CN110903998A

CN110903998A - Intestinal tract separated fusobacterium nucleatum Wenzeri strain and application thereof

Info

Publication number: CN110903998A
Application number: CN201911013474.9A
Authority: CN
Inventors: 秦环龙; 蔚青; 毕德玺; 李豪
Original assignee: Shanghai Tenth Peoples Hospital
Current assignee: Shanghai Tenth Peoples Hospital
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2020-03-24
Anticipated expiration: 2039-10-23
Also published as: CN110903998B

Abstract

The invention provides a Fusobacterium nucleatum Wedelia strain THCT14A3 separated from intestinal tracts, which is classified and named as Fusobacterium nucleatum subsp. M2019363, the preservation date is 2019, 05 and 17, and the preservation unit is the China center for type culture Collection. The sequence of the 16SrRNA gene of the strain is shown as SEQ ID NO. 1. The invention also provides a specific DNA sequence for identifying the fusobacterium nucleatum Wen subspecies and a corresponding primer sequence thereof. The result of co-culture of the colorectal cancer cell line and THCT14A3 shows that THCT14A3 can remarkably promote colorectal cancer cell proliferation. Therefore, the THCT14A3 provided by the invention is helpful for microecological study of colorectal cancer intestinal tracts.

Description

Intestinal tract separated fusobacterium nucleatum Wenzeri strain and application thereof

Technical Field

The invention relates to the field of fusobacterium nucleatum, in particular to a fusobacterium nucleatum Wen subspecies strain separated from an intestinal tract and application thereof in intestinal microecological research of colorectal cancer.

Background

Colorectal cancer is not only the third most advanced cancer in the world, but also the fourth leading cause of cancer death in the world, immediately following lung, liver and stomach cancers. Colorectal carcinogenesis is a malignant proliferation of cells caused by a dual hit of both genetics and environment. The discovery of the association of gut microbes with colorectal cancer has prompted researchers to investigate the mechanisms by which microbial carcinogenesis occurs. A variety of microorganisms associated with colorectal cancer have been sequentially discovered so far, including Citrobacter, Streptococcus bovis, Pasteurella multocida, helicobacter pylori, human papilloma virus, enterotoxigenic fragile bacillus, pathogenic Escherichia coli, and Fusobacterium nucleatum, and the like.

The fusobacterium nucleatum is the most common pathogenic bacterium causing extraoral infection in oral symbiosis bacteria, has strong invasive capability for infecting various eukaryotic cells, is closely related to gingivitis, periodontitis, tonsillitis, liver abscess, acute appendicitis and the like, and along with the deepening of research, more and more evidences support the generation and development of the fusobacterium nucleatum and colorectal cancer.

Fusobacterium nucleatum includes four subspecies: nucleated, polymorphic, venule and subspecies animals. However, only ATCC25586 and ATCC 23726, which belong to the species Setaria nucleata, were used as the subjects in the study of colorectal cancer; and both are not from the intestinal tract but are isolated from the oral cavity (ATCC 25586) and the vagina (ATCC 23726), respectively. The intestinal flora composition is diverse, the subspecies composition of the fusobacterium nucleatum in the intestinal tract is not clear, a single standard strain is difficult to have enough representativeness, and different subspecies isolates isolated from the tumor microenvironment are not common.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides an intestinal tract separated fusobacterium nucleatum subsp Venturi strain and application thereof in intestinal tract microecological research of colorectal cancer.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a fusobacterium nucleatum Wedney subspecies strain separated from intestinal tract, which is named as THCT14A3, and the 16SrRNA gene sequence of the strain is shown as follows:

