阅读说明：本技术 Nfe2l1的截短蛋白及其应用 (Truncated protein of NFE2L1 and application thereof ) 是由 邱露 高艳锋 祁元明 于 2020-06-13 设计创作，主要内容包括：本发明涉及对转录因子NFE2L1的截短蛋白的原创性发现及应用。本发明中发现的截短蛋白序列如SEQ ID NO.1所示,其具有抑制肿瘤生长的作用。(The invention relates to the original discovery and application of a truncated protein of a transcription factor NFE2L 1. The truncated protein sequence found in the invention is shown in SEQ ID NO.1, and has the function of inhibiting tumor growth.)

1. The protein has an amino acid sequence shown as SEQ ID NO. 1.

2. A nucleic acid molecule encoding the protein of claim 1.

3. The nucleic acid molecule of claim 2, having the sequence shown in SEQ ID No. 2.

4. A vector comprising the nucleic acid molecule of claim 2 or 3.

5. A cell comprising the nucleic acid molecule of claim 2 or 3, or comprising the vector of claim 4.

6. A pharmaceutical composition comprising the protein of claim 1.

7. Use of the protein of claim 1 for the preparation of a medicament against liver cancer.

The technical field is as follows:

the invention belongs to the technical field of biomedicine, and particularly relates to a truncated protein independent of the activity of a transcription factor of NFE2L1, application of the truncated protein in related diseases such as tumors and the like.

Background art:

the redox homeostasis formed by oxidative stress and an antioxidant system is a necessary prerequisite for maintaining normal physiological homeostasis, and long-term oxidative damage caused by imbalance of the redox homeostasis is a cause for the occurrence of various chronic systemic diseases (such as chronic inflammation, diabetes, obesity, neurodegenerative diseases, aging, tumors, and the like). The cap-ring-alkaline leucine zipper (cap 'n' collarbasic-leucoine zipper, CNC-bZIP) transcription factor family is the most powerful transcription factor group for regulating the intracellular redox homeostasis in the currently known eukaryotes; of the 6 members of the mammalian CNC-bZIP transcription factor, the nuclear factor erythrokine 2-like factor 1(NFE2L1/Nrf1) is the most protein subtype and most complex in regulation, and is a lethal antioxidant transcriptional activator knocked out in mice.

The human NFE2L1 gene contains 7 exons and 23 transcripts, the proteins mainly comprise subtypes TCF11, Nrf1 alpha and the like, the full-length NFE2L1 protein is about 120-140kDa, and the amino acids are 772 in total. By analyzing the molecular structure and functional characteristics of the NFE2L1 protein, the NFE2L1 protein contains main functional structural regions such as a DNA Binding Domain (DBD) and a C-terminal domain (CTD).

The DBD domain of human NFE2L1 is an essential domain for entering into nucleus and binding with DNA, is an essential prerequisite for NFE2L1 protein to perform transcription factor function, comprises CNC structure and bZIP structure, is a region where NFE2L1 protein and sMAF protein form heterodimer and are mutually combined, performs transcription factor function on DNA by indirect combination of small MAF protein, the bZIP domain is an amino acid characteristic sequence peculiar to bZIP family, the bZIP family protein can form homodimer or heterodimer through the bZIP domain, and is an essential structure for protein to enter nucleus; the CTD domain is the C-terminal transmembrane domain of the NFE2L1 protein, and the transcriptional activity enhancement of the NFE2L1 protein with the deletion of the CTD domain indicates that the CTD domain has a negative regulation effect on the NFE2L 1.

