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Zika virus vaccine based on chimpanzee adenovirus vector and preparation method thereof

A Zika virus and adenovirus technology, applied in microorganism-based methods, viruses/phages, botanical equipment and methods, etc., can solve the problems of technical bottlenecks, restricted use, etc., and achieve a simple production process, low toxicity, and easy expansion. Enhancement and purification effect

Active Publication Date: 2018-05-04
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commonly used type 5 human adenovirus (HuAd5) pre-existing immunity in the population is as high as 74.2% (Wang Xiang, Xing Man, Zhang Chao et al. Neutralizing antibody responses to enterovirus and adenovirus in healthy adults in China. Emerg Microbes Infect, 2014, 3( 5):e30.), which restricts the use of this carrier
In addition, due to the relatively large genome of adenovirus, about 36kb, direct cloning of recombinant adenovirus vectors has become a technical bottleneck

Method used

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  • Zika virus vaccine based on chimpanzee adenovirus vector and preparation method thereof
  • Zika virus vaccine based on chimpanzee adenovirus vector and preparation method thereof
  • Zika virus vaccine based on chimpanzee adenovirus vector and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1, construction of pAdC7-M / E vector

[0062] Acquisition of the target gene: the signal peptide sequence derived from JEV is used for protein expression, the M / E gene sequence is derived from the FSS13025 virus strain, the signal peptide and the M / E gene sequence are codon optimized, and the optimized signal peptide sequence is shown in SEQ ID NO As shown in .1, the optimized M / E gene sequence is shown in SEQ ID NO.2. After being synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., it was cloned into the pCAGGS vector to obtain pCAGGS-M / E.

[0063]The acquisition of the recombinant adenovirus shuttle vector: using pCAGGS-M / E as a template, design a forward primer to access the XbaI restriction site and Kozak sequence (GCCACC), design a reverse primer to access the KpnI restriction site, use High-fidelity DNA polymerase is used for PCR, and the PCR product is subjected to gel electrophoresis, and then recovered and purified with a kit. Both the PCR product a...

Embodiment 2

[0065] Embodiment 2 Packaging AdC7-M / E

[0066] After linearizing pAdC7-M / E with PacI enzyme and inactivating it, transfected HEK 293 cells, plaques could be seen in about 7 days ( image 3 ), indicating that the adenovirus has been successfully packaged, wait another 2-3 days for all the cells to fall off, and collect the cells and supernatant in a cryopreservation tube.

Embodiment 3

[0067] Example 3 Western blot detection of protein expression

[0068] Spread HEK293T cells on a 12-well plate one day in advance, and add 10 9 , 10 8 , 10 7 For vpAdC7-M / E, the cells and supernatant were collected after 48 hours, and GAPDH was used as an internal reference to detect the expression of E protein by Western blot. It was found that in the cells and supernatant, the amount of E protein was positively correlated with the scale of the added adenovirus ( Figure 4 A, B).

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Abstract

The invention discloses a zika virus vaccine based on a chimpanzee adenovirus vector and a preparation method thereof, which belong to the field of a biotechnology and virology. A chimpanzee adenovirus AdC 7 is constructed as a replication defective type recombinant expression vector with favorable immunogenicity and hereditary stability, and the novel zika virus vaccine is prepared based on the vector. After mice are immunized through the vaccine, a neutralizing antibody aiming at a zika virus can be effectively induced, so that the mice are protected against being infected, and meanwhile, tissues and organs of the mice can be protected against being damaged.

Description

technical field [0001] The invention relates to a Zika virus vaccine based on a chimpanzee adenovirus vector and a preparation method thereof, belonging to the fields of biotechnology and virology. Background technique [0002] Zika virus is a mosquito-borne virus belonging to the Flaviviridae family. The Zika virus was first isolated from monkeys in the Zika forest in Uganda in 1947. It has not spread widely in the past 70 years, and the symptoms of human infection are relatively mild. The Zika virus, which broke out in 2015-2016, became a global crisis. So far, the epidemic has spread to 84 countries, including China. There is growing evidence that Zika virus infection in pregnant women is likely to cause abnormal brain development in fetuses, leading to microcephaly. Zika virus infection may cause Guillain-Barré syndrome in adults. Zika virus is mainly transmitted by the Aedes aegypti mosquito, but can also be transmitted from mother to child, blood and sexually. Zika...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N7/01A61K39/12A61P31/14C12R1/93
CPCA61K39/12A61K2039/5256C12N7/00C12N15/86C12N2710/10321C12N2710/10343C12N2770/24134Y02A50/30
Inventor 高福严景华徐坤戴连攀周东明
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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