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Recombinant rabies virus carrying deoptimized M gene and two G genes

A rabies virus, de-optimized technology, applied in the direction of viruses, viral peptides, antiviral agents, etc., can solve the problems of high production costs, limited production scale, and high prices

Active Publication Date: 2017-09-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country's current veterinary inactivated rabies vaccine, due to the limitation of production scale and high production cost, the domestic veterinary inactivated vaccine has not yet been widely used in China
Most economically developed cities still prefer to import inactivated vaccines. These inactivated vaccines are too expensive for rural and economically backward areas, and it is difficult to promote them on a large scale. Although domestic inactivated vaccines are cheap, their immune effect is not good. good

Method used

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  • Recombinant rabies virus carrying deoptimized M gene and two G genes
  • Recombinant rabies virus carrying deoptimized M gene and two G genes
  • Recombinant rabies virus carrying deoptimized M gene and two G genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Optimization of M gene based on mouse codon bias

[0071] By referring to the Codon Usage Database, considering the coordination of nucleic acid sequences before and after optimization, the ratio of CpG and UpA after optimization, and the AU enrichment sequence, the M gene of RABV was optimized (underrepresented) based on mouse-derived codons. , and the reverse bias tropism-optimized M gene was named M-min. The results of M gene deoptimization are shown in Table 1.

[0072] Table 1 Codon preference optimization of RABV M gene

[0073]

[0074] Specifically, the sequence of the original M gene (RABV-M) of RABV is shown in SEQ ID NO.1, and the sequence of the deoptimized M gene (RABV-M-Min) is shown in SEQ ID NO.2.

Embodiment 2

[0075] Example 2 Homologous recombination seamless cloning of M-min fragment and pHEP-3.0 plasmid

[0076] 1. Construction of recombinant plasmid pHEP-3.0-M min

[0077] (1) The M gene was amplified using primers M-min-F / M-min-R (Table 2), and the vector pHEP-3.0 was linearized using primers vHEP-3.0-F / vHEP-3.0-R.

[0078] Table 2 Primers for recombinant rabies virus gene cDNA cloning

[0079]

[0080]

[0081] (2) After the reaction, 5 μL of PCR products were taken for electrophoresis detection on 1.0% agarose gel. M-min amplification PCR obtained a specific DNA electrophoresis band with a size of about 644bp, which was consistent with the test expectation, see figure 2 shown. The vector pHEP-3.0 was linearized to obtain a specific DNA electrophoresis band, which was about 16088bp, which was in line with the experiment expectation, see image 3 shown.

[0082] (3) The PCR amplification product was recovered from the gel DNA, and the recovered M-min fragment was con...

Embodiment 3

[0089] Embodiment 3 utilizes recombinant plasmid pHEP-3.0-M min Construction of recombinant plasmid pHEP-dG-M min

[0090] 1. Using the recombinant plasmid pHEP-3.0-M constructed in Example 2 min Construction of pHEP-dG-M min , see the construction scheme Figure 4 .

[0091] (1) Using the pHEP-3.0 plasmid as a template, use primers RV-G-F / RV-G-R to carry out PCR amplification on the recombinant plasmid pHEP-3.0, and add BsiWI and NheI restriction sites at both ends to obtain rabies virus G gene The complete open reading frame, the amplified length is 1575bp.

[0092] The G gene was amplified using the pHEP-3.0 plasmid as a template, and the reaction system was shown in Table 4 below:

[0093] Table 4 reaction system

[0094] 5×Phusion HF buffer

10.0 μL

dNTPs (10mM)

6μL

Mgcl 2 (10mM)

3.5 μL

pHEP-3.0 plasmid

5.5ng

RV-G-F (10μM)

2.5μL

RV-G-R (10μM)

2.5μL

Phusion polymerase (2U / μL)

0.5μL

...

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Abstract

The invention discloses a recombinant rabies virus carrying a deoptimized M gene and two G genes. Firstly, a reading frame part of the M gene is deoptimized, wherein the sequence after deoptimization is as shown in SEQ ID NO.2; secondly, a rabies virus HEP-Flury strain is taken as a skeleton, an M gene in the HEP-Flury is replaced by the deoptimized M gene, and an additional rabies virus G gene is inserted to obtain recombinant rabies virus pHEP-dG-Mmin plasmid carrying the deoptimized M gene and two G genes; finally, rescuing and screening are conducted to obtain a recombinant rabies virus strain rHEP-dG-Mmin. The recombinant virus has higher virus titer, and the cost of canine vaccine can be reduced. Under the condition of multiplicity of infection, G protein with higher level can be expressed, virus replication and transcription of each structural gene are increased, and the recombinant virus has the potential of serving as a rabies vaccine candidate strain.

Description

technical field [0001] The invention belongs to the technical field of virus molecular biology. More specifically, it relates to a recombinant rabies virus carrying a deoptimized M gene and two G genes. Background technique [0002] Rabies is a deadly, highly neurotropic zoonotic infectious disease caused by Rabies virus (RABV). 99% of human rabies infections come from sick dogs, and rabies is transmitted to humans by infecting domestic and wild animals through bites or scratches (usually through saliva). About 60,000 people die from rabies every year in the world, and Asia and Africa account for more than 95%. The incidence of rabies in my country is second only to India, ranking second in the world. At present, there is no effective treatment plan, and immunoprophylaxis is the main measure to prevent and control the disease. Therefore, it is particularly important to explore the replication and pathogenic mechanism of rabies virus and develop a cheap and efficient vacci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/47C12N7/01A61K39/205A61P31/14
CPCA61K39/12A61K2039/5252C07K14/005C12N7/00C12N2760/20121C12N2760/20122C12N2760/20134C12N2760/20151
Inventor 郭霄峰张琼罗均
Owner SOUTH CHINA AGRI UNIV
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