>16S_rRNA_gene THCT14A3

AGAGTTTGATCCTGGCTCAGGATGAACGCTGACAGAATGCTTAACACATGCAAGTCAACTTGAATTTGGGTTTTTAACTTAGGTTTGGGTGGCGGACGGGTGAGTAACGCGTAAAGAACTTGCCTCACAGCTAGGGACAACATTTGGAAACGAATGCTAATACCTAATATTATGATAATAGGGCATCCTATAATTATGAAAGCTATAAGCGCTGTGAGAGAGCTTTGCGTCCCATTAGCTAGTTGGAGAGGTAACGGCTCACCAAGGCGATGATGGGTAGCCGGCCTGAGAGGGTGATCGGCCACAAGGGGACTGAGACACGGCCCTTACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGACCAAGAGTCTGATCCAGCAATTCTGTGTGCACGATGAAGTTTTTCGGAATGTAAAGTGCTTTCAGTTGGGAAGAAAGAAATGACGGTACCAACAGAAGAAGTGACGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCACGAGCGTTATCTGGATTTATTGGGCGTAAAGCGCGTCTAGGTGGTTATGTAAGTCTGATGTGAAAATACAGGGCTCAACTCTGTATTGCGTTGGAAACTGTATAACTAGAGTACTGGAGAGGTAAGCGGAACTACAAGTGTAGAGGTGAAATTCGTAGATATTTGTAGGAATGCCGATGGGGAAGCCAGCTTACTGGACAGATACTGACGCTGAAGCGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATTACTAGGTGTTGGGGGTCGAACCTCAGCGCCCAAGCAAACGCGATAAGTAATCCGCCTGGGGAGTACGTACGCAAGTATGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGCAACGCGAGGAACCTTACCAGCGTTTGACATCTTAGGAATGAGACAGAGATGTTTCAGTGTCCCTTCGGGGAAACCTAAAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTTTCGTATGTTACCATCATTAAGTTGGGGACTCATGCGATACTGCCTACGATGAGTAGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATACGCTGGGCTACACACGTGCTACAATGGGTAGAACAGAGAGTTGCAAAGCTGTGAAGTGGAGCTAATCTCAGAAAACTATTCTTAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGCGAATCAGCAATGTCGCGGTGAATACGTTCTCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTGGTTGCACCTGAAGTAGCAGGCCTAACCGTAAGGAGGGATGCTCCGAGGGTGTGATTAGCGATTGGGGTGAAGTCGTAACAAGGTATCCGTACGGGAACGTGCGGATGGATCACCT(SEQID NO:1)。

further, the strain is classified and named as Fusobacterium subclavus. vincenii, and the preservation number is CCTCC NO: m2019363, wherein the preservation date is 2019, 05 and 17, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university in Wuhan City.

Further, the strain was isolated from a fresh sigmoid colon elevated adenocarcinoma surgical resection specimen.

Further, the strain forms relatively large, round-edged, flat, translucent white colonies after 7 days of culture on Columbia medium.

Furthermore, the strain is red and fusiform under the mirror after gram staining.

The second aspect of the invention provides the application of the strain in intestinal microecological research of colorectal cancer.

In a third aspect, the invention provides a specific DNA sequence for identifying Fusobacterium nucleatum Wedney subspecies, comprising the sequence shown in SEQ ID NO. 2 and a fragment having BLASTn similarity with any fragment of the sequence in SEQ ID NO. 2.

>vincentii_specific_region

GGCAAGCACAAGATAGGGCATAATTATTAATTAAACAATTAAAAATAAAATATAAAAAATGGAGGAAAAATGAAAAAAAAATTTTTATTAATGGTTGTTTTAGGAATGTTGATAGTTC(SEQ ID NO:2)。

Further, the sequences of the DNA amplification primers used for detecting and identifying the Wenzene subspecies are as follows:

Vin-F：5’-GGCAAGYACAAGATAGGGCA-3’(SEQ ID NO:3)；

Vin-R：5’-GAACTATCAACATTCCTAAAACAACCA-3’(SEQ ID NO:4)。

further, the pair of primers can be applied to general PCR and real-time quantitative PCR.

Compared with the prior art, the invention has the following beneficial effects:

the fusobacterium nucleatum Wenyun subspecies THCT14A3 provided by the invention is directly derived from colon tumor tissues, has more representativeness under the condition of simulated intestinal tract experiment (such as co-culture), and is beneficial to the research on intestinal microecology of colorectal cancer. In addition, the invention firstly provides a molecular detection and identification method of fusobacterium nucleatum venture subspecies on the basis of THCT14A3 in combination with comparative genomics, and the provided specific DNA sequence and corresponding primer are suitable for single thallus samples or samples containing mixed thallus.

