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Adenovirus high expression vector for improving expression of hantavirus fusion protein G2S0.7

A fusion protein, hantavirus technology, applied in the biological field, can solve the problem that the gene can not get satisfactory expression effect and so on

Inactive Publication Date: 2012-10-31
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

Although the widely used CMV promoter is a relatively effective expression system promoter, some genes usually cannot be expressed satisfactorily in practical applications.

Method used

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  • Adenovirus high expression vector for improving expression of hantavirus fusion protein G2S0.7
  • Adenovirus high expression vector for improving expression of hantavirus fusion protein G2S0.7
  • Adenovirus high expression vector for improving expression of hantavirus fusion protein G2S0.7

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Embodiment Construction

[0020] 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, which contains CMV promoter.

[0021] The Hantaan virus used in the present invention is Hantaan virus 76-118 strain. Its genome consists of three gene segments, L, M and S. The L segment encodes RNA-dependent RNA polymerase; the M segment encodes envelope glycoproteins (GP) G1 and G2; the S segment encodes nucleocapsid protein (NP). The chimeric gene involved in this patent is obtained by PCR amplification of the G2 part of the M segment and the 0.7 part of the S segment respectively, and is fused by gene fusion technology, and Xba I is designed at its 5' end and 3' end The NotI restriction site was cloned into the corresponding restriction site of the adenovirus transfer vector pShuttle to obtain a recombinant plasmid, which was named G2S0.7-pShuttle.

[0022] The replaced CAG promoter / enhanc...

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Abstract

The invention relates to reconstruction of an adenovirus transfer vector, in particular to reconstruction of a pShuttle transfer vector of mosaic gene G2S0.7 of hantavirus (HV) serving as hemorrhagic fever with renal syndrome (HFRS) pathogen for improving the expression of fusion protein G2S0.7. The adenovirus transfer vector G2S0.7-pShuttle containing the mosaic gene G2S0.7 is reconstructed by using the gene recombination technology. The reconstruction comprises the following steps: replacing a CAG promoter / enhancer for a CMV promoter; or inserting a WPRE transcriptional control element at the 3' end of the promoter; or replacing the promoter and inserting a WPRE control element. The vector expresses the hantavirus fusion proteins G1S0.7 respectively, and compares the expression level offoreign protein in each recombinant adenovirus. The result shows that the vector can effectively improve the expression of the fusion protein G2S0.7 compared with the primary transfer vector pShuttle, and has most obvious effect of replacing a promoter group.

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 the adenovirus recombination transfer vector and its expression to the chimeric gene G2S0.7 of Hantavirus (HV), the causative agent 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 high mortality rate. So far, there is still a lack of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/861C12N15/67C12R1/93
Inventor 徐志凯张芳琳白文涛李璞媛吴兴安李凯胡刚
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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