CN117230075A - Nucleotide sequence related to bovine tuberculosis - Google Patents
Nucleotide sequence related to bovine tuberculosis Download PDFInfo
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- CN117230075A CN117230075A CN202311070504.6A CN202311070504A CN117230075A CN 117230075 A CN117230075 A CN 117230075A CN 202311070504 A CN202311070504 A CN 202311070504A CN 117230075 A CN117230075 A CN 117230075A
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- 206010006049 Bovine Tuberculosis Diseases 0.000 title claims abstract description 13
- 239000002773 nucleotide Substances 0.000 title claims abstract description 13
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 108091081062 Repeated sequence (DNA) Proteins 0.000 claims 1
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- 230000002365 anti-tubercular Effects 0.000 abstract description 5
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- 229940072185 drug for treatment of tuberculosis Drugs 0.000 abstract 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a nucleotide sequence related to bovine tuberculosis, belonging to the technical field of bovine tuberculosis related gene sequence screening, wherein the nucleotide sequence is shown as SEQ ID NO. 1. The difference of 108bp between the CD43 genes of the Hainan cattle and the common cattle is found by comparing the CD43 genes of the Hainan cattle and the common cattle, and the 108bp is considered to be related to bovine tuberculosis, so that a theoretical basis is laid for identifying anti-tuberculosis cattle and researching and developing anti-tuberculosis drugs in the future.
Description
Technical Field
The invention relates to the technical field of nucleotide sequence screening related to bovine tuberculosis, in particular to a nucleotide sequence related to bovine tuberculosis.
Background
Bovine tuberculosis (Bovine tuberculosis, bTB) is a zoonosis caused by infection with mycobacterium bovis (Mycobacterium bovis, m.bovis), and the primary infectious source is sick cattle. Studies have shown a broad range of mycobacterium bovis infections, including humans, domestic animals and wild animals. The main route of human infection is to consume non-sterilized milk products and inhale aerosols in contact with the diseased animals. bTB is a group B animal epidemic that is listed by the world animal health Organization (OIE). At present, the specific prevalence rate of bovine tuberculosis in various places in China is not completely clear. Shaanxi, xinjiang and inner Mongolia are provinces (autonomous areas) reporting a large number of epidemic situations, and the overall trend is shown to be significantly higher in northern areas than in other areas. Thus, the treatment of bovine tuberculosis has been an important goal for the cattle industry. Searching for dominant disease resistance genes has become a major issue by utilizing genomics technology.
CD43 (SPN) is a sialoglycoprotein that is ubiquitous on monocytes, neutrophils and T lymphocytes, where CD43 extends 45nm from the phospholipid bilayer, becoming the largest glycoprotein on the cell surface. Cell-to-cell contact repulsion is limited as a barrier molecule due to its negative charge, ubiquity, size and rigidity; as a signaling molecule, regulate T cell migration, activation, and survival; at the same time, the mycobacterium is involved in the processes of adhesion, invasion and infection, etc.
With the rapid development of cattle farming, proteomics, genomics and other technologies are applied to production practice, and the health and diseases of cattle are continuously and deeply studied. Therefore, searching for nucleotide sequences related to anti-tuberculosis in different species of bovine SPN genes using genomic sequencing techniques is a technical problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention obtains genetic data by using whole genome sequencing technology, wherein it is found that SPN gene has a specific variation of a certain sequence between normal cattle and Hainan cattle, and the structural variation is determined to be related to the function of invasion of Mycobacterium tuberculosis into macrophages by constructing SPN/SPN-HN vector.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a nucleotide sequence associated with bovine tuberculosis, said nucleotide sequence being shown in SEQ ID No. 1:
AGCTCTCTGGAGATCTCTGGTGGGACCAGTAGACCTCCTGTCTCCATGGCAATGAGCTCTTTGGACACCTCCAGTGGGACCAGCGAAGTCCTGTTAACTATGGCAACT, as shown in SEQ ID NO. 1.
