Street virus strains of rabie virus HN10 complete genome sequence, and preparation and use thereof

A virus genome and whole genome technology, applied in the field of rabies street virus sequence, can solve the problem of difficult determination

Inactive Publication Date: 2009-06-24
中国疾病预防控制中心病毒病预防控制所
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  • Claims
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AI Technical Summary

Problems solved by technology

At present, the determination of the full-length genome sequence of the rabies street strain is a difficult technology, mainly because the total length of the rabies virus genome is about 12kb, and the proportion of base mutations is relatively large. It has been difficult to balance the two to obtain the full-length genome sequence with as few steps as possible

Method used

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  • Street virus strains of rabie virus HN10 complete genome sequence, and preparation and use thereof
  • Street virus strains of rabie virus HN10 complete genome sequence, and preparation and use thereof
  • Street virus strains of rabie virus HN10 complete genome sequence, and preparation and use thereof

Examples

Experimental program
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Embodiment 1

[0028] Example 1 Preparation of whole genome sequence of street rabies strain HN10

[0029] (1) Isolation of virus:

[0030] Take about 0.3 g of the hippocampus sample of the postmortem brain tissue of a rabies patient (the sample comes from a human brain tissue that died of rabies in Hunan Province, with the consent of the patient's family), and add penicillin (500 U / mL) and streptomycin (2 mg / mL) ) and 2% fetal bovine serum in PBS (pH7.4) were ground into a 30% suspension, centrifuged at 2000rpm at 4°C for 20min, and then the supernatant was inoculated on the BHK-21 cells that had formed a monolayer in the cell culture flask. After 2h, Supplement DMEM solution containing 2% fetal bovine serum, 37°C 5% CO 2 nourish.

[0031] (2) Extraction of viral RNA:

[0032] The virus HN10 passed to the fourth generation by BHK-21 cells was taken, and the virus was repeatedly frozen and thawed 3 times. Centrifuge at 12000r / min at 4°C for 10min, take the virus suspension and put it in ...

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Abstract

The invention discloses a whole genome sequence of a rabies street virus HN10, shown in SEQ ID No. 1. The invention also provides an amplimer of the whole genome sequence, shown in SEQ ID No.2 and SEQ ID No. 49. In the invention, the virus genome sequence is divided into 24 segments, corresponding cDNA is amplified by PCR method, a fragment primer is used for direct sequencing, the used primer is designed based on a nucleotide sequence in the conservative area of rabies viruses, the end of a primer 3' is terminated in the position of little amino acid degenerating codons, and a last basic group is terminated at a second-place basic group of an amino-acid codon. In the invention, the target fragment is effectively amplified, a whole genome sequence is provided. Therefore, the sequence is favorable for understanding the classification, evolution and pathogenicity of the virus and molecular epidemiology, etc. The virus is possibly used as the vector of a reverse genetic system, and the determination of the virus sequence lays a foundation for the construction of the virus vector.

Description

technical field [0001] The present invention relates to a rabies street virus sequence, specifically a whole genome sequence of a rabies street virus strain HN10, and a preparation method and use thereof. Background technique [0002] Rabies is a disease with a mortality rate of 100%, which poses a great threat to people's health. Understanding the molecular epidemiology of rabies virus is helpful to control the prevalence of rabies virus, and the nucleotide sequence and analysis of rabies street strains A powerful tool for molecular epidemiology. At present, the determination of the full-length genome sequence of the rabies street strain is a difficult technology, mainly because the total length of the rabies virus genome is about 12kb, and the proportion of base mutations is relatively large. It has always been difficult to balance between obtaining the full-length genome sequence with as few steps as possible. [0003] In view of the problems existing in the above-menti...

Claims

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Application Information

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IPC IPC(8): C12N15/47C12N15/11C12N15/10C12N15/86
Inventor 唐青李浩
Owner 中国疾病预防控制中心病毒病预防控制所
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