The NFE2L1 gene is generally expressed in various tissues and organs of human and is very sensitive to environmental factors, and the different expression of the NFE2L1 gene can be caused by radiation, nutrition, metabolites, oxidative stimulation of various medicines or heavy metals and even environmental temperature. The molecular function of NFE2L1 relates to regulation of oxidation-reduction homeostasis, glycolipid metabolism homeostasis and protein turnover, and the physiological function relates to oxidation resistance, detoxification, cell proliferation and apoptosis, tissue and organ development and function specialization, carcinogenesis, inflammatory response, immune response and the like, and the function of the NFE2L1 in chronic systemic diseases such as chronic inflammation, cancer, diabetes, obesity, neurodegenerative diseases and the like indicates that the protein has potential clinical application value. However, since the transcription factor directly regulates the expression of a large number of downstream genes, the related biological functions are wide, and the transcription factor cannot be used as a target molecule for clinical application and research. Similarly, simple overexpression or knockdown (activation or inhibition) of NFE2L1 can cause systemic diseases, such as dysplasia of tissues and organs, neurodegenerative diseases, diabetes, etc.

The existing molecular theory models such as NFE2L1 protein modification and cleavage regulation theory and multi-subtype models are based on the functions of antioxidant transcription factors, and the research on the functions of non-transcription factors possibly existing in NFE2L1 is not found in the consultable range.

The invention content is as follows:

in this patent, we have developed a novel approach by constructing NFE2L1 without transcription factor activity^1-620The protein (the sequence is shown as SEQ NO.1, and is identical to the 1 st-620 th amino acid region of NFE2L1 protein, namely the NFE2L1 full-length protein loses 152 amino acid residues at the C end and loses a DBD structure domain, and NFE2L1 is obtained^1-620Protein) and verified NFE2L1 by western immunoblotting method^1-620The protein can be normally expressed, and the non-transcription factor function of NFE2L1, namely the anti-cancer function, is originally innovatively discovered. We convert NFE2L1^1-620The stable over-expressed HepG2 cell line is subjected to an immunodeficiency mouse tumor-bearing experiment to verify whether the cell line has the effect of inhibiting tumor progression, and as a result, the NFE2L1 without transcription factor activity is found^1-620Has an effect of remarkably inhibiting tumor growth, thereby completing the invention.

In a first aspect, the present invention provides a novel protein NFE2L1^1-620The amino acid sequence is shown in SEQ NO. 1.

In a second aspect, the present invention provides a method for encoding the aforementioned NFE2L1^1-620The sequence of the nucleic acid molecule of (1) can be shown as SEQ NO. 2.

In a third aspect, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecule.

In a fourth aspect, the invention provides a host cell transformed with the aforementioned nucleic acid molecule or recombinant vector.

In a fifth aspect, the present invention provides NFE2L1 comprising the aforementioned protein^1-620The pharmaceutical composition can comprise the protein and a pharmaceutically acceptable carrier or excipient thereof, and can be an injection, such as a powder injection or an injection.

In a sixth aspect, the present inventors have found that the aforementioned protein NFE2L1^1-620Can be used for treating hepatocarcinoma.

The invention develops a new way, finds that NFE2L1 has a new function which is not known by people and is independent of the activity of a transcription factor, and proves that the truncated protein of NFE2L1 has the effect of inhibiting tumors through animal experiments.

It should be noted that the truncated form of the protein of this patent is an anti-cancer, and may be a whole function, or a part of the amino acid region may function, either directly or indirectly, and all constitute an equivalent infringement of this patent based on a simple variation of the idea of this patent.

Description of the drawings:

FIG. 1NFE2L1^1-620And (4) detecting the protein expression effect.

FIG. 2NFE2L1^1-620Effect of stable overexpression on the ability of tumor cells to form tumors subcutaneously, wherein fig. 2A shows tumor-bearing volume comparison and fig. 2B shows tumor-bearing mouse body weight comparison; FIG. 2C shows tumor-bearing tissues taken out at 30 days after tumor-bearing, and photographed for comparison, wherein the scale in the figure is 0.5 cm; FIG. 2D shows subcutaneous tumor mass statistics at 30 days tumor bearing; the cross-references in the figures indicate p < 0.01.

The specific implementation mode is as follows:

embodiments of the present invention will be described in detail below with reference to examples, but the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. Unless otherwise specified, the reagents, biological materials, culture media and solutions used below are all commonly used, publicly available or commercially available in the art.