As the same invention concept as the technical proposal, the invention also claims the amino acid sequence expressed by the nucleotide sequence, and the amino acid sequence is
TSEVLLTMATSSLDISGGTSRPPVTMATSSLETSSG, a repeat sequence as shown in SEQ ID NO. 2.
Compared with the prior art, the invention discovers that the CD43 genes of common cattle and Hainan cattle are only 108bp different, more specifically 108bp different, by constructing the SPN/SPN-repeat vector, the repetition number of the segment of sequence is related to the anti-tuberculosis capability of the cattle, therefore, the segment of gene sequence is used as a nucleotide locus to screen cattle with strong anti-tuberculosis capability, and provides a theoretical basis for animal husbandry cultivation.
Meanwhile, the invention discovers that over-expression of SPN or SPN-repeat causes the expression level of IL-6, IL-12 and IL-10 genes on the surface of macrophages to be increased, which indicates that SPN improves the anti-tuberculosis capability of cattle by improving the immunity of immune cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the structure of amino acids expressed by different bovine genomes;
FIG. 2 is a diagram showing the effect of SPN-repeat structure on bacterial adhesion; a: after 48h transfection of SPN/SPN-delta repeat vector, the MTB with moi=10 was infected with RAW264.7 and the number of MTBs was counted using immunofluorescent staining; b: MTB adhered to RAW264.7 number statistics;
FIG. 3 is a diagram showing the structural function of SPN-repeat; 48h after transfection of SPN/SPN-HN vector, MTB with MOI=10 was infected with RAW264.7, and changes in the expression levels of immune factors IL-6 (A), IL-12 (B), IL-10 (C) and TNF- α (D) were detected by qRT-PCR.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 comparison of genomes of Normal cattle and Hainan cattle
Collection of samples
Collecting blood samples of common cattle, american bison and Hainan cattle by using a jugular vein blood sampling mode;
extraction of blood samples
The whole genomic DNA of each sample was extracted using standard phenol chloroform procedure as follows:
(1) Thawing frozen blood sample at room temperature, adding 1mL of blood into a 2mL sterilizing centrifuge tube, adding 1:1 Phosphate Buffer Solution (PBS), mixing, shaking, mixing for 15min, centrifuging (12000 r/min,5min, 4deg.C), discarding supernatant, and repeating the above steps until the supernatant and precipitate are transparent;
(2) Adding 1mLDNA extraction buffer, blowing blood cells to precipitate, and water-bathing at 37 ℃ for 1h;
(3) Adding proteinase K to final concentration of 6ug/mL, mixing, and digesting in water bath at 55deg.C until flocculent precipitate is obtained for several hours, and clarifying overnight;
(4) After the temperature of the reaction liquid is reduced to room temperature, adding Tris saturated phenol in a ratio of 1:1, gently shaking for 20min until the mixture is fully and uniformly mixed, centrifuging (12000 r/min,10min and 4 ℃), transferring the upper liquid into a new sterilization centrifuge tube, and repeating the steps (1) - (3);
(5) Adding 1:1 volume of chloroform, gently shaking for 20min to fully mix, centrifuging (12000 r/min,10 min), and transferring the upper water phase to a new centrifuge tube;
(6) Adding 1:1 volume of chloroform to isoamyl alcohol (24:1), gently shaking for 15min to fully mix, centrifuging (12000 r/min,10min,4 ℃) and transferring the upper water phase to a new sterilizing centrifuge tube;
(7) 1:2 volumes of pre-chilled absolute ethanol at-20deg.C was added, and after 30min at-20deg.C the tube was gently inverted to visualize flocculent precipitated DNA. After centrifugation (12000 r/min,10min,4 ℃) the ethanol was discarded;
(8) Adding 1mL of 70% ethanol precooled at-20 ℃, vibrating for 10min, centrifuging (12000 r/min,5min,4 ℃) and rinsing the DNA precipitate for 1-2 times;
(9) Centrifuging (12000 r/min,10min, 4deg.C), discarding supernatant, and standing at room temperature to volatilize ethanol;
(10) 200-400 uL TE solution is added to dissolve DNA, and the solution is kept at 4 ℃ until DNA precipitation is completely dissolved.
