector capable of performing rapid flux display and a construction method of the vector. The HVR1 position in a neighboring hypervariable region of the vector 6 contains SfiI-pAD-CCDB1-Chloramphenicol resistance-SfiI in the sequence SEQ ID NO:1. The construction method comprises the following steps: inserting two SfiI sites opposite to each other at the HVR1 site of the neighboring hypervariable region of the human type 3 adenovirus 6, inserting an exogenous nucleic acid sequence into the HVR1 site of the neighboring hypervariable region of the human type 3 adenovirus 6 by virtue of a Golden gate method, inserting pAD-CCDB1-Chloramphenicol Cassette between the two SfiI sites opposite to each other so as to serve as negative screening genes of plasmid construction, and performing fusion expression. The invention belongs to the technical field of biomedicine.
Description
Technical field
The present invention relates to a kind of viral display field, especially a kind of human 3-type adenovirus display carrier; Belong to field of biomedicine technology.
Background technology
Human 3-type adenovirus particle is a kind of without togavirus, its be a kind of have 20 body symmetrical structures virion.Its housing is made up of 252 subunits, and wherein 240 subunits are the tripolymer that 6 adjacent bodies are formed, and body can be stimulated to produce neutralizing antibody and combined specific antibody.
Human 3-type adenovirus capsid protein comprises six adjacent bodies, penton pedestal, fine prominent and protein I X.Wherein six adjacent bodies are main capsid structural protein, and there are 7 hypervariable regions (HVRs) pinnacle of a pagoda being wherein positioned at surface, includes type specificity epi-position.Bioinformatic analysis shows that HVR1, HVR2, HVR5 and HVR7 carry potential Neutralization and crystallization, is applicable to merging Surface Display of Foreign Epitopes by engineered method, and evades the already present immune response for adenovirus carrier of host self simultaneously.
Summary of the invention
The object of the present invention is to provide the construction process of a kind of display carrier of human 3-type adenovirus fast and efficiently and this carrier thereof.
For solving the problems of the technologies described above, last technical scheme provided by the invention is as follows:
This human 3-type adenovirus display carrier, is characterized in that, SfiI-pAD-CCDB1-Chloramphenicol resistance – SfiI sequence SEQ ID NO:1 is contained in HVR1 position, described carrier 6 adjacent body hypervariable region.
Above-mentioned human 3-type adenovirus display carrier, described human 3-type adenovirus 6 adjacent body hypervariable region HVR1 is containing Golden gate site.
Further, above-mentioned human 3-type adenovirus display carrier, two reverse SfiI sites are contained in described HVR1 site.
Further, above-mentioned human 3-type adenovirus display carrier, described Golden gate site is containing, for example sequence SEQ ID NO:2Chloramphenicol resistance screening-gene.
Further, above-mentioned human 3-type adenovirus display carrier, described Golden gate site is containing, for example sequence SEQ ID NO:3 negative sense screening-gene CCDB1.
Another technical scheme provided by the invention is as follows:
The construction process of this human 3-type adenovirus display carrier, the method is by the reverse SfiI site of insertion two on HVR1 site, human 3-type adenovirus 6 adjacent body hypervariable region, by the method for Golden gate, exogenous nucleic acid sequences is inserted on HVR1 site, human 3-type adenovirus 6 adjacent body hypervariable region, between two reverse SfiI sites, insert pAD-CCDB1-Chloramphenicol Cassette, as the negative sense screening-gene of plasmid construction, carry out amalgamation and expression.
Compared with prior art, human 3-type adenovirus display carrier provided by the invention the present invention selects HVR1 region, by cloning two reverse SfiI sites of entering, thus can by the method for Golden gate, the peptide section of external source is cloned into this site, carry out amalgamation and expression, can carrier construction fast and efficiently.Can be used for the exploitation of vaccine.
Embodiment
In order to understand the present invention better, below in conjunction with specific embodiment, set forth the present invention further.Should be understood that embodiment only illustrates the present invention below, instead of be used for limiting the scope of the invention.In embodiment, NM experimental technique, conventionally operates usually.
The invention provides a kind of human 3-type adenovirus display carrier, this human 3-type adenovirus display carrier, SfiI-pAD-CCDB1-Chloramphenicolresistance – SfiI sequence SEQ ID NO:1 is contained in HVR1 position, described carrier 6 adjacent body hypervariable region.
Specifically, this human 3-type adenovirus 6 adjacent body hypervariable region HVR1 containing Golden gate site, two reverse SfiI sites.Described Golden gate site is containing, for example sequence SEQ ID NO:2 screening-gene Chloramphenicol resistance; Described Golden gate site is containing, for example sequence SEQ ID NO:3 negative sense screening-gene CCDB1.
Embodiment 2
Another technical scheme provided by the invention is as follows:
The construction process of this human 3-type adenovirus display carrier, the method is by the reverse SfiI site of insertion two on HVR1 site, human 3-type adenovirus 6 adjacent body hypervariable region, by the method for Golden gate, exogenous nucleic acid sequences is inserted on HVR1 site, human 3-type adenovirus 6 adjacent body hypervariable region, between two reverse SfiI sites, insert pAD-CCDB1-Chloramphenicol Cassette, as the negative sense screening-gene of plasmid construction, carry out amalgamation and expression.
