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Synthesizing virus genome of West Nile fever, and kit for detecting virus

A detection kit, West Nile fever technology, used in gene therapy, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2007-12-26
BAIOUSAIDI BIOTECH TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although no cases of West Nile fever virus infection have been reported in my country so far, there are mosquito species that transmit West Nile fever virus and birds as hosts in China.

Method used

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  • Synthesizing virus genome of West Nile fever, and kit for detecting virus
  • Synthesizing virus genome of West Nile fever, and kit for detecting virus
  • Synthesizing virus genome of West Nile fever, and kit for detecting virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1, preparation of the whole genome sequence of synthetic West Nile fever virus

[0066] 1. Artificially synthesized fragments of the whole genome sequence of West Nile fever virus. The fragments were synthesized in segments of 35-45mer length with reference to the genome cDNA sequence of West Nile fever virus NY99 strain reported in NCBI, and formed 35-45bp after annealing The cDNA fragments are spliced ​​into four cDNA fragments (AD(1-4830), DF(3601-7230) and FH(6001-9630) with a length of about 3-4kb through overlapping parts between two adjacent fragments. , HJ (8401-11925).The West Nile virus NY99 strain genome cDNA sequence reported in the NCBI is the sequence shown in AF196835 for the acceptance number in the NCBI;

[0067] 2. Perform PCR enzymatic reaction on the fragment, and introduce restriction sites into both ends of the fragment.

[0068] 3. Insert the four cDNA fragments into the rebuilt pGEM-5zf (promega company) (insert the Not I, Xba I, BamH...

Embodiment 2

[0069] Embodiment 2, the assay of West Nile fever virus full-length cDNA clone

[0070] 1. Enzyme digestion assay of the full-length cDNA clone of West Nile fever virus:

[0071] Table 3. Enzyme digestion system used for enzyme digestion assay of West Nile fever virus full-length cDNA clone

[0072] H and B buffers are the most suitable reaction buffers (Biolab Company) for different domestic endonucleases.

[0073] Digestion conditions: 37°C, 4 hours.

[0074] Results of enzyme digestion: After two double enzyme digestions and three single enzyme digestions, electrophoresis on 0.8% agarose gel proved that the recombinant plasmid pBR322 / WNV contained the full-length cDNA of West Nile fever virus. The restriction map is consistent with theoretical speculation (see Figure 5, where, 1: pBR3-WNV / XbaI+BamHI; 2: pBR3-WNV / NotI+XbaI; 3: pBR3-WNV / BamHI; 4: pBR3-WNV / XhoI; 5: pBR3-WNV / EcoRI; M: 1 kb gradient marker).

[0075] 2. PCR detection of the full-length cDNA clone of West ...

Embodiment 3

[0082] PCR results (see FIG. 6 ): PCR amplification and electrophoresis on 1% agarose gel proved that the full-length cDNA clone of West Nile fever virus contained all ORFs of the whole genome of West Nile fever virus. Embodiment 3, subcloning in vitro transcription experiment of West Nile fever virus full-length cDNA

[0083] Transcription system: subcloned plasmid pGEM-5zf-AD (2 μg) linearized with Not I, NTP (10 mM) 10 μl, RNase inhibitor 1 μl (Takara Company), 10×T7 RNA polymerase buffer 5 μl, T7 RNA polymerase (50U) (Takara Company), add water to make up the total system to 50μl.

[0084] Transcription conditions: 37°C, after 2 hours, add DNase I (Takara Company) for 30 minutes.

[0085] Transcription results (see Figure 8): The size of the transcribed RNA conforms to the theoretical value without degradation and can be used for the following experiments.

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Abstract

This invention discloses chemical splicing of full-length cDNA of western Nile virus, RT-PCR and real-time quantitative PCR techniques for detecting western Nile virus, and test kit for detecting western Nile virus. The full-length cDNA of western Nile virus can be used for detecting western Nile virus, developing anti-western Nile virus vaccines, and preventing western Nile virus infection. The test kit has such advantages as high sensitivity, easy operation and simple apparatus, and is suitable for high-throughput western Nile virus screen at frontier ports.

Description

technical field [0001] The invention relates to the whole genome sequence of the artificially synthesized West Nile virus and a preparation method thereof, and also relates to a detection method and a detection kit of the West Nile virus. Background technique [0002] West Nile virus (English full name is Western Nile Virus, abbreviated as WNV) infection is a natural foci disease transmitted by mosquitoes, with birds as the main animal host. Infection and immunity epidemiological surveys show that about 80% of West Nile fever virus-infected patients are asymptomatic, and the other 20% have fever as the main symptom. Other common symptoms include headache, extreme fatigue, nausea, vomiting, rash, lymph node inflammation. If there are no serious complications, the above symptoms usually resolve spontaneously in about a week, but fatigue symptoms usually last longer. Central nervous system infection is more common in the elderly, mainly meningitis and encephalitis, is the mos...

Claims

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Application Information

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IPC IPC(8): C12N15/33C07H21/04C12Q1/68A61K48/00
Inventor 彭小忠李莲心
Owner BAIOUSAIDI BIOTECH TECH DEV
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