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A herpes simplex virus thymidine kinase mutant and its preparation method and use

A technology of herpes simplex virus and thymidine kinase, which is applied in the field of genetic engineering, can solve the problems of large side effects and poor curative effect, and achieves the effect of enhancing the effect opportunity, good clinical application prospect and reducing the competitive interference.

Active Publication Date: 2017-04-19
北京爱施凯健康管理发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Therefore, the use of natural herpes simplex virus thymidine kinase to treat tumors has the disadvantages of poor curative effect and large side effects.

Method used

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  • A herpes simplex virus thymidine kinase mutant and its preparation method and use
  • A herpes simplex virus thymidine kinase mutant and its preparation method and use
  • A herpes simplex virus thymidine kinase mutant and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 Preparation of herpes simplex virus thymidine kinase mutant of the present invention

[0031] 1. Recombinant expression

[0032] 1.1 Gene cloning

[0033] a. Synthetic recombinant target gene:

[0034] (1) Natural tk gene: chemical synthesis method synthetic GenBank number is the HSV-1tk gene of AB032875, and its nucleotide sequence is as shown in SEQ NO: 1:

[0035]

[0036] (2) tk gene variant 1: the 160th amino acid is mutated from isoleucine (I) to leucine (L) by chemical synthesis method, and the 161st amino acid is mutated from phenylalanine (F) to leucine Acid (L), the 167th position is mutated from alanine (A) to tyrosine (Y), and the 168th position is mutated from alanine (A) to tyrosine (Y) 4 amino acid residues are mutated simultaneously Mutant TK (I160L, F161L, A167Y, A168Y), abbreviated as mtk1608, its nucleotide sequence is shown in SEQ NO: 2:

[0037]

[0038]

[0039] Its amino acid sequence is shown in SEQ NO: 4:

[0040]

...

Embodiment 2

[0080] Construction of embodiment 2 pBES-mtk1608

[0081] 1. Experimental method

[0082] (1) Preparation of pBES vector

[0083]The first step is to replace the sequence region of the Ampicillin resistance gene of the pGEX-5x-1 plasmid 1344-2220nt with the kanamycin resistance gene, that is, (a) first use the PCR method from the plasmid pEASY-T1 whose GenBank number is EU233623 Amplify the 1203-2114nt fragment to obtain the kanamycin resistance gene. The forward PCR primer is: agtagacgtcctggtaag gttgggaag, the position is at 1203-1219nt, and an AatII restriction site is added at the 5' end. The reverse primer is: acgtcagtggctgcaattcagaagaactcgtc, the position is at 2114-2085nt, with an AlwN1 restriction site added at the 5' end; the PCR amplification conditions are: 95°C for 4min, followed by 25 cycles of 95°C for 40Sec, 55°C for 30Sec, and 72°C for 1min; (b) the above PCR purified product and pGEX-5x-1 plasmid were double-digested with Aat II and AlwN1 respectively, and th...

Embodiment 3

[0089] Embodiment 3 The preparation of the recombinant bifidobacterium containing pBES-mtk1608 recombinant vector

[0090] The recombinant bifidobacterium of the pBES-mtk1608 recombinant vector prepared in Example 2 (also known as Bifidobacterium-mTK1608) can be prepared as an injection for vacuum safe packaging at room temperature and for injection, and is constructed according to the following steps:

[0091] a) The recombinant bifidobacterium of the pBES-mtk1608 recombinant vector prepared in Example 2 (i.e., the bifidobacterium-mTK1608 solid tumor gene therapy system), which was preserved after freeze-drying with 20% skimmed milk in a vacuum, was Bifidobacterium-mTK1608 Seed bacteria for gene therapy systems for solid tumors.

[0092] b) Resuscitate the seed bacteria described in a) above, spread it on the resistant MRS medium containing kanamycin and culture it anaerobically for 48 hours, then pick a single resistant colony and culture it in the resistant MRS liquid Bact...

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Abstract

The invention discloses a nucleotide sequence represented by SEQ ID NO:2, and discloses a recombinant vector and recombinant bacteria containing the nucleotide sequence, and a hrpesvirus hominis thymidine kinase mutant coded by the nucleotide sequence represented by SEQ ID NO: 2 as well as a preparation method and use thereof. The herpesvirus hominis thymidine kinase mutant can be used for effectively treating solid tumor, and is good in security and good in clinical application prospect.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a herpes simplex virus thymidine kinase mutant and its preparation method and application. Background technique [0002] Tumor is one of the diseases that seriously threaten human health. More than 90% of common malignant tumors in human body tumors are solid tumors; with the aggravation of environmental pollution, its incidence rate is increasing year by year. The traditional treatment methods for solid tumors are mainly surgery, chemotherapy and radiotherapy, but the tumor metastasis rate and recurrence rate after treatment are high, and the survival rate is still unsatisfactory. Therefore, it is of great social and economic significance to further research and develop new and efficient solid tumor treatment methods combined with the latest research results in tumor physiology and medical molecular biology. [0003] The use of suicide genes to treat solid tumors has become a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/63C12N1/21C12N9/12A61K38/45A61P35/00
Inventor 马永平宋方洲唐伟
Owner 北京爱施凯健康管理发展有限公司
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