Genome sequencing
Carrying out double-end library establishment and whole genome sequencing with the sequencing length of 150bp according to the requirements of an Illumina Hiseq 2000 sequencing platform;
data analysis
The gene sequence data of common cattle, bison and Hainan cattle were obtained by genomic sequencing. Obtaining structural variation of the American bison and Hainan cattle relative to the CD43 gene compared with common cattle; see fig. 1.
As shown below, the genome sequence of the common cattle is shown as SEQ ID NO. 3; the genome sequence of Hainan cattle is shown as SEQ ID NO. 4;
as shown in SEQ ID NO. 3;
as shown in SEQ ID NO. 4.
Constructing an SPN/SPN-HN vector of common cattle, infecting mycobacterium tuberculosis, and verifying the function of SPN structural variation.
RAW264.7 cells from the laboratory were cultured, the SPN/SPN-HN vector was overexpressed, mycobacterium tuberculosis was infected with moi=10, and immune responses of macrophages were detected using immunofluorescent staining and fluorescent quantitative PCR techniques.
Immunofluorescent staining:
(1) Incubating the mycobacterium tuberculosis with FITC dye solution for 1h at 37 ℃, and washing twice with PBS;
(2) Co-culturing for 4h with different MOI and RAW264.7, and washing twice with PBS;
(3) Fixing the groveling piece by using 4% paraformaldehyde;
(4) Incubation is carried out for 10min at normal temperature by using DAPI dye liquor, and TBS is washed twice;
(5) Placing the groveling piece on a glass slide in which an anti-fluorescence quenching agent is dripped in advance, and sealing the glass slide;
(6) Observations were made using a laser confocal microscope and photographed.
Fluorescent quantitative PCR technique:
extraction of total RNA from cells Using Trizol reagent according toII reverse transcription kit procedure reverse transcription was performed to synthesize cDNA. According to->qPCR/>The Green Master Mix kit instructions perform a real-time fluorescent quantitative PCR reaction. The GAPDH gene is used as an internal reference, the corresponding gene sequence of GenBank is referred to, and primers 5.0 are adopted to design primers, and the information of the primers is shown in Table 1.PCR reaction procedure at 95℃for 10min;95℃for 10s and 60℃for 30s, 40 cycles total.
TABLE 1 primer sequences
Results:
by constructing SPN/SPN-HN vector, immunofluorescence staining was used to determine the relationship of repeat adhesion to MTB. The results are shown in fig. 2, in which the bacteria entering the cells are increased by infection with the bound bacillus with moi=10, SPN-HN.
Relation between SPN-repeat Structure and immune response
The effect of SPN-repeat on the MTB-infected RAW264.7 immune response was examined using qRT-PCR using the SPN/SPN-HN vector. As a result, as shown in FIG. 3, over-expression of both SPN and SPN-HN resulted in a significant increase in the expression levels of IL-6, IL-10 and TNF- α, with no change in the expression levels of IL-10.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (2)
1. A nucleotide sequence related to bovine tuberculosis, which is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1:
AGCTCTCTGGAGATCTCTGGTGGGACCAGTAGACCTCCTGTCTCCATGG CAATGAGCTCTTTGGACACCTCCAGTGGGACCAGCGAAGTCCTGTTAAC TATGGCAACT, as shown in SEQ ID NO. 1.
2. The amino acid sequence expressed by the nucleotide sequence of claim 1, wherein the amino acid sequence is TSEVLLTMATSSLDISGGTSRPPVTMATSSLETSSG as a repeated sequence shown in SEQ ID NO. 2.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311070504.6A CN117230075A (en) | 2023-08-24 | 2023-08-24 | Nucleotide sequence related to bovine tuberculosis |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311070504.6A CN117230075A (en) | 2023-08-24 | 2023-08-24 | Nucleotide sequence related to bovine tuberculosis |
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2023
- 2023-08-24 CN CN202311070504.6A patent/CN117230075A/en active Pending
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