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Expression vector based on adenovirus AdC68 and construction method thereof

An expression vector and adenovirus technology, applied in the fields of biotechnology and virology, can solve the problems of cumbersome screening of positive clones, easy mutation of adenovirus genome, and increased genetic changes of adenovirus

Inactive Publication Date: 2014-07-23
INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the construction strategy of adenovirus vectors generally adopts the method of homologous recombination, including: (1) intracellular homologous recombination, the disadvantage of this method is that it is easily contaminated by the parental adenovirus, and must go through 2-3 times of virus plaque purification, And identify the recombinant virus through multiple plaque formation methods; in addition, the recombination efficiency is low, and the recombination time needs at least two weeks
(2) Site-specific recombination, the disadvantage is that it is also susceptible to contamination by parental viruses, and requires multiple virus plaque purifications. Due to the introduction of the Cre / loxp recombination system, the probability of genetic changes in adenoviruses will increase, resulting in unstable adenovirus progeny
(3) homologous recombination in bacteria (such as AdEasy TM System), its disadvantage is that it is cumbersome to screen positive clones, and at the same time due to the introduction of homologous recombination method, the adenovirus genome is prone to mutation in Escherichia coli

Method used

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  • Expression vector based on adenovirus AdC68 and construction method thereof
  • Expression vector based on adenovirus AdC68 and construction method thereof
  • Expression vector based on adenovirus AdC68 and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0115] Example 1. Amplification of wild-type adenovirus AdC68 and its genome isolation

[0116] The wild-type adenovirus AdC68 purchased from ATCC was amplified in HEK293 cells, the supernatant of the virus was collected, and then purified by cesium chloride gradient centrifugation to obtain a large amount of wild-type adenovirus AdC68. Isolate the genome of adenovirus AdC68, digest it with Mfe I and Bgl II, and go through 1% agarose gel electrophoresis. The digested DNA band proves that the genome of AdC68 is complete and correct, see figure 1 .

Embodiment 2

[0117] Example 2, construction of replication-deficient adenovirus AdC68

[0118] According to the analysis of the nucleotide sequence of the adenovirus AdC68 genome, it was divided into four fragments by using the properties of the restriction site ( FIG. 2A ), and then cloned into the pNEB193 vector respectively in sequence. Among them, the E1 part of the adenovirus AdC68 is connected with a Linker fragment containing the restriction sites I-Ceu I and PI-Sce I through the two restriction sites of SnaB I and Nde I, which will replace the about 3.5kb fragment of the E1 part . The identification of restriction endonucleases Xho I, Bgl II, Mfe I and Xma I proved that the constructed replication-defective adenovirus vector AdC68 was correct, as shown in Fig. 2B-C.

Embodiment 3

[0119] Example 3. Construction of the replication-deficient adenoviral vector pNEB193-E1-deleted-EGFP containing the reporter gene EGFP

[0120] PI-Sce I and I-Ceu I double digest the pShuttle-CMV-EGFP plasmid, recover the target fragment of CMV-EGFP from agarose gel, and connect it to the pAdC68- The E1-deleted vector was transformed into Stbl2 competent cells, the positive clones were screened, and the reporter gene CMV-EGFP was identified to be completely correct after enzyme digestion, as shown in Figure 3A-B.

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Abstract

The invention relates to an expression vector based on an adenovirus AdC68 and a construction method thereof. By improving a construction method, the vaccine vector based on a chimpanzee adenovirus AdC68 genome is constructed. The vaccine vector can be applied to preparation of a vaccine capable of high-efficiency expression and having good immunogenicity.

Description

technical field [0001] The invention belongs to the fields of biotechnology and virology; more specifically, the invention relates to an expression vector based on adenovirus AdC68 and a construction method thereof. Background technique [0002] Adenovirus (Adenovirus) is a non-enveloped, linearized double-stranded DNA virus, which can efficiently infect various mammalian cells and is easy to amplify and purify. It is widely used as a gene carrier tool in molecular and cell biology research. Adenovirus can effectively induce strong humoral immunity and cellular immune response in vivo, and is considered to be one of the most promising vaccine vectors, including for the development of HPV, HIV, hepatitis B, hepatitis C, influenza, malaria, rabies and other diseases vaccine. Traditional adenovirus vectors are mainly based on human serotypes AdHu2 and AdHu5, among which AdHu5 was once used in clinical gene therapy. However, according to the serotype survey, about 40-60% of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K39/125A61P31/14
Inventor 周东明杨勇丁妙
Owner INST PASTEUR OF SHANGHAI CHINESE ACADEMY OF SCI
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