Drawings

The invention discloses an intestinal tract separated Fusobacterium nucleatum Wenychii strain THCT14A3 which is preserved and is classified and named as Fusobacterium nucletusubsp. M2019363, the preservation date is 2019, 05 and 17 months, the preservation unit is China center for type culture Collection, and the preservation unit address is Wuhan university in Wuhan City.

FIG. 1 is a graph showing the proliferation of the HCT116 cell line after co-culture with THCT14A3 in one embodiment of the present invention (in which the left graph is the growth curve of HCT116 cells; and the right graph is the comparison of proliferation at 72 hours of co-culture (two-tailed t-test p < 0.01));

FIG. 2 is a graph showing the proliferation of a LoVo cell line after co-culture with THCT14A3 in accordance with one embodiment of the present invention (in which the left graph is a LoVo cell growth curve; and the right graph is a comparison of proliferation at 72 hours of co-culture (two-tailed t-test p < 0.01));

FIG. 3 is a diagram showing the result of agarose gel electrophoresis for identifying a Fusobacterium nucleatum Wen subspecies strain in a Fusobacterium strain using Vin-F/Vin-R primers in an embodiment of the present invention.

Detailed Description

The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

Example one

This example provides an enterolytically isolated Fusobacterium nucleatum subsp, THCT14a3, classified and designated Fusobacterium nucleatum subsp. M2019363, the preservation date is 2019, 05 and 17, and the preservation unit is the China center for type culture Collection.

The 16S rRNA gene sequence of the strain is specifically shown as SEQ ID NO:1, and has 99.59% consistency with the 16S rRNA gene sequence (NCBI accession number NR _113040.1) of the F.nuclear susbsp.vintentii strain ATCC 51190 and 98.58% consistency with the 16S rRNA gene sequence (NCBI accession number NR _114702.1) of the F.nuclear subsp.nuclear strain ATCC 25586.

The complete genome of THCT14A3 was determined, and it was found that the THCT14A3 genome comprises a circular chromosome, has a size of 2052554bp, a GC content of 27.46%, and contains 1,898 protein-encoding genes and 63 non-encoding genes (46 tRNA genes, 5 sets of 5S-16S-23SrRNA genes, and 2 sRNA genes). The whole genome of this strain was analyzed by Jspecies software, and found to have a whole genome ANIb value of 92.13 with respect to F.null subsp.null strain ATCC25586 (NCBI genome accession No.: GCA-003019295.1) and 97.83 with respect to F.null subsp.vintensii strain 3-1-36A 2(NCBI genome accession No.: GCA-000162235.2). ANIb, averagenucleotide identity using BLAST (based on the average nucleotide sequence identity of BLAST), is an index for evaluating the affinity of strains, and generally, 95 or more is considered as the same genus. Indicating that the strain belongs to the subspecies of animals (vincentii).

Example two

This example provides a specific DNA sequence and corresponding primer sequence for identifying fusobacterium nucleatum venoms based on the sequence of THCT14a3 in combination with comparative genomics. The specific method comprises the following steps:

combining the F.nucleatum genome downloaded from NCBI on the basis of THCT14A3 genome; performing subspecies classification according to the ANIb value; sequences conserved in the genome of the Wenyujin subspecies were analyzed by BLASTn; these conserved sequences were then compared with the genomes of other subspecies by BlASTn, where a region not present in the genomes of the other subspecies, i.e.the Venturi subspecies-specific DNA sequence, was selected (see below). (the DNA signature sequences referred to herein include the following sequences and unlisted fragments having BLASTn similarity to any of the following sequences, which are not exhaustive because of unavoidable polymorphisms in the DNA sequence)

>vincentii_specific_region

On the basis of the sequence shown in SEQ ID NO. 2, DNA amplification primers for detecting and identifying the Wen subspecies are designed, and the sequences are as follows:

Vin-F：5’-GGCAAGYACAAGATAGGGCA-3’(SEQ ID NO:3)；

Vin-R：5’-GAACTATCAACATTCCTAAAACAACCA-3’(SEQ ID NO:4)。

the pair of primers can be applied to common PCR and real-time quantitative PCR. In addition, the specific DNA sequences and corresponding primers provided in this example are suitable for use in a single cell sample or a sample containing mixed cells.

