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WO2014075445A1 - Fragment de gène de la polyhédrose avec les 180 première paires de base et son application - Google Patents

Fragment de gène de la polyhédrose avec les 180 première paires de base et son application Download PDF

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Publication number
WO2014075445A1
WO2014075445A1 PCT/CN2013/077528 CN2013077528W WO2014075445A1 WO 2014075445 A1 WO2014075445 A1 WO 2014075445A1 CN 2013077528 W CN2013077528 W CN 2013077528W WO 2014075445 A1 WO2014075445 A1 WO 2014075445A1
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WIPO (PCT)
Prior art keywords
gene
fragment
vector
egfp
fusion protein
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Ceased
Application number
PCT/CN2013/077528
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English (en)
Chinese (zh)
Inventor
张耀洲
耿文杰
毕臻乐
舒特俊
陈剑清
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TIANJIN YAOYU BIO-TECH Co Ltd
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TIANJIN YAOYU BIO-TECH Co Ltd
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Publication of WO2014075445A1 publication Critical patent/WO2014075445A1/fr
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Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • the invention relates to the field of genetic engineering technology, and particularly relates to a 180 bp fragment of a polyhedrin gene and an application thereof. Background technique
  • the Bombyx mori Polyhedrin (Poh) gene has a total of 738 nucleotides and encodes a polypeptide of 29 kD consisting of 245 amino acids. It is the major structural component of the polyhedron.
  • the protein crystals composed of polyhedrin proteins have a protective effect on the virions embedded therein, which keeps the virions stable and infective in the natural environment.
  • the polyhedrin gene polh is a super-expressing very late gene. In the late stage of viral infection, when other viral genes and host genes are closed, the gene can continue to express at a high level, and the amount of polyhedrin produced in the infected cells can reach 25 to 50% of the total amount of cellular protein.
  • the polyhedrin protein of Bombyx mori nuclear polyhedrosis virus has its unique properties.
  • the polyhedrin expressed in E. coli exists in the form of inclusion bodies under neutral pH conditions, only in alkaline conditions (pH 10.8 and above). It is soluble, and this polyhedrin has the same properties as the natural polyhedrin crystal. Therefore, the target protein gene can be expressed in fusion with one another, and on the other hand, the expression level of the target protein can be increased.
  • a simple fusion protein can be obtained by simple pH gradient elution and differential centrifugation, and then The protease is digested and the pure protein of interest is recovered.
  • the protease reaction is most suitable in the process of digesting the fusion protein with a protease to obtain a separate protein of interest.
  • the pH value is generally neutral (pH 7 to 9), which results in the inability to simply obtain the desired protein by enzymatic cleavage under the optimal conditions of the enzyme, and when the enzyme is cleaved under alkaline conditions, the protease is protease. The activity is lost or the maximum activity is not achieved, and the amount of the target protein obtained is also limited.
  • the present invention provides a fragment of a polyhedrin gene of Bombyx mori, which is fused with a gene of interest, and in addition to satisfying a large amount of expression, the solubility property thereof is also changed, and is within a suitable pH range of the protease. It is soluble, which greatly simplifies the digestion and purification steps.
  • the first 180 bp fragment of the polyhedrin gene of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1. The application of the first 180 bp fragment of the above polyhedrin gene in protein fusion expression.
  • An expression vector is constructed by inserting a multiple cloning site of the polyhedrin gene into the first 180 bp fragment of the polyhedrin gene and a protease cleavage site fragment.
  • the fusion protein is expressed and digested with the corresponding protease to obtain the protein of interest.
  • the starting vector is a pET series vector, a pcDNA3 series vector, a pFastBacTM series vector, a pKK series vector or a pBAD series vector.
  • pET-28a of pET series vector is the best.
  • the protease cleavage site fragment is a thrombin cleavage site fragment.
  • the nucleotide sequence of the thrombin cleavage site fragment is shown in SEQ ID NO: 2.
  • a fusion protein expression vector is constructed by inserting the first 180 bp fragment of the polyhedrin gene, the protease cleavage site fragment and the target gene fragment in turn at the multiple cloning site of the starting vector.
  • the gene of interest is an EGFP (Enhanced Green Fluorescent Protein) gene, a LZM (lysozyme) gene or a MMgT (membrane magnesium transporter protein) gene.
  • a genetically engineered bacterium comprising the above fusion protein expression vector.
  • the genetically engineered bacteria is Escherichia coli.
  • the present invention has the following beneficial effects:
  • the present invention finds that the partial sequence has more excellent properties than the full-length sequence based on the polyhedrin gene polh, and can express the fusion protein together with other target genes, thereby not only achieving the effect of increasing the expression amount, but also increasing the fusion protein.
