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

A nuclease and kit technology, applied in the field of HSV-1 alkaline nuclease, can solve the problems of no alkaline nuclease, high cost, and difficulty in industrial application

Inactive Publication Date: 2011-02-23
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 (HSV)-1 alkali nuclease gene and method for expressing HSV-1 alkali nuclease
  • Kit for amplifying herpes simplex virus (HSV)-1 alkali nuclease gene and method for expressing HSV-1 alkali nuclease
  • Kit for amplifying herpes simplex virus (HSV)-1 alkali nuclease gene and method for expressing HSV-1 alkali nuclease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1 amplifies UL12 gene fragment

[0057] (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;

[0058] (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;

[0059] (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:

[0060] reaction system:

[0061] Template 1μl

[0062] Nucleotide sequence as shown in SEQ ID NO: 1 Primer 1μl

[0063] Nucleotide sequence as shown in SEQ ID NO: 2 Primer 1μl

[0064] GC buffer 12.5μl

[0065] dNTP 2μl

[0...

Embodiment 2

[0071] Embodiment 2 further amplifies the UL12 gene fragment

[0072] (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.

[0073] (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:

[0074] reaction system:

[0075] Template 1μl

[0076] Nucleotide sequence as shown in SEQ ID NO: 1 Primer 1μl

[0077] Nucleotide sequence as shown in SEQ ID NO: 2 Primer 1μl

[0078] GC buffer 12.5μl

[0079]2 μl of dNTPs

[0080] 0.25 μl of LAtaq

[0081] wxya 2 7.25 μl of O

[0082] 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...

Embodiment 3

[0086] Expression of embodiment 3 alkaline nuclease and the identification of existing form

[0087] (1) Construction of recombinant expression plasmid

[0088] The UL12 gene fragment obtained in Example 1 or Example 2 is 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, The constructed recombinant plasmid contains T7 RNA polymerase promoter, controls the expression of alkaline nuclease, and contains 2 His tags, which can be used for protein purification.

[0089] (2) Recombinant expression plasmid induced expression in Escherichia coli BL21

[0090] Pass the pET28a-UL 12 recombinant expression plasmid that step (1) obtains through CaCl 2 Mediate the transformation into E.coli BL21 competent cells, culture at 37°C on LB culture plates containing 50 μg / ml karamycin, shake at 220r / min overnight;

[0091] Pick the Escherichia coli c...

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Abstract

The invention provides a kit for amplifying a gene segment UL12 of herpes simplex virus (HSV)-1 alkali nuclease. The kit comprises a primer pair of which the nucleotide sequence is shown as SEQ ID NO:1-2 and other relevant reagents. The invention also provides a method for obtaining HSV-1 alkali nuclease with bioactivity by using the kit. A large amount of HSV-1 alkali nuclease with high bioactivity can be obtained by a genetic engineering method, the alkali nuclease play an important role in further finding an anti-virus medicament and clarifying a medicament action mechanism and an experimental basis is laid for in-vitro construction of a medicament screening molecular model of the HSV-1 alkali nuclease.

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): C12N15/10C12N15/55C12N9/22C12N15/70
Inventor 陈恬张磊曹康郭洪秀代富英
Owner CHENGDU MEDICAL COLLEGE
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