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Protein and coding gene and suicide vector thereof

A protein and carrier technology, applied in the field of bioengineering, can solve the problems of multiple screening steps and high false positive rate, and achieve the effects of reducing production costs, improving growth status, and simplifying operations

Inactive Publication Date: 2010-12-22
JIERUI BIOENG SHANGHAI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved in the present invention is to provide a new carrier for the blue-white spot screening method of positive clones in the existing gene cloning technology, which has many screening steps and high false positive rate. Fewer screening steps, low false positive rate

Method used

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  • Protein and coding gene and suicide vector thereof
  • Protein and coding gene and suicide vector thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Design and function of embodiment 1 cytotoxic protein gene

[0024] The inventors unexpectedly discovered that the core functional region of the snake neurotoxic protein whose original coding sequence is ABX58152 is the 37th to 107th positions. Therefore, the inventors further optimized the core functional region and obtained the amino acid sequence of the protein shown in SEQ ID NO: 3 in the sequence listing. Then, according to the codon preference of Escherichia coli, the nucleotide sequence of the amino acid sequence was optimally designed, as shown in the 4th to 267th positions of SEQ ID NO: 2 in the sequence listing. The inventor synthesized the sequence shown in the 4th to 267th positions in SEQ ID NO: 2, cloned it into the plasmid pTZ19R (MBI Fermentas), and transformed the recombinant plasmid into Escherichia coli DH5a competent cells. to grow any transformants.

Embodiment 2

[0025] Example 2 Design and synthesis of β-galactosidase gene-cytotoxic protein gene fusion gene

[0026] The DNA fragment of the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing was synthesized in vitro by Shanghai Jierui Bioengineering Co., Ltd. by chemical synthesis. Among them, the 4th to 267th positions are the optimized toxic protein coding sequence of the present invention. Positions 268-552 are β-galactosidase genes including multiple cloning sites and blocking sequences. Positions 468-508 are multiple cloning sites, and positions 483-494 are an exogenous blocking fragment. The foreign fragment blocks the reading frame of the fusion gene of β-galactosidase gene-cytotoxic protein gene-multicloning site.

Embodiment 3

[0027] Example 3 Construction of suicide vector

[0028] Carry out following polymerase chain reaction (PCR) respectively: With the DNA fragment synthesized in embodiment 1 as template, with following primer F and R1 as primer pair; With plasmid pTZ19R (MBI Fermentas) as template, with following primer F1 and R is a primer pair; then using the above two amplification products as templates, using primers F and R as a primer pair, the resulting PCR product contains a ScaI (agtact) restriction site at the 5'-end and a HindIII (agtact) at the 3'-end. aagctt) restriction site fragment, spare.

[0029] The primer sequences used in the PCR reaction were:

[0030] Primer F: 5'-agtactcaaccaagtcattctgagaatagtgtat-3',

[0031]Primer R1: 5'-atcatggatcacccatgaagggtgatggttcacgtagtg-3',

[0032] Primer F1: 5'-cactacgtgaaccatcacccttcatgggtgatccatgata-3',

[0033] Primer R: 5'-aagcttcacgtgatatcgcctggg-3'.

[0034] The PCR reaction system is:

[0035] template DNA

1μl

...

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Abstract

The invention discloses a protein, a coding gene thereof and a vector containing the gene, wherein the amino acid sequence of the protein is shown as SEQ ID NO:2 in a sequence list; the protein has cytotoxicity; when no exogenous segment is inserted into the vector containing the coding genes of the protein, the coding gene expression of the protein causes the death of transformants; the insertion of the exogenous segments destroys reading frames of the coding gene of the protein; cytotoxicity genes are not expressed, and the transformants grow normally; and the vector is a suicide vector. The invention can ensure that the vector is used in a clone technology; the quantity of non-purpose clone is reduced; the work load of screening is greatly reduced; the trouble caused by false positive is effectively overcome; the false positive rate is low; and the screening course in a conventional molecular biology operation is greatly simplified.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a protein, its coding gene and a suicide vector. Background technique [0002] In order to quickly screen positive clones containing target gene fragments, many scientific researchers have invented a variety of detection tools, such as blue-white screening using β-galactosidase gene, antibiotic screening, polymerase chain reaction screening, and tool enzyme screening. filter and more. But there are some defects. The rate of positive clones in blue-white screening is low, generally <80%. When the β-galactosidase gene fails or the vector is self-ligated, there will be false positives, and the white clones may not contain the target fragment, and the false positive rate is high, > 15%. Antibiotic screening is only an auxiliary of other screening and cannot Use independently. PCR screening takes a long time and is prone to cross-contamination. The screening time of tool enzym...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12C12N15/63C12N1/21C12N15/64C12R1/19
Inventor 肖爱军邵永胜严引娣
Owner JIERUI BIOENG SHANGHAI
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