Verification experiment I

The following demonstrates that F.nucleatum THCT14E2 obtained from colorectal cancer tumor tissues can promote proliferation of colorectal cancer cell lines by co-culturing the colorectal cancer cell lines with THCT14E 2.

THCT14a3 co-cultured with a colorectal cancer HCT116 cell line, the specific procedure was as follows:

freshly prepared HCT116 cell suspension was seeded into 24-well plates (1X 10)⁵Individual cells/well). After culturing for 12-24 hours until the cells are attached, the cells are washed with PBS and the non-resistant medium is replaced. Freshly prepared THCT14a3 bacterial suspension (bacteria suspended using PBS) was co-cultured (1 × 10) with HCT116 cells⁸CFU/well); equal volumes of PBS were used as blanks, each in 3 replicate wells. Culturing under normal conditions, and detecting cell proliferation activity by using a CCK8 method (according to the steps given in the specification) at 6 hours, 24 hours, 48 hours and 72 hours. Wherein freshly prepared bacterial suspension was replaced every 24 th.

As can be seen from fig. 1, the proliferation activity of HCT116 cells at 72 hours is significantly higher than that of the control group (p <0.01), indicating that THCT14a3 can significantly promote the proliferation of HCT116 cells.

The THCT14A3 and colorectal cancer LoVo cell line are cultured together, and the specific operation steps are as follows:

freshly prepared LoVo cell suspension was inoculated into 24-well plates (1X 10)⁵Individual cells/well). After culturing for 12-24 hours until the cells are attached, the cells are washed with PBS and the non-resistant medium is replaced. Get newFreshly prepared THCT14a3 bacterial suspension (bacteria suspended using PBS) was co-cultured (1 × 10) with LoVo cells⁸CFU/well); an equal volume of PBS was used as a blank. Each was done in 3 replicate wells. Culturing under normal conditions, and detecting cell proliferation activity by using a CCK8 method (according to the steps given in the specification) at 6 hours, 24 hours, 48 hours and 72 hours. Wherein freshly prepared bacterial suspension was replaced every 24 th.

From fig. 2, it can be seen that the proliferation activity of the LoVo cells at 72 hours is significantly higher than that of the control group (p <0.01), indicating that THCT14a3 can significantly promote the proliferation of the LoVo cells.

The verification experiment shows that the fusobacterium nucleatum Wedneri strain THCT14A3 can promote the proliferation of colorectal cancer cells, so that THCT14A3 can be used for the research of colorectal cancer.

Verification experiment 2

The specificity of the primers Vin-F/Vin-R for identifying the Wenzin subspecies in the second embodiment is verified by the verification experiment.

The specific operation steps are as follows:

total DNA of the strains listed in Table 1 was prepared, PCR-amplified using Vin-F/Vin-R primers, and subjected to agarose gel electrophoresis.

TABLE 1 Clostridium strains used in the experiments

Bacterial strains	Bacterial strain
		1Fn2①	F.varium
1Fn2②	F.varium
		4FN2②	F.varium
4FN2④	F.varium
		5CB④	F.nucleatumsubsp.animalis
5CB⑤	F.nucleatumsubsp.animalis
		6CVE②	F.mortiferum
6CB3	F.nucleatumsubsp.animalis
		7Fn2②	F.nucleatumsubsp.polymorphum
7CB②	F.nucleatumsubsp.animalis
		13Fn2①	F.varium
THCT14A3	F.nucleatumsubsp.vincentii
		14CVE③	F.nucleatumsubsp.vincentii
14Fn2②	F.nucleatum
		15Fn2①	F.nucleatumsubsp.polymorphum
18Fn2①	F.periodonticum
		23CVE①	F.varium
23Fn2①	F.varium
		23Fn2③	F.varium
ATCC 26686	F.nucleatumsubsp.nucleatum

As can be seen from FIG. 3, the pair of primers only amplified positively at the corresponding position (118bp) in the strain of Fusobacterium nucleatum Wen subspecies, but not amplified in other Fusobacterium species. Thus, the primer has good specificity and no non-specific amplification.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Sequence listing

<110> tenth people hospital in Shanghai City

<120> intestinal tract separated fusobacterium nucleatum Wenzeri subspecies strain and application thereof

<160>4

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<210>1

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<213>16S_rRNA_gene THCT14A3 [CCTCC M 2019363]