Inventor's reference to NFE2L1^1-620The discovery process of the function and antitumor effect of (A) is briefly described as follows:

1.NFE2L1^1-620construction and expression verification of expression plasmid:

a) extracting human cell line total RNA by using a total RNA extraction kit, and performing reverse transcription on the total RNA into cDNA by using a reverse transcription kit to serve as a template of PCR reaction.

b) The NFE2L1 gene amplification primers (NFE2L1-F: 5'-CTCGAGGAATTCATGCTTTCTCTGAAGAAATAC-3'; NFE2L1-R:5'-GGCCGCTCTAGATCACTTTCTCCGGTCCTTT-3') to obtain the CDS region of the NFE2L1 gene, and inserting the CDS region into a pLVX-puro vector to obtain the NFE2L1 overexpression plasmid pNFE2L 1.

c) Using pNFE2L1 plasmid as a template, a primer set (NFE2L1)^1-620-F:5'-CTCGAGGAATTCATGCTTTCTCTGAAGAAATAC-3',NFE2L1^1-6205'-GAAAGGTCTAGATCAGGCTCGGGCTCGG TGCTCAT-3') amplified NFE2L1^1-620The nucleotide sequence of the protein (the sequence is shown as SEQ ID NO. 2) is inserted into a pLVX-puro vector to obtain NFE2L1^1-620Overexpression plasmid pNFE2L1^1-620。

d) pLVX-puro, pNFE2L1, pNFE2L1^1-620Plasmids were transfected into HepG2 cells, total protein was harvested 24 hours later, and NFE2L1 and NFE2L1 were detected by Western blotting^1-620The protein was normally expressed (FIG. 1).

2.NFE2L1^1-620Effects of overexpression on subcutaneous tumor bearing in tumor cells nude mice:

a) packaging of NFE2L1 and NFE2L1 Using a Lentiviral packaging System^1-620Overexpression of lentivirus and empty vector lentivirus, infection of HepG2 cells, and selection of control cell line (pLVX), NFE2L1 overexpression cell line (NFE2L1), NFE2L1 cell line (NFE2L1) by culturing in puromycin-containing medium^1-620Over-expression cell line (NFE2L1)^1-620)。

b) pLVX, NFE2L1, NFE2L1^1-620The cell lines were digested, washed 2 times with PBS, and resuspended in PBS to a density of 500 million cells per ml.

c) Nude mice of 7 weeks old were randomly divided into three groups of 5 mice each, each nude mouse was subcutaneously injected with 100 ten thousand of the cells obtained in the previous step, the length and width of subcutaneous tumor were recorded every other day starting from day 4 after subcutaneous tumor, according to the formula V-1/2 × a (length) × b²(Width)²) Changes in tumor volume in mice (as shown in FIG. 2A) and changes in body weight in tumor-bearing mice (as shown in FIG. 2B) were calculated and recorded.

d) The volume of the tumor to be maximized reaches 1cm³On the left and right, tumor bearing mice were sacrificed, tumors were removed for photography (as shown in fig. 2C) and weighed (as shown in fig. 2D). As a result, it was found that NFE2L1 lost the activity of transcription factor^1-620The over-expression still has obvious tumor inhibition effect. Three groups are small during the experimentThe body weight of the mice was not significantly different and the mental state was good.

Sequence listing

<110> Zhengzhou university

Truncated protein of <120> NFE2L1 and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>620

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Met Leu Ser Leu Lys Lys Tyr Leu Thr Glu Gly Leu Leu Gln Phe Thr

