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Enhance-like element gene for enhancing foreign protein expression and application of gene

An enhancer-like, exogenous protein technology, applied in the direction of using vectors to introduce foreign genetic material, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of undeveloped and less used regulatory gene expression sequences, and achieve increased protein The effect of expressing horizontal characteristics, increasing possibility, and improving practicality

Inactive Publication Date: 2013-03-13
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the development of improved protein expression systems, methods such as adding protein expression tags, supplementing rare codons required for exogenous gene expression, and increasing the copy number of exogenous genes in the expression system are commonly used, and enhancer-like gene sequences are used to improve protein expression. The system is currently used less, and many unknown regulatory gene expression sequences may not have been developed

Method used

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  • Enhance-like element gene for enhancing foreign protein expression and application of gene
  • Enhance-like element gene for enhancing foreign protein expression and application of gene
  • Enhance-like element gene for enhancing foreign protein expression and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Construction of L1-CAT-pET21a enhancer-like sequence detection vector

[0036] The tumor tissue of the cervical cancer patient in the First People's Hospital of Yunnan Province was taken, and the DNA genome of the sample was prepared according to the DNA extraction method of the animal tissue genome in the "Guidelines for Molecular Biology Experiments" (Science Press). The sample was identified as belonging to the human papillomavirus HPV58 HPV58 type infection, using primers pv58p1: 5'-ATGGTGCTGATTTTATGTTGCACCCT-3'; PV58P2: 5'-TTATTTTTTAACCTTTTTGCGTTTG-3', HPV58 type L1 gene was obtained by PCR amplification, and constructed into L1-pMD18-T vector (pMD18-T vector was purchased from TAKARA company, CK5601C). The recombinant plasmid L1-pMD18-T was transformed into Escherichia coli DH5α (DH5α was purchased from TAKARA company, D9057), and the L1-pMD18-T / DH5α strain was shaken at 37°C for 16 hours, and then the plasmid mini-extraction kit (purchased from Beiji...

Embodiment 2

[0037] Example 2: Detection of Recombinant Anti-chloramphenicol Threshold and L1-CAT Fusion Protein Expression Level

[0038] Take 10ng of enhancer-like sequence detection plasmid L1-CAT-pET21a and 100μl E. coLi BL21(DE3) (purchased from Novagen, 69450-3) was competently mixed, placed on ice for 30 minutes, then placed in a 42°C water bath for 2 minutes for heat shock, then added 1ml LB culture medium, incubated at 37°C for 1 hour, and discarded by centrifugation Most of the supernatant, coated with Amp + -In LB solid petri dish, cultured at 37°C for 12~16h to obtain the screening strain L1-CAT-pET21a / BL21(DE3); pick a single colony of the bacteria and inoculate it into Amp + -In LB liquid medium, shake culture overnight at 37°C, and inoculate Amp respectively at 1% the next day + - LB liquid medium, cultured by shaking until OD600 is about 0.5, dilute the bacterial solution by 10 5 times and 10 6 times, take 100μl and directly spread it on the medium containing 1mmoL I...

Embodiment 3

[0040] Example 3: Genome extraction, digestion and library construction

[0041] The domestic sewage samples from residents in Chenggong District, Kunming City and the sewage treatment plant near the 43rd People's Liberation Army General Hospital in Kunming City were shaken and mixed, and 150 μl was added to 15ml beef extract peptone liquid culture medium for shaking at 37°C for 16 hours. Dispense into 10ml centrifuge tubes, centrifuge at 12000g for 2min, discard the supernatant; add 2ml TE buffer to the bacterial precipitate, resuspend it by pipetting repeatedly with a pipette gun, add 180μl 10% SDS and 12μl 20mg / ml proteinase K, Mix well and incubate at 37°C for 1h; add 600μl 5moL / L NaCl, mix thoroughly, then add 400μl CTAB / NaCl, incubate at 65°C for 10min; add an equal volume of chloroform, mix well, centrifuge at 12000g for 5min, and supernatant Transfer to a new centrifuge tube; add an equal volume of phenol / chloroform, mix well, centrifuge at 12000g for 5min, and divid...

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Abstract

The invention discloses an enhance-like element gene ER1 for enhancing foreign protein expression in prokaryotic cells. The enhance-like element gene has a nucleotide sequence as shown in SEQ ID NO: 1 (Sequence Identity No) or a truncated sequence of the nucleotide sequence, wherein the gene sequence is screened by establishing a fusion expression reporter gene recombinant vector; the fusion reporter gene consists of major capsid protein L1 gene of human papilloma virus and chloramphnicol acetyltransferase (CAT); the gene fragments to be selected are linked with the sieving recombinant vector and is transformed so as to establish a gene library; and the recombinant bacterial strain containing the enhance-like element gene sequence is screened based on the chloramphenicol resistance, so as to obtain the enhance-like element sequence which can improve the activity of chloramphenicol of the fusion reporter gene of the strain and promotes the foreign protein expression.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enhancer-like gene sequence for enhancing the expression of foreign proteins, which is used for improving the protein expression level of foreign genes in prokaryotic cells. Background technique [0002] With the development of molecular biology and genetic engineering, exogenous gene expression provides broad application prospects for the development of protein expression systems. Currently commonly used protein expression systems include Escherichia coli expression system, yeast expression system, insect cell expression system and mammalian cell expression system, etc. The Escherichia coli expression system is relatively mature in research at present, but the expression level of some expressed target products is still very low; the yeast expression system also often has low copy number of foreign genes; the mammalian cell expression system, foreign genes cannot be expressed persis...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/70
Inventor 井申荣王应明曾韦锟黄芬
Owner KUNMING UNIV OF SCI & TECH
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