Recombinant adenovirus expression vector SAd23-L

A technology of recombinant adenovirus and expression vector, which is applied in the direction of antiviral agents, viral antigen components, and diseases transmitted by vectors. The success rate, virus titer increase, and the effect of saving manpower and material resources

Inactive Publication Date: 2019-05-24
广州佰芮慷生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventors have found in practice that the carrier Sad23 constructed by this method encountered difficulties in amplifying the virus and preparing for purification after the virus was successfully packaged. After the virus infected many HEK293 cells, the cells were recovered and frozen. Melt the cells, and the virus purified from the supernatant cesium chloride always fails to reach the ideal virus titer. After Sad23-eGFP virus infects 90 75T HEK293 cells, the recombinant virus obtained is purified, and the final titer is determined by plaque formation experiments. degrees only 10 9 PFU / ml
That is, in the process of amplifying the virus with the Sad23 virus vector, the amount of virus produced is too low, which then affects its actual clinical application, so further improvement is needed

Method used

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  • Recombinant adenovirus expression vector SAd23-L
  • Recombinant adenovirus expression vector SAd23-L
  • Recombinant adenovirus expression vector SAd23-L

Examples

Experimental program
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Effect test

Embodiment 1

[0093] Example 1. Construction of a replication-deficient adenovirus vector.

[0094] according to Figure 4 As shown in the flow chart, the LITR fragment (about 450bp) of SAd23 genome was amplified by PCR, the pNEB193 plasmid and the PCR product were digested with PmeI and BstAPI, and ligated to form plasmid pNEB193-PB.

[0095] The pUC57-Linker and pNEB193-PB plasmids were digested with SnaBI and NdeI, and ligated into plasmid pNEB193-PB+Linker.

[0096] The genome of SAd23 was digested with NdeI and MluI, and the excised target fragment was inserted into the NdeI and MluI sites of the pNEB193-PB+Linker plasmid to obtain the plasmid pNEB193-PB+Linker+NM.

[0097] PCR amplified the end 36181 to 36604 fragment of SAd23, and PCR amplified the E4 coding region of Ad5 at the same time, and connected the two DNA fragments by overlapping PCR amplification to obtain the EK-Ad5-E4 gene fragment, using EcoRI and KpnI double enzymes cut and cloned into the pNEB193 vector to obtain th...

Embodiment 2

[0101] Example 2. Construction of recombinant adenovirus SAd23-L-eGFP.

[0102] After the eGFP gene fragment was cloned into SAd23-L, the recombinant adenoviral vector SAd23-L-eGFP was digested with BglII and there were twelve DNA bands of different sizes: 6985bp, 5235bp, 4489bp, 3955bp, 3900bp, 2334bp, 2052bp, 1740bp, 1416bp, 863bp, 635bp, 495bp, after comparing with Marker, are all correct bands, such as figure 2 As shown in A. At the same time, send the plasmid sequencing (BGI) to identify the correct band by enzyme digestion. The sequencing confirms that the gene of SAd23 is correctly connected to the pNEB193 plasmid. At the same time, the E1 and E3 coding regions are deleted, and the E4 coding region of SAd23 is replaced by the E4 coding region of Ad5. Instead, the exogenous gene of eGFP is connected to the SAd23-L vector to form a recombinant vector: SAd23-L-eGFP.

[0103] After the recombinant vector SAd23-L-eGFP was sequenced and identified correctly, HEK293 cells w...

Embodiment 3

[0104] Example 3. Construction of recombinant adenovirus SAd23-L-ZIKV-E.

[0105] Cloning the envelope protein (E) gene of ZIKV into the SAd23-L vector to obtain the plasmid: SAd23-L-ZIKV-E, and digesting the SAd23-L-ZIKV-E plasmid with HindIII will form 3 DNA bands: 16335bp , 11789bp and 6400bp, such as figure 2 As shown in A, compared with Marker, the sizes of the three DNA bands are completely correct. Enzyme digestion identified the correct plasmid sequencing (BGI), and the sequencing confirmed that the SAd23-L-ZIKV-E plasmid correctly connected the SAd23 gene to the pNEB193 plasmid, and deleted the E1 and E3 coding regions, and the E4 coding region of SAd23 Replaced by the E4 coding region of Ad5, the foreign gene of ZIKV-E protein was connected to the SAd23-L vector to form a recombinant vector: SAd23-L-ZIKV-E.

[0106] After the recombinant adenovirus vector SAd23-L-ZIKV-E was sequenced and identified correctly, it was linearly transfected into HEK293 cells, and the ...

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Abstract

The invention discloses a recombinant adenovirus expression vector, which is characterized in that an E1 coding region and an E3 coding region are deleted through a molecular cloning method based on agenome of a wild-type adenovirus SAd23, I-Ceu I and PI-Sce I cleavage sites are inserted in the E1 deletion region, and meanwhile, an E4 coding region of SAd23 is replaced with the E4 coding region of an adenovirus human serotype Ad5, so that the success rate of packaging the recombinant virus and the titer of the recombinant virus in human kidney embryo cells (HEK293) are improved, and the replication-defective recombinant adenovirus expression vector is obtained. The vector has the advantages that: 1. a large fragment of plasmids are constructed through a direct molecular cloning method, the method is simple and easy to operate, and can be used for constructing any large fragment of the vector; 2. the adenovirus expression vector can be constructed through multiple cloning connections,the steps are simple; and 3. screening positive clones at the bacterial level is easier and faster, and the construction time of the adenovirus vector is shortened.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a recombinant adenovirus vector used for gene therapy and vaccine vectors; specifically, the invention relates to a recombinant adenovirus expression vector SAd23-L. Background technique [0002] The development of adenovirus (adenovirus, Ad) as a gene expression vector began in the early 1960s. Adenoviruses can stimulate and induce effective T cell and B cell immune responses in different animals, but different types of adenoviruses have different inducing abilities. Adenoviruses in subgroup C induced the strongest specific T cell and B cell immune responses against foreign genes. Expression vectors based on adenovirus human serotype 5 (Ad5) and 2 (Ad2) are widely used in vaccine research and treatment, but due to the ubiquitous immune response against Ad5 adenovirus itself in the human body, this pre-existing immune response Including anti-adenovirus neutralizing antibody and kil...

Claims

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Application Information

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IPC IPC(8): C12N15/861A61K39/12A61P31/14
CPCY02A50/30
Inventor 罗升学刘博超马晓瑞张攀丽
Owner 广州佰芮慷生物科技有限公司
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