Concrete preparation method is as follows:
Build the human 3-type adenovirus carrier containing foreign DNA.
1, human 3-type modifies adenovirus vector construct.Gene chemical synthesis comprises the Modified-Hexon of SfiI-pAD-CCDB1-Chloramphenicol-SfiI; Then with DNA fragmentation and the shuttle vectors pBR322-L/R of the synthesis of ClaI and BamHI double digestion, after agarose gel electrophoresis reclaims, the two is reclaimed product T4DNA ligase enzyme and connects transformed competence colibacillus cell E.coli TOP10, obtain shuttle plasmid pBR322-L/R-mHexon through qualification.
2, the structure of recombinant adenovirus plasmid pBRADdeltaE3GFP-mHexon, shuttle plasmid pBR322-L/R-mHexon obtains HexonL-mHexon-HexonR fragment through EcoRI and SalI double digestion, pBRADdeltaE3GFP removes original six adjacent body genes through PacI and AvrII double digestion, DNA fragmentation after reclaiming two is transformed into E.coli BJ5183 simultaneously and recombinates, and obtains recon pBRADdeltaE3GFP-mHexon.Finally Positive recombinant clones pBRADdeltaE3GFP-mHexon is transformed into E.coli TOP10 to extract plasmid, and order-checking qualification, as shown in SEQ ID NO:1.
Claims (6)
1. a human 3-type adenovirus display carrier, is characterized in that, SfiI-pAD-CCDB1-Chloramphenicol resistance – SfiI sequence SEQ IDNO:1 is contained in HVR1 position, described carrier 6 adjacent body hypervariable region.
2. human 3-type adenovirus display carrier according to claim 1, is characterized in that, described human 3-type adenovirus 6 adjacent body hypervariable region HVR1 is containing Golden gate site.
3. human 3-type adenovirus display carrier according to claim 1, is characterized in that, two reverse SfiI sites are contained in described HVR1 site.
4. human 3-type adenovirus display carrier according to claim 2, is characterized in that, described Goldengate site is containing, for example sequence SEQ ID NO:2Chloramphenicol resistance screening-gene.
5. human 3-type adenovirus display carrier according to claim 2, is characterized in that, described Goldengate site is containing, for example sequence SEQ ID NO:3 negative sense screening-gene CCDB1.
6. the construction process of human 3-type adenovirus display carrier according to claim 1, it is characterized in that, the method is by the reverse SfiI site of insertion two on HVR1 site, human 3-type adenovirus 6 adjacent body hypervariable region, by the method for Golden gate, exogenous nucleic acid sequences is inserted on HVR1 site, human 3-type adenovirus 6 adjacent body hypervariable region, between two reverse SfiI sites, insert pAD-CCDB1-ChloramphenicolCassette, as the negative sense screening-gene of plasmid construction, carry out amalgamation and expression.
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| CN201510125605.8A CN104651404A (en) | 2015-03-20 | 2015-03-20 | Human type 3 adenovirus display vector and construction method thereof |
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| CN201510125605.8A CN104651404A (en) | 2015-03-20 | 2015-03-20 | Human type 3 adenovirus display vector and construction method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013036791A2 (en) * | 2011-09-09 | 2013-03-14 | Beth Israel Deaconess Medical Center, Inc. | Modified adenoviral vectors and methods of treatment using same |
| CN104109683A (en) * | 2014-05-19 | 2014-10-22 | 武汉金开瑞生物工程有限公司 | Primer-free gene synthesis method |
| CN104419717A (en) * | 2013-08-23 | 2015-03-18 | 长春百克生物科技股份公司 | Recombinant adenovirus for escaping pre-existing immunity, and construction method and use thereof |
-
2015
- 2015-03-20 CN CN201510125605.8A patent/CN104651404A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013036791A2 (en) * | 2011-09-09 | 2013-03-14 | Beth Israel Deaconess Medical Center, Inc. | Modified adenoviral vectors and methods of treatment using same |
| CN104419717A (en) * | 2013-08-23 | 2015-03-18 | 长春百克生物科技股份公司 | Recombinant adenovirus for escaping pre-existing immunity, and construction method and use thereof |
| CN104109683A (en) * | 2014-05-19 | 2014-10-22 | 武汉金开瑞生物工程有限公司 | Primer-free gene synthesis method |
Non-Patent Citations (4)
| Title |
|---|
| CHUNYAN XUE ET.AL.: "Construction and characerization of a recombinant human adenovirus type 3 vector containing two foreign neutralizing epitopes in hexon", 《VIRUS RESEARCH》 * |
| INVITROGEN: "pAd/CMV/V5-DESTTM and pAd/PL-DESTTM Gateway® Vectors", 《INVITROGEN》 * |
| TIANHUA ZHONG ET.AL.: "Characterization of malleability and immunological properties of human adenovirus type 3 hexon hypervariable region 1", 《ARCH VIROL》 * |
| 庄国庆等: "人SNAP25基因重组腺病毒的构建与表达", 《中国生物工程杂志》 * |
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Application publication date: 20150527 |
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