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Preparation method, expression gene, recombinant expression vector and application of gp32 single-strand binding protein

A single-chain binding protein and expression vector technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as unfavorable promotion and use of gp32 single-chain binding protein, low purity, etc.

Active Publication Date: 2018-11-30
深圳市艾伟迪生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the domestically produced gp32 single-chain binding protein generally has the problem of low purity, which is not conducive to the popularization and use of gp32 single-chain binding protein

Method used

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  • Preparation method, expression gene, recombinant expression vector and application of gp32 single-strand binding protein
  • Preparation method, expression gene, recombinant expression vector and application of gp32 single-strand binding protein
  • Preparation method, expression gene, recombinant expression vector and application of gp32 single-strand binding protein

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preparation example Construction

[0039] The preparation method of the gp32 single-chain binding protein of one embodiment, comprises the following steps:

[0040] Step S110: Using the nucleotide sequence shown in SEQ ID No.1 as an amplification template, and adding upstream primers and downstream primers, PCR amplification is performed to obtain PCR amplification products.

[0041] Specifically, step S110 includes the following steps:

[0042] Step S111: obtaining the gene coding sequence of the gp32 single-chain binding protein.

[0043] Furthermore, the gene coding sequence of the gp32 single-chain binding protein can be obtained by screening the gene database. Furthermore, the gene coding sequence of gp32 single-chain binding protein was obtained from NCBI database. Specifically, the genbank sequence number of the gp32 single-chain binding protein is: NC_000866.4.

[0044] Step S112: redesign the nucleotide sequence encoding the gp32 single-chain binding protein to obtain the nucleotide sequence shown i...

Embodiment 1

[0095] The preparation steps of the gp32 single-chain binding protein (gp32) of the present embodiment are as follows:

[0096] (1) Construction of cloning vector

[0097] Look up the gene sequence of T4 bacteriophage gene 32 encoding protein on NCBI, the GenBank sequence number is NC_000866.4. The gene sequence was optimized according to the codon preference of Escherichia coli, and the optimized sequence is shown in SEQ ID No.1. Then the sequence shown in SEQ ID No.1 was spliced ​​by PCR method, then cloned into the PMD19-T SIMPLE vector and transformed into competent cell TOP10, and sequenced to verify whether the inserted sequence in the cloned vector was consistent with the requirements, and the inserted sequence was obtained Correct cloning vector.

[0098] Partial sequencing results of the cloning vector of this embodiment are as follows: Figure 1 ~ Figure 3 shown. in, figure 1 is the partial forward sequencing map of the cloning vector, Figure 2 ~ Figure 3 is t...

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Abstract

The invention relates to a preparation method, expression gene, recombinant expression vector and application of gp32 single-strand binding protein. The expression gene of the gp32 single-strand binding protein comprises a nucleotide sequence shown in SEQ ID No.1. The expression gene of the gp32 single-strand binding protein can achieve a large amount of soluble expression in escherichia coli, andthe purity of the expressed gp32 single-strand binding protein is greater than 95%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method, expression gene, recombinant expression vector and application of a gp32 single-chain binding protein. Background technique [0002] T4 phage gene 32 encoded protein (T4gene 32protein) is a single-stranded DNA (ssDNA) binding protein, referred to as gp32 single-stranded binding protein. The gp32 single-strand binding protein is required for T4 phage DNA replication and repair, it coordinately binds and stabilizes the transiently formed ssDNA region, and plays an important structural role in the process of T4 phage DNA replication. This gp32 single-strand binding protein is also widely used to stabilize and label ssDNA regions for electron microscopy to visualize the structure of DNA in cells. The latest report shows that gp32 single-strand binding protein can promote restriction enzyme digestion reaction, reverse transcription efficiency in RT-PCR, enhance T4 D...

Claims

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Application Information

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IPC IPC(8): C12N15/34C12N15/70C07K14/01A61K38/16A61P43/00
CPCA61P43/00C07K14/005C12N15/70C12N2795/10022C12N2795/10033
Inventor 周娇娇李泓彦莫颜瑛张敏
Owner 深圳市艾伟迪生物科技有限公司
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