<400>1

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tgaatttggg tttttaactt aggtttgggt ggcggacggg tgagtaacgc gtaaagaact 120

tgcctcacag ctagggacaacatttggaaa cgaatgctaa tacctaatat tatgataata 180

gggcatccta taattatgaa agctataagc gctgtgagag agctttgcgt cccattagct 240

agttggagag gtaacggctc accaaggcga tgatgggtag ccggcctgag agggtgatcg 300

gccacaaggg gactgagaca cggcccttac tcctacggga ggcagcagtg gggaatattg 360

gacaatggac caagagtctg atccagcaat tctgtgtgca cgatgaagtt tttcggaatg 420

taaagtgctt tcagttggga agaaagaaat gacggtacca acagaagaag tgacggctaa 480

atacgtgcca gcagccgcgg taatacgtat gtcacgagcg ttatctggat ttattgggcg 540

taaagcgcgt ctaggtggtt atgtaagtct gatgtgaaaa tacagggctc aactctgtat 600

tgcgttggaa actgtataac tagagtactg gagaggtaag cggaactaca agtgtagagg 660

tgaaattcgt agatatttgt aggaatgccg atggggaagc cagcttactg gacagatact 720

gacgctgaag cgcgaaagcg tgggtagcaa acaggattag ataccctggt agtccacgcc 780

gtaaacgatg attactaggt gttgggggtc gaacctcagc gcccaagcaa acgcgataag 840

taatccgcct ggggagtacg tacgcaagta tgaaactcaa aggaattgac ggggacccgc 900

acaagcggtg gagcatgtgg tttaattcga cgcaacgcga ggaaccttac cagcgtttga 960

catcttagga atgagacaga gatgtttcag tgtcccttcg gggaaaccta aagacaggtg 1020

gtgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080

acccctttcg tatgttacca tcattaagtt ggggactcat gcgatactgc ctacgatgag 1140

taggaggaag gtggggatga cgtcaagtca tcatgcccct tatacgctgg gctacacacg 1200

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gtgtgattag cgattggggt gaagtcgtaa caaggtatcc gtacgggaac gtgcggatgg 1500

atcacct 1507

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ggcaagcaca agatagggca taattattaa ttaaacaatt aaaaataaaa tataaaaaat 60

ggaggaaaaa tgaaaaaaaa atttttatta atggttgttt taggaatgtt gatagttc 118

<210>3

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<213> DNA amplification primers for detection and identification of Wen subspecies (Forward)

<400>3

ggcaagyaca agatagggca 20

<210>4

<211>27

<212>DNA

<213> DNA amplification primers for detection and identification of Wenzi subspecies (reverse)

<400>4

gaactatcaa cattcctaaa acaacca 27

Claims

1. A fusobacterium nucleatum Wenzene strain separated from intestinal tracts is characterized in that the 16SrRNA gene sequence of the strain is shown as SEQ ID NO. 1.

2. The F.nucleatum Wehneri strain of claim 1, wherein the strain is named THCT14A3, classified and named Fusobacterium nuclear subsp. M2019363, the preservation date is 2019, 05 and 17, and the preservation unit is the China center for type culture Collection.

3. The fusobacterium nucleatum subsp venturi strain of claim 1, isolated from a fresh sigmoid colonic bulge adenocarcinoma surgical resection specimen.

4. The fusobacterium nucleatum subsp venus strain of claim 1, wherein the red, fusiform rod-shaped strain appears under the mirror after gram staining.

5. The fusobacterium nucleatum venus strain of claim 1, wherein a relatively large, round, flat, translucent white colony with irregular edges is formed after 7 days of culture on columbia medium.

6. Use of an enterically isolated fusobacterium nucleatum subsp venus strain according to any one of claims 1-5 in the microecological study of colorectal cancer.

7. A specific DNA sequence for identifying Fusobacterium nucleatum Venezetian subspecies, which is characterized in that the specific DNA sequence comprises a sequence shown as SEQ ID NO. 2 and a fragment with BLASTn similarity with any sequence in the SEQ ID NO. 2.

8. The specific DNA sequence of claim 7, wherein the primer sequence corresponding to the sequence shown in SEQ ID NO. 2 is shown in SEQ ID NO. 3-4.