  • the solubility under neutral pH conditions makes it suitable for the optimal pH of the protease digestion function, which greatly facilitates the acquisition of the target protein.
  • DRAWINGS Figure 1 is a map of the pET-28a-polhl80-EGFP plasmid.
  • Figure 2 is an electrophoresis map of the PCR product of the first 180pb fragment of the polyhedrin gene; wherein, M: nucleic acid molecular weight standard; 1: target gene PCR product.
  • Figure 3 is a PCR amplification diagram of EGFP, M: marker; 1: EGFP amplification product.
  • Figure 4 is a diagram showing the identification of a recombinant expression vector, wherein M: nucleic acid molecular weight standard; 1: EGFP gene PCR product; 2: recombinant expression vector pET-28a-polhl80-EGFP was digested with EcoR l ⁇ WXho l product strip.
  • Figure 5 shows the expression of pET-28a-polhl80-EGFP protein, M: protein molecular weight standard; 1: pET-28a-polhl80-EGFP uninduced; 2: pET-28a-polhl80-EGFP induction; 3: pET-28a-polh -EGFP induction; 4: pET-28a-EGFP induction.
  • Figure 6 is a graph showing the solubility analysis of the polhl80-EGFP fusion protein; wherein, 1, 3, 5, 7, 9, 11, 13 are the supernatants at pH 8.0, 8.4, 9.2, 9.6, 10.0, 10.4, 10.8; 2, 4, 6, 8, 10, 12, 14 are precipitates at pH 8.0, 8.4, 9.2, 9.6, 10.0, 10.4, 10.8.
  • Figure 7 is an electropherogram of the pH value of the polhl80-EGFP protein solution using Gly; wherein, M: protein molecular weight standard; 5: polhl80-EGFP supernatant (pH 10.8); 6: polhl80-EGFP supernatant (pH 9.0) 7: polhl80-EGFP supernatant (pH 8.0); 8: polhl80-EGFP precipitation (pH 8.0); 9: polh-EGFP supernatant (pH 10.8); 10: polh-EGFP precipitation (pH 10.8) .
  • Figure 8 is a graph showing the results of electrophoresis after oxidase digestion of polhl80-EGFP fusion protein, wherein M: protein molecular weight standard; 1: supernatant at pH 7.6; 2: sample after thrombin digestion at pH 7.6 . detailed description
  • biomaterials used in the following examples such as Bombyx mori nuclear polyhedrosis virus, vector pET-28a, plasmids PGEX-4T-1-EGFP and E. coli TGI were purchased from Tefi (Tianjin) Biomedical Technology Co., Ltd.
  • Example 1 Construction of expression vector pET28a-jw/zl80
  • the gene sequence is known, and any plasmid containing the gene fragment or such as a virus can be used.
  • this embodiment uses the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome as a template to amplify the first 180 bp fragment of the polyhedrin gene.
  • BmNPV Bombyx mori nuclear polyhedrosis virus
  • Primer design is as follows: Upstream primer F : 5'-CGC
  • PCR reaction system In the centrifuge tube of ⁇ , add the following components:
  • the reaction parameters are designed as: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, refolding at 72 °C for 20 s, 30 cycles, 72 °C extended for 3 min. 30 cycles were designed according to the above reaction parameters.
  • the amplified fragment is identified by electrophoresis (see Fig. 2), and the target fragment is recovered by gelatinization.
  • the amplified product was 180 bp in size and sequenced, which was identical to the first 180 bp in GenBank accession number JQ291703.1.
  • the EGFP gene ORF sequence primers designed with Primer5.0 are as follows:
  • Upstream arch 1 F 5'-GCClGAATT ⁇ ATGGTGAGCAAGGGCGAGGA -3' (SEQ ID NO: 5, within the box is the EcoR I restriction site);
  • Downstream arch I R' 5'-CCG
  • the components were added according to the PCR reaction system, mixed, and placed in a PCR machine.
  • the PCR reaction parameters were designed as follows: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, refolding at 72 °C for 50 s, 30 cycles, and extension at 72 °C for 7 min. 30 cycles were designed according to the above reaction parameters.
  • the amplified fragment is identified by electrophoresis (see Fig. 3), and the target fragment is recovered by gelatinization and identified by sequencing.
  • the recombinant expression vector pET-28a-polhl80-EGFP was transferred to E. coli TGI, and cultured in an LB plate medium containing 50 ⁇ ⁇ / ⁇ kanamycin sulfate (Kan), and single colonies were picked, in LB. The culture was continued to expand, and the recombinant plasmid pET-28a-polhl80-EGFP was extracted for PCR and double enzyme digestion, and identified by sequencing. Transfer to E.coli BL21 and add IPTG to a final concentration of 1 mM. The expression was induced at 37 °C. After 4 hours, the cells were collected, centrifuged at 12000 rpm for 10 min, and the precipitate was collected.
  • the cells after induction of expression were collected, and the cells were sonicated and centrifuged at 12000 rpm for 10 min.
  • the precipitate was collected, resuspended in a buffer of pH 8.0, stirred at 4 ° C for 30 min, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken as The supernatant of pH 8.0; the collected precipitate was resuspended in an equal volume of buffer of pH 8.4, stirred at 4 ° C for 30 min, and the resuspension was taken as the precipitation component of the previous pH 8.0, 12,000 rpm. After centrifugation for 10 min, the supernatant was taken as the supernatant of pH 8.4.
  • fusion protein is mostly in the supernatant in the buffer with a pH of 10.8, and some of it is still in the precipitate.
  • the polh-EGFP protein solution was found to have turbidity at a pH of 10.8. At 12,000 rpm, it was centrifuged for 10 min, and a large amount of precipitate was observed at the bottom of the centrifuge tube.
  • the polhl80-EGFP fusion protein solution of Example 3 was adjusted back to pH 8.0, and the fusion protein was subjected to a digestion reaction in a 1.5 mL centrifuge tube according to the Novagen THROMBIN product specification.
  • the response system is as follows:

