Enhanced subsample gene capable of improving expression of foreign protein and application thereof

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 problem of undiscovered enhancer-like sequences, so as to increase the possibility and increase The effect of practicality

Inactive Publication Date: 2014-02-26
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The methods commonly used to increase the expression level of exogenous proteins mainly include adding protein expression tags, optimizing the codons required for exogenous gene expression, and increasing the copy number of exogenous genes in the expression system. Enhancer-like gene sequences are used to increase the expression of exogenous proteins. Levels are less used, and many unknown enhancer-like sequences have not yet been discovered

Method used

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  • Enhanced subsample gene capable of improving expression of foreign protein and application thereof
  • Enhanced subsample gene capable of improving expression of foreign protein and application thereof
  • Enhanced subsample gene capable of improving expression of foreign protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Construction of pET21a-CAT-L11 enhancer-like sequence detection vector

[0035] The tumor tissue of the cervical cancer patient in the First People's Hospital of Yunnan Province was taken, and the genome of the sample was extracted according to the DNA extraction method of animal tissue genome in the "Guidelines for Molecular Biology Experiments" (Science Press). After identification, the sample was HPV58 type Infection, HPV58 type L1 was amplified by PCR using primers L11P1: 5'-GGA ATT CCA TAT GTC TGT TTG GAG ACC TTC-3'; L11P2: 5'-CCG CTC AGA CAG TGA GTC TCC ATA AGG TTC-3' The truncated sequence L11 of the gene was constructed into a pMD18T-L11 recombinant plasmid (the pMD18T vector was purchased from TAKARA Company, CK5601C), and the plasmid was transformed into Escherichia coli DH5α (DH5α was purchased from TAKARA Company, D9057), and the recombinant plasmid was cultured with shaking at 37°C Bacteria pMD18T-L11 / DH5α, after 16 hours, use the plasmid mini-...

Embodiment 2

[0038] Example 2: Detection of Recombinant Bacteria Chloramphenicol Resistance Threshold and L11-CAT Fusion Protein Expression Level

[0039] Take 10ng of the enhancer-like sequence detection plasmid pET21a-CAT-L11 and 100 μL E.coli BL21 (DE3) (purchased from Novagen, 69450-3) and mix competently, bathe in ice water for 30 minutes, and heat shock in a warm water bath at 42°C for 2 minutes. Then add 1ml of LB culture solution, shake and culture at 37°C for 45 minutes, discard most of the supernatant after centrifugation and resuspend the bacteria, and spread on Amp + -In LB solid petri dish, cultivate at 37°C for 12-16h to obtain the recombinant screening strain pET21a-CAT-L11 / BL21 (DE3); pick the single colony and inoculate it into Amp + -In LB liquid medium, shake culture overnight at 37°C, then inoculate Amp at a ratio of 1% + -LB liquid medium, cultured with shaking at 37°C until OD 600 About 0.5, 10 for the bacterial solution 5 times and 10 6 After doubling dilution,...

Embodiment 3

[0041] Example 3: Genome extraction, digestion and library construction

[0042] After shaking and mixing, 150μL was added to 15ml beef extract peptone liquid medium, and shake cultured at 37°C. After 16 hours, divide the culture solution into 10ml centrifuge tubes, centrifuge at 12000g×2min, discard the supernatant, and resuspend the bacteria with 2ml TE buffer, add 180μL of 10% SDS and 12μL of 20mg / ml proteinase K, and mix well at 37 Incubate at ℃ for 1 h, then add 600 μL of 5M NaCl and mix well, add 400 μL of CTAB / NaCl solution, incubate at 65 °C for 10 min; add an equal volume of chloroform, mix well, centrifuge at 12000 g×5 min, take the supernatant, add etc. Mix the volume of phenol / chloroform mixture evenly, centrifuge at 12000g×5min, take the supernatant and distribute it into 1.5ml centrifuge tubes, 700μL / tube, add an equal volume of isopropanol, invert and mix well, store at -40℃ Let stand for 30min, centrifuge at 12000g×10min, discard the supernatant, and then ri...

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Abstract

The invention discloses an enhanced subsample gene capable of improving expression of foreign protein in a prokaryotic cell. The gene has a nucleotide sequence or a truncated sequence thereof shown as SEQ ID NO:1. The gene is screened by constructing a fusion expression report gene recombinant carrier. The fusion expression report gene consists of a truncated sequence L11 of a main capsid protein L1 of a human papilloma virus and a chloramphnicol acetyl transferase gene (CAT). A to-be-screened gene segment and the recombinant carrier for screening are connected and converted for constructing a gene library, and a recombinant strain comprising the gene sequence of the enhanced subsample is screened by improving the resistance of chloramphnicol, so that the enhanced subsample sequence is obtained. The sequence can be used for improving the resistance of chloramphnicol of the fusion expression report gene recombinant carrier and the expression level of the foreign protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enhancer-like gene sequence for improving the expression of foreign proteins, which is used for improving the expression level of foreign proteins in prokaryotic cells. Background technique [0002] Currently, a variety of protein expression systems have been developed, such as Escherichia coli expression system, yeast expression system, insect cell expression system and mammalian cell expression system. Although the Escherichia coli expression system is relatively mature, the expression level of some target proteins is still very low; the yeast expression system also has the problem of low copy number of foreign genes; the mammalian cell expression system has the problem that foreign genes cannot last Expressive, costly and technically complex issues. Therefore, in these commonly used expression systems, the low expression level of certain exogenous genes is still an urgent proble...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/70
Inventor 井申荣闫沁男王应明曾韦锟黄芬
Owner KUNMING UNIV OF SCI & TECH
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