CN118703565A - Construction method and application of goat mammary epithelial cells overexpressing Ltf gene - Google Patents

Abstract

The present invention provides a construction method and application of goat mammary epithelial cells with overexpression of Ltf gene, a gRNA targeting the Ltf gene of goat mammary epithelial cells, and construction of goat mammary epithelial cells with overexpression of Ltf gene. The method comprises: using gene editing technology to replace the promoter encoding the Ltf gene with the promoter of the endogenous strong expression b-actin gene. The present invention designs a gRNA specifically targeting the Ltf gene, and uses the Cas9 protein to insert an endogenous strong promoter before the 5'UTR of the Ltf gene, thereby achieving the purpose of gene overