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Kit for amplifying herpes simplex virus 1 (HSV-1) alkaline nuclease gene and method for expressing HSV-1 alkaline nuclease

A nuclease and kit technology, applied in the field of HSV-1 alkaline nuclease, can solve the problems of no alkaline nuclease, complex technical solutions, difficulty in obtaining a large amount of alkaline nuclease, etc.

Inactive Publication Date: 2012-07-04
CHENGDU MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The technical scheme of this method is complicated, and it is difficult to obtain a large amount of alkaline nuclease, the cost is high, and industrial application is difficult
There is no commercially available alkaline nuclease

Method used

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  • Kit for amplifying herpes simplex virus 1 (HSV-1) alkaline nuclease gene and method for expressing HSV-1 alkaline nuclease
  • Kit for amplifying herpes simplex virus 1 (HSV-1) alkaline nuclease gene and method for expressing HSV-1 alkaline nuclease
  • Kit for amplifying herpes simplex virus 1 (HSV-1) alkaline nuclease gene and method for expressing HSV-1 alkaline nuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] (1) Virus genome extraction: culture vero cells with RPMI 1640 medium containing 10% fetal bovine serum, inoculate HSV-1 until the cells are damaged, that is, the CPE reaches +++~++++, harvest the cells, and extract them with viral DNA Extraction kit to extract the whole genome of HSV-1;

[0091] (2) According to the nucleotide sequence of the UL12 gene fragment published by GenBank, primers were designed with PRIMER5.0 software, and the primer sequences are shown in SEQ ID NO: 1-2;

[0092] (3) Amplify the target gene: take the extracted HSV-1 whole genome as a template, and use the nucleotide sequence as primer pair 1 shown in SEQ ID NO: 1~2 to amplify the target gene UL12 gene fragment:

[0093] reaction system:

[0094]

[0095] Reaction conditions: pre-denaturation at 96°C for 3min, denaturation at 95°C for 40s, annealing at 68°C for 2min and 30s, 31 cycles, extension at 72°C for 10min.

[0096] (4) Product recovery: PCR products were separated by 0.8% conven...

Embodiment 2

[0099] (1) Template preparation: The UL12 gene fragment obtained in Example 1 was ligated with the pMD-19T vector under the action of T4 DNA ligase to construct a recombinant pMD19T vector.

[0100] (2) Amplify the target gene: use the recombinant pMD19T vector connected with the UL12 gene fragment obtained in step (1) as a template, and use the nucleotide sequence such as the primer pair shown in SEQ ID NO: 1-2 as primers to amplify the target gene UL12 gene fragment:

[0101] reaction system:

[0102]

[0103]

[0104] Reaction conditions: pre-denaturation at 96°C for 3min, denaturation at 95°C for 40s, annealing at 68°C for 2min and 30s, 31 cycles, extension at 72°C for 10min.

[0105] (3) Product recovery: PCR products were separated by 0.8% conventional agarose gel electrophoresis, and amplified products were recovered with a gel recovery kit.

[0106] (4) Result detection: The UL12 gene fragment recovered from the gel was connected to the pMD-19T vector to const...

Embodiment 3

[0109] (1) Construction of recombinant expression plasmid

[0110] The UL12 gene fragment obtained in Example 1 or Example 2 was connected with pET28a (+) plasmid or pET32a (+) under the action of EcoRI and HindIII endonuclease to form pET28a-UL12 recombinant expression plasmid or pET32a-UL12 recombinant expression plasmid, and construct The recombinant plasmid contains the T7 RNA polymerase promoter, which controls the expression of the protein, and contains 2 His tags, which can be used to purify the protein.

[0111] (2) Recombinant expression plasmid induced expression in Escherichia coli BL21

[0112] The pET28a-UL12 recombinant expression plasmid obtained in step (1) was passed through CaCl 2 Mediate the transformation into E.coliBL21 competent cells, culture at 37°C on LB culture plates containing 50μg / ml kalamycin, shake at 220r / min overnight;

[0113] Pick the Escherichia coli clone that obtained the recombinant expression plasmid;

[0114] Inoculate the Escherichi...

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Abstract

The invention provides a kit for amplifying a herpes simplex virus 1 (HSV-1) alkaline nuclease gene fragment UL12. The kit comprises a primer pair of which the nucleotide sequences are shown as SEQ ID NO:1-2, and other related reagents. The invention also provides a method for preparing HSV-1 alkaline nuclease by using the kit. By a genetic engineering method, a great amount of HSV-1 alkaline nuclease with high bioactivity is obtained, and the method has important significance for further searching anti-virus medicines and illustrating action mechanisms of the medicines, and lays an experimental basis for constructing medicine screening molecular models for the HSV-1 alkaline nuclease in vitro.

Description

technical field [0001] The invention relates to a method for obtaining viral protein, in particular to a method for amplifying HSV-1 alkaline nuclease gene by PCR technology and obtaining HSV-1 alkaline nuclease through gene expression. Background technique [0002] Herpes simplex virus type 1 (HSV-1) is an enveloped DNA virus, which is a common pathogen that endangers human health and easily causes recurrent infection and latent infection. HSV-1 has a wide range of infection sites, can cause cold sores, genital herpes, encephalitis, keratitis, etc., and is related to cervical cancer and lip cancer. HSV-1 is neurotoxic and can invade the central nervous system and destroy nerve cells. [0003] Alkaline nuclease is encoded by the UL12 gene in the HSV-1 genome. It has 5'-3' exonuclease and endonuclease activities. It is considered to be an alkali because of its high optimum pH value. sexual nuclease. It plays an important role in the replication of HSV-1. Studies have confi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/55C12N15/63C12N9/22
Inventor 陈恬张磊曹康郭洪秀代富英
Owner CHENGDU MEDICAL COLLEGE
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