Expression vector based on recombinant adenoviruses and construction method thereof

A recombinant adenovirus and expression vector technology, which is applied in antiviral agents, viral antigen components, recombinant DNA technology, etc., can solve the problems of difficult acquisition of recombinant adenovirus, easy mutation, complicated operation, etc., and achieve easy screening of positive clones , easy operation, and the effect of improving the success rate

Inactive Publication Date: 2018-01-12
SOUTHERN MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional method is to carry out homologous recombination of exogenous gene and wild-type virus in the packaging virus cell line to obtain recombinant adenovirus, but this method is complicated to operate and it is not easy to obtain a single recombinant adenovirus; the second method is Based on

Method used

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  • Expression vector based on recombinant adenoviruses and construction method thereof
  • Expression vector based on recombinant adenoviruses and construction method thereof
  • Expression vector based on recombinant adenoviruses and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0109] Example 1. Amplification and purification of adenovirus and extraction of adenovirus genome

[0110] Wild-type AdC6 proliferated in 293A cells, and the purified virus was obtained by CsCl density gradient centrifugation, and then the viral genomic DNA was extracted, digested with AgeI, and subjected to agarose electrophoresis. The results showed that the amplified and purified virus was AdC6 adenovirus, For experimental results, please refer to figure 1 .

Embodiment 2

[0111] Example 2 Construction of a replication-deficient adenoviral vector

[0112] according to Figure 5 As shown in the flow chart, the LITR fragment (about 450 bp) of AdC6 genome was amplified by PCR, the pNEB193 plasmid and the PCR product were digested with PmeI and BstAPI, and ligated to form the plasmid pNEB193-PB.

[0113] PUC57-linker and pNEB193-PB plasmids were digested with SnaBI and NdeI, and ligated into plasmid pNEB193-PB+linker.

[0114] The genome of AdC6 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.

[0115] The DNA fragments from 30137 to 33841 and from 34746 to 36604 of the AdC6 genome were amplified by PCR, and the DNA fragments from 33193 to 34077 of the Ad5 genome were amplified by PCR. The three genomes were amplified by Overlapping PCR, and connected to the PNEB193 vector through the EcoRI and PacI restriction s...

Embodiment 3

[0120] Example 3, Construction of recombinant adenovirus AdC6-Ad5ORF6-eGFP

[0121] After cloning the eGFP gene fragment into AdC6-Ad5 ORF6, the enzyme digestion and identification results of the recombinant adenoviral vector AdC6-Ad5ORF6-eGFP can be found in image 3 a.

[0122] refer to image 3 B, After the recombinant vector AdC6-Ad5ORF6-eGFP was linearized and transfected into 293A cells, cytopathy appeared on the eighth day, and then the cytopathy gradually expanded.

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Abstract

The invention discloses a method for preparing an expression vector of adenoviruses. The method comprises the following steps: using a genome of wild type adenoviruses AdC6 as a base, deleting an E1 coding region and an E3 coding region according to a genome direct cloning method, and inserting I-Ceu I and PI-Sce I digestion sites in the E1 deleted region. Simultaneously, OFR6 of human-derived adenoviruses Ad5 is used for replacing ORF6 of AdC6, the success rate of packaging recombinant viruses and the titer of the packaged recombinant viruses in human-derived kidney blastocyte (293A) are improved, and the replication-deficient expression vector of the recombinant adenovirus is obtained. According to the direct molecular cloning method, long-fragment plasmids are constructed, the method issimple and easy to operate, and the method can be used for construction of any long-fragment vectors. The construction of the expression vector of the adenoviruses can be completed through several times of cloning connection, and the steps are simple; the E1 and E3 regions are deleted, the Ad5 ORF6 is replaced, and the deletion and replacing can be completed in the PCR amplification process of DNA fragments; the method is easier and quicker in horizontal bacteria screening and positive cloning, and the construction time of the adenovirus vector is shortened.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant adenovirus vector for gene therapy and vaccine vector and its construction method. Background technique [0002] The development of adenovirus (adenovirus, Ad) as a gene expression carrier began in the early 1960s, when virologists observed that the adenovirus genome can hybridize with the simian virus 40 (SV40) genome, indicating that the adenovirus genome can carry heterogeneity. source gene. Since then, adenovirus has gradually developed into an important vector system. [0003] At present, 57 different adenovirus serotypes have been identified. Recombinant adenovirus vectors have been widely used in gene therapy and vaccine vectors, especially human serotype 5 adenovirus, which is currently used in tumor gene therapy and human immunodeficiency The primary viral vector for viral (HIV) vaccine research. In addition, the development of vaccines for pathogens such as...

Claims

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Application Information

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IPC IPC(8): C12N15/66C12N15/861A61K39/12A61P31/12
Inventor 李婷婷罗升学王文敬黎诚耀
Owner SOUTHERN MEDICAL UNIVERSITY
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