1 5 10 15

Ile Leu Leu Ser Leu Ile Gly Val Arg Val Asp Val Asp Thr Tyr Leu

20 25 30

Thr Ser Gln Leu Pro Pro Leu Arg Glu Ile Ile Leu Gly Pro Ser Ser

35 40 45

Ala Tyr Thr Gln Thr Gln Phe His Asn Leu Arg Asn Thr Leu Asp Gly

50 55 60

Tyr Gly Ile His Pro Lys Ser Ile Asp Leu Asp Asn Tyr Phe Thr Ala

65 70 75 80

Arg Arg Leu Leu Ser Gln Val Arg Ala Leu Asp Arg Phe Gln Val Pro

85 90 95

Thr Thr Glu Val Asn Ala Trp Leu Val His Arg Asp Pro Glu Gly Ser

100 105 110

Val Ser Gly Ser Gln Pro Asn Ser Gly Leu Ala Leu Glu Ser Ser Ser

115 120 125

Gly Leu Gln Asp Val Thr Gly Pro Asp Asn Gly Val Arg Glu Ser Glu

130 135 140

Thr Glu Gln Gly Phe Gly Glu Asp Leu Glu Asp Leu Gly Ala Val Ala

145 150 155 160

Pro Pro Val Ser Gly Asp Leu Thr Lys Glu Asp Ile Asp Leu Ile Asp

165 170 175

Ile Leu Trp Arg Gln Asp Ile Asp Leu Gly Ala Gly Arg Glu Val Phe

180 185 190

Asp Tyr Ser His Arg Gln Lys Glu Gln Asp Val Glu Lys Glu Leu Arg

195 200 205

Asp Gly Gly Glu Gln Asp Thr Trp Ala Gly Glu Gly Ala Glu Ala Leu

210 215 220

Ala Arg Asn Leu Leu Val Asp Gly Glu Thr Gly Glu Ser Phe Pro Ala

225 230 235 240

Gln Val Pro Ser Gly Glu Asp Gln Thr Ala Leu Ser Leu Glu Glu Cys

245 250 255

Leu Arg Leu Leu Glu Ala Thr Cys Pro Phe Gly Glu Asn Ala Glu Phe

260 265 270

Pro Ala Asp Ile Ser Ser Ile Thr Glu Ala Val Pro Ser Glu Ser Glu

275 280 285

Pro Pro Ala Leu Gln Asn Asn Leu Leu Ser Pro Leu Leu Thr Gly Thr

290 295 300

Glu Ser Pro Phe Asp Leu Glu Gln Gln Trp Gln Asp Leu Met Ser Ile

305 310 315 320

Met Glu Met Gln Ala Met Glu Val Asn Thr Ser Ala Ser Glu Ile Leu

325 330 335

Tyr Ser Ala Pro Pro Gly Asp Pro Leu Ser Thr Asn Tyr Ser Leu Ala

340 345 350

Pro Asn Thr Pro Ile Asn Gln Asn Val Ser Leu His Gln Ala Ser Leu

355 360 365

Gly Gly Cys Ser Gln Asp Phe Leu Leu Phe Ser Pro Glu Val Glu Ser

370 375 380

Leu Pro Val Ala Ser Ser Ser Thr Leu Leu Pro Leu Ala Pro Ser Asn

385 390 395 400

Ser Thr Ser Leu Asn Ser Thr Phe Gly Ser Thr Asn Leu Thr Gly Leu

405 410 415

Phe Phe Pro Pro Gln Leu Asn Gly Thr Ala Asn Asp Thr Ala Gly Pro

420 425 430

Glu Leu Pro Asp Pro Leu Gly Gly Leu Leu Asp Glu Ala Met Leu Asp

435 440 445

Glu Ile Ser Leu Met Asp Leu Ala Ile Glu Glu Gly Phe Asn Pro Val

450 455 460

Gln Ala Ser Gln Leu Glu Glu Glu Phe Asp Ser Asp Ser Gly Leu Ser

465 470 475 480

Leu Asp Ser Ser His Ser Pro Ser Ser Leu Ser Ser Ser Glu Gly Ser

485 490 495

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ala Ser Ser

500 505 510

Ser Ala Ser Ser Ser Phe Ser Glu Glu Gly Ala Val Gly Tyr Ser Ser

515520 525

Asp Ser Glu Thr Leu Asp Leu Glu Glu Ala Glu Gly Ala Val Gly Tyr

530 535 540

Gln Pro Glu Tyr Ser Lys Phe Cys Arg Met Ser Tyr Gln Asp Pro Ala