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  • Physics & Mathematics (AREA)
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Abstract

Font l'objet de cette invention un fragment de gène de la polyhédrose avec les 180 premières paires de base et son application, la séquence nucléotidique dudit fragment étant SEQ ID N°:1. Ce fragment peut servir à l'expression d'une protéine de fusion. Fait aussi l'objet de cette invention un vecteur d'expression formé d'une région de clivage de protéase et dudit fragment permettant l'insertion séquentielle d'un fragment dans une région de clonage multiple d'un vecteur de départ. Le rapport de séquence du gène de la polyhédrose avec les 180 premières paires de base de cette invention et la séquence sur toute sa longueur sont avantageusement caractérisées en ce qu'elles peuvent lier un autre gène cible à une protéine de fusion, tout en améliorant les résultats des niveaux d'expression et la capacité de dissolution de la protéine de fusion avec un pH neutre, et ce, afin d'assurer un pH optimal pour le clivage de la protéase et obtenir ainsi aisément une protéine cible.
PCT/CN2013/077528 2012-11-13 2013-06-20 Fragment de gène de la polyhédrose avec les 180 première paires de base et son application Ceased WO2014075445A1 (fr)

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CN201210453344.9 2012-11-13
CN201210453344.9A CN102952807B (zh) 2012-11-13 2012-11-13 多角体基因前180bp片段及其应用

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Publication number Priority date Publication date Assignee Title
CN102952807B (zh) * 2012-11-13 2015-07-08 天津耀宇生物技术有限公司 多角体基因前180bp片段及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN86107784A (zh) * 1985-11-14 1987-06-03 第一制药株式会社 生产肽的方法
CN101629187A (zh) * 2009-08-17 2010-01-20 浙江大学 提高家蚕杆状病毒表达系统外源蛋白质表达水平的方法
CN102516369A (zh) * 2011-12-21 2012-06-27 天津耀宇生物技术有限公司 一种突变多角体蛋白及其制备方法
CN102517319A (zh) * 2011-12-27 2012-06-27 天津耀宇生物技术有限公司 一种基于家蚕杆状病毒多角体溶解特性的融合蛋白纯化方法
CN102952807A (zh) * 2012-11-13 2013-03-06 天津耀宇生物技术有限公司 多角体基因前180bp片段及其应用

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CN101481702B (zh) * 2009-01-22 2013-05-01 天津耀宇生物技术有限公司 含多角体基因的重组载体及其表达与纯化蛋白质的方法

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CN86107784A (zh) * 1985-11-14 1987-06-03 第一制药株式会社 生产肽的方法
CN101629187A (zh) * 2009-08-17 2010-01-20 浙江大学 提高家蚕杆状病毒表达系统外源蛋白质表达水平的方法
CN102516369A (zh) * 2011-12-21 2012-06-27 天津耀宇生物技术有限公司 一种突变多角体蛋白及其制备方法
CN102517319A (zh) * 2011-12-27 2012-06-27 天津耀宇生物技术有限公司 一种基于家蚕杆状病毒多角体溶解特性的融合蛋白纯化方法
CN102952807A (zh) * 2012-11-13 2013-03-06 天津耀宇生物技术有限公司 多角体基因前180bp片段及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CAO, CUIPING ET AL.,: "IMMOBOLIZATION OF FOREIGN PROTEIN INTO BMNPV POLYHEDRA THROUGH FUSION WITH PARTIAL POLYHEDRIN FRAGMENT", SCIENCE OF SERICULTURE, vol. 36, no. 2, 2010, pages 0282 - 0289 *
GENG, WENJIE ET AL.,: "RESEARCH ON THE SOLUBILITY OF FRAGMENTS OF POLYHEDRON GENE FUSED EGFP", SCIENCEPAPER ONLINE, 30 November 2012 (2012-11-30), pages 1 - 12 *

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CN102952807B (zh) 2015-07-08

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