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Expression vector based on adenovirus AdC7 and its construction method

An expression vector and adenovirus technology, applied in the fields of biotechnology and virology, can solve the problems of difficulty in direct cloning and large adenovirus genome

Active Publication Date: 2014-07-16
中国科学院上海免疫与感染研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because the adenovirus genome is relatively large, with a size of about 36kb, it is difficult to directly clone its genome

Method used

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  • Expression vector based on adenovirus AdC7 and its construction method
  • Expression vector based on adenovirus AdC7 and its construction method
  • Expression vector based on adenovirus AdC7 and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1. Amplification and purification of adenovirus and extraction of adenovirus genome

[0101] After wild-type AdC7 was amplified in HEK293 cells, it was purified by CsCl density gradient centrifugation, and then the viral genomic DNA was extracted, and the genome was digested with Bgl II for agarose electrophoresis. The results showed that the amplified and purified virus was AdC7 adenovirus. See figure 1 .

Embodiment 2

[0102] Example 2, Construction of a replication-defective adenoviral vector

[0103] According to the flowchart ( Figure 5 ), the fragments the ori, LITR, and NdeI-AgeI were obtained by PCR; these three fragments were fused into a fragment OLN by fusion PCR; OLN was digested with Spe I and Age I, and the AdC7 genome was digested with Hind III, Age I, and Spe I , followed by ligation to form plasmid pLITR.

[0104] The AdC7 genome was digested with Hind III and Avr II, and then the large fragment obtained by digestion was cloned into pLITR to form plasmid pOINH.

[0105] The fragment RsrII-AsisI obtained by PCR was cloned into pOINH to form pOINHR.

[0106] Finally, the fragment AvrII-RsrII obtained by PCR was cloned into the plasmid pOINHR to obtain pAdC7-ΔE1ΔE3.

[0107] Digest pAdC7-ΔE1ΔE3 with Bgl II, Xho I, and Mfe I, and perform agarose electrophoresis. The results show that pAdC7-ΔE1ΔE3 is the correct destination vector. See figure 2 .

Embodiment 3

[0108] Example 3, Construction of recombinant adenovirus AdC7-eGFP

[0109] After cloning eGFP into pAdC7, the enzyme digestion identification of the recombinant adenovirus vector pAdC7-eGFP is as follows: image 3 a.

[0110] The recombinant plasmid DNA was linearized and transfected into HEK293 cells. Viral plaques appeared on the tenth day, and then the plaques gradually expanded. Such as image 3 b.

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Abstract

The invention relates to an expression vector based on adenovirus AdC7 and its construction method. An effective strategy is designed for successfully construct the new expression vector based on chimpanzee adenovirus AdC7. The vaccine vector can be applied to the preparation of high expression viral vaccines with 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 AdC7 and a construction method thereof. Background technique [0002] Recombinant adenovirus vector has the characteristics of high transduction efficiency, high expression level, long duration, easy purification, etc., and can induce exogenous gene-specific cellular and humoral immune responses after immunization, so it is an ideal vaccine carrier. Expression vectors based on adenovirus human serotype 2 (AdHu2) and 5 (AdHu5) have been widely used in vaccine research and gene therapy, but since 40-60% of individuals in the population have been infected with the corresponding adenovirus, there are The corresponding pre-stored neutralizing antibodies limit the clinical application of AdHu2 and AdHu5 vectors. To overcome the effect of pre-existing neutralizing antibodies, one approach is to modify the cap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861A61K39/145A61P31/16
Inventor 周东明成涛
Owner 中国科学院上海免疫与感染研究所
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