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Promoter-like gene for efficiently promoting and expressing heterologous protein and application thereof

An exogenous protein, high-efficiency start-up technology, applied in the field of molecular biology, can solve the problems of low protein expression and low specificity, and achieve the effects of clear genetic background, strong continuous expression ability, and increased application scope.

Active Publication Date: 2017-12-15
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The current constitutive promoter-like sequence (Constitutive Promoter, CP) generally has the characteristics of low protein expression and low specificity

Method used

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  • Promoter-like gene for efficiently promoting and expressing heterologous protein and application thereof
  • Promoter-like gene for efficiently promoting and expressing heterologous protein and application thereof
  • Promoter-like gene for efficiently promoting and expressing heterologous protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Construction of screening vector

[0037]Primers catF / catR were synthesized by Kunming Shuoqing Biotechnology Co., Ltd. (upstream primer catF: 5ˊ- CGC CAT ATGGAG AAA AAA ATC ACT GGA TAT -3ˊ, downstream primer catR: 5ˊ-CCG CTC GAG TTA CGC CCC GCCCTG CCA CTC -3ˊ) , CAT was amplified on vector pACYC184 (purchased from New England BioLabs, VKN0287) by primers catF / catR, and then CAT was amplified with Fd Nde I (purchased from TAKARA, FD0584) and Fd xho I (purchased from TAKARA, FD0694) was digested and connected to the pET-28a vector (purchased from Novagen, 69864-3) to form pET-28a-CAT; primers M28a_F / M28a_R were synthesized by Kunming Shuoqing Biotechnology Co., Ltd.: ( Upstream primer M28a_F: 5ˊ-AGATCT GCG GCC GCA AGC TTG TCG ACG GAG CTC GGA TCC AAG GAG ATA TAC ATA TGA CACGAG-3ˊ; Downstream primer M28a_R: 5ˊ-TCT AGA CGC CGG CGT TCG AAC AGC TGC CTC GAG CCT AGGTTC CTC TAT ATG TAT ACT GAG CTC-3ˊ), the two primers denatured and annealed to form a Bgl II ...

Embodiment 2

[0038] Example 2: Construction of gene library and preliminary screening of promoters

[0039] Vero (purchased from ATCC, CL28) cells were revived, and cultured in a carbon dioxide incubator with DMEM (purchased from GE Healthcare Life Sciences, AAE202588) medium containing fetal bovine serum (purchased from PAN-Biotech, P141101) Day, the sample DNA genome was prepared with a kit (purchased from Tiangen Biotechnology, DP304), and then used Sau 3A Ⅰ (purchased from TAKARA, 1082A) digested the genome at 37°C for 3 hours, and recovered fragments of about 500 bp or less with an ordinary agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., DP209). use Bam H Ⅰ (purchased from ThermoFisher FD0055) digested the pCMR8a vector for 15 minutes, and used the characteristics of the isotail enzyme to digest the digested pCMR8a vector and the genomic fragment below 500bp recovered from the gel, and used T4 DNA ligase (purchased from TAKARA, SD026...

Embodiment 3

[0041] Embodiment 3: compare and screen 3 promoters to start CAT ability, select the promoter gene sequence with the strongest starting ability

[0042] The 3 strains obtained by screening were picked and cultured in 5mL LB liquid medium (Kan antibiotics, 50μg / mL, Yeast Extract 2.5g, Tryptone 5g, NaCI 5g, ddH 2 O constant volume 500mL), when the bacterial solution OD 600 When the value reaches 0.6, transfer it to a new 5mL liquid LB medium containing Kan antibiotics at a ratio of 1:100. 600 Sampling of the value, using SDS-PAGE electrophoresis to detect the expression of CAT protein, such as image 3 , using Image J software for quantitative analysis, the results are as follows Figure 4 shown. In 24h, the protein expression level first increased and then decreased with the prolongation of time. The CAT protein expression level reached the highest level at 12h, and then decreased at 24h. Therefore, 12h was selected for sampling and detection in subsequent experiments. At t...

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Abstract

The invention discloses a constitutive promoter-like gene VP2 which is found from a eukaryotic cell African green monkey kidney cell (Vero) and can be used for promoting heterologous protein expression in a prokaryotic cell, and a truncating sequence Vh2 of the constitutive promoter-like gene VP2. A nucleotide sequence of the promoter-like gene VP2 is shown as SEQ ID NO: 1; the nucleotide sequence of the truncating sequence is shown as 60bp to 107bp in SEQ ID NO: 1; the gene is constructed to form an expression reporter gene chloramphenicol acetyl transferase (CAT) gene recombinant vector for screening; the screened vector and a gene segment to be screened are recombined, and a gene library is constructed; a promoter-like sequence is obtained by a recombinant strain containing a promoter-like gene sequence through a method for screening the resistance of chloramphenicol; the sequence can be used for efficiently starting and expressing heterologous protein in the prokaryotic cell.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a constitutive promoter-like gene sequence for highly expressing foreign proteins in prokaryotic cells and its application. Background technique [0002] With the continuous development of molecular biology and genetic engineering, protein expression is a difficult and hot spot in the fields of modern industry, medical treatment and basic research. Although some eukaryotic expression systems, such as yeast protein expression system and insect cell expression system, can express heterologous proteins, their expression products are easily degraded, the expression level is uncontrollable, and the production cost is high. Therefore, the expression of foreign proteins in prokaryotic cells is particularly important, and when expressing foreign proteins, the promoter is a key factor in constructing an expression vector for high-level expression of heterologous proteins. It is known that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/70
CPCC12N9/1029C12N15/70C12Y203/01
Inventor 井申荣仉秋实苑荣亮陈伟金维维
Owner KUNMING UNIV OF SCI & TECH
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