545 550 555 560

Gln Leu Ser Cys Leu Pro Tyr Leu Glu His Val Gly His Asn His Thr

565 570 575

Tyr Asn Met Ala Pro Ser Ala Leu Asp Ser Ala Asp Leu Pro Pro Pro

580 585 590

Ser Ala Leu Lys Lys Gly Ser Lys Glu Lys Gln Ala Asp Phe Leu Asp

595 600 605

Lys Gln Met Ser Arg Asp Glu His Arg Ala Arg Ala

610 615 620

<210>2

<211>1860

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

atgctttctc tgaagaaata cttaacggaa ggacttctcc agttcaccat tctgctgagt 60

ttgattgggg tacgggtgga cgtggatact tacctgacct cacagcttcc cccactccgg 120

gagatcatcc tggggcccag ttctgcctat actcagaccc agttccacaa cctgaggaat 180

accttggatg gctatggtat ccaccccaag agcatagacc tggacaatta cttcactgcc 240

cggcggctcc tcagtcaggt gagggccctg gacaggttcc aggtgccaac cactgaggta 300

aatgcctggc tggttcaccg agacccagag gggtctgtct ctggcagtca gcccaactca 360

ggcctcgccc tcgagagttc cagtggcctc caagatgtga caggcccaga caacggggtg 420

cgagaaagcg aaacggagca gggattcggt gaagatttgg aggatttggg ggctgtagcc 480

cccccagtca gtggagactt aaccaaagag gacatagatc tgattgacat cctttggcga 540

caggatattg atctgggggc tgggcgtgag gtttttgact atagtcaccg ccagaaggag 600

caggatgtgg agaaggagct gcgagatgga ggcgagcagg acacctgggc aggcgagggc 660

gcggaagctc tggcacggaa cctgctagtg gatggagaga ctggggagag cttccctgca 720

caggtgccta gtggggagga ccagacggcc ctgtccctgg aagagtgcct taggctgctg 780

gaagccacct gcccctttgg ggagaatgct gagtttccag cagacatttc cagcataaca 840

gaagcagtgc ctagtgagag tgagccccct gctcttcaaa acaacctctt gtctcctctt 900

ctgaccggga cagagtcacc atttgatttg gaacagcagt ggcaagatct catgtccatc 960

atggaaatgc aggccatgga agtgaacaca tcagcaagtg aaatcctgta cagtgcccct 1020

cctggagacc cactgagcac caactacagc cttgccccca acactcccat caatcagaat 1080

gtcagcctgc atcaggcgtc cctggggggc tgcagccagg acttcttact cttcagcccc 1140

gaggtggaaa gcctgcctgt ggccagtagc tccacgctgc tcccgttggc ccccagcaat 1200

tctaccagcc tcaactccac cttcggctcc accaacctga cagggctctt ctttccaccc 1260

cagctcaatg gcacagccaa tgacacagca ggcccagagc tgcctgaccc tttggggggt 1320

ctgttagatg aagctatgtt ggatgagatc agccttatgg acctggccat tgaagaaggc 1380

tttaaccctg tgcaggcctc ccagctggag gaggaatttg actctgactc aggcctttcc 1440

ttagactcga gccatagccc ttcttcccta agcagctctg aaggcagttc ttcctcttct 1500

tcctcctcct cttcctcttc ttcctctgct tcttcctctg cctcttcctc cttttctgag 1560

gaaggtgcgg ttggctacag ctctgactct gagaccctgg atctggaaga ggccgagggt 1620

gctgtgggct accagcctga gtattccaag ttctgccgca tgagctacca ggatccagct 1680

cagctctcat gcctgcccta cctggagcac gtgggccaca accacacata caacatggca 1740

cccagtgccc tggactcagc cgacctgcca ccacccagtg ccctcaagaa aggcagcaag 1800

gagaagcagg ctgacttcct ggacaagcag atgagccggg atgagcaccg agcccgagcc 1860