Recombinant adenovirus high-expression vector for promoting effective presentation of Hantavirus fusion protein G2S0.7

A recombinant adenovirus, fusion protein technology, applied in molecular biology, immunology and immunization applications, recombinant adenovirus high expression vector field, can solve the problems of low protein expression and unsatisfactory immune level.

Inactive Publication Date: 2012-08-01
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

[0005] However, we also found some problems in the research. For example, although the complete fusion protein can be expressed by using various systems, the overall protein expression is still low.
In addition, after immunizing animals with fusio

Method used

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  • Recombinant adenovirus high-expression vector for promoting effective presentation of Hantavirus fusion protein G2S0.7
  • Recombinant adenovirus high-expression vector for promoting effective presentation of Hantavirus fusion protein G2S0.7
  • Recombinant adenovirus high-expression vector for promoting effective presentation of Hantavirus fusion protein G2S0.7

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[0024] The adenovirus expression system used in the present invention was purchased from Adeno-X of CLONTECH company TM system (Product No. K1650-1), the transfer vector is pShuttle.

[0025] The recombinant adenovirus transfer vector G2S0.7-pCAG used in the present invention was previously constructed by the applicant. (see patent application number 200910254563.2)

[0026] The recombinant adenovirus rAd-G2S0.7-pCAG and rAd-G2S0.7 used in the present invention were packaged and purified by the applicant in the early stage.

[0027] The bivalent HFRS inactivated vaccine used in the present invention was purchased from Zhejiang Tianyuan Company.

[0028] The Ub gene sequence inserted in the present invention is Ub up primer, Ub down primer upstream and downstream primers, pCMV-F3Ub as template, and 55°C as annealing temperature for PCR amplification, as shown in the attached figure 1 shown. The PCR product was ligated with the T-easy carrier overnight at 14°C, and the ligat...

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Abstract

The invention relates to a recombinant adenovirus high-expression vector for promoting effective presentation of Hantavirus fusion protein G2S0.7. A G2S0.7-pCAG transfer vector containing G2S0.7 mosaic gene of Hantavirus 76-118 strain and a CAG promoter/enhancer is mainly transformed to promote the effective presentation of the high-expression Hantavirus fusion protein Hantavirus in an organism. By a gene recombination technology, the recombinant adenovirus transfer vector G2S0.7-pCAG containing the mosaic gene G2S0.7 is transformed, and Ub gene is connected to the vector. The recombinant transfer vector and the adenovirus vector are respectively subjected to double digestion, and a recombinant fragment Ub-G2S0.7-pCAG is integrated into the DNA of the adenovirus vector; and after packaging, purification and titer detection, rAd-Ub-G2S0.7-pCAG and recombinant adenovirus rAd-G2S0.7-pCAG containing the G2S0.7 mosaic gene and the CAG promoter/enhancer, which is constructed early by the inventor are respectively used for immunizing C57BL/6 mice, immunological characteristics are researched, and results show that compared with the conventional recombinant adenovirus, the recombinant adenovirus can effectively improve partial humoral immune response level and partial cellular immune response level of organisms of experimental animals.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to related fields such as molecular biology, immunology and immune application. The invention relates to the reconstruction of an adenovirus recombinant transfer vector and a recombinant adenovirus high-expression vector for promoting the effective presentation of hantavirus fusion protein G2S0.7 to the pathogen of hemorrhagic fever with renal syndrome (HFRS). Background technique [0002] Research status of hemorrhagic fever with renal syndrome and its genetic engineering vaccine: [0003] Hemorrhagic fever with renal syndrome (HFRS) is caused by Hantavirus (HV) and is an acute viral infectious disease transmitted by rodents. Clinically, it is characterized by fever, bleeding and acute renal failure. Damage is the main feature. China is the country with the most severe HFRS epidemic in the world. It has the characteristics of a wide range of epidemics, a large number of patients, and a...

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Application Information

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IPC IPC(8): C12N15/861
Inventor 徐志凯张芳琳白文涛李璞媛程林峰李凯吴兴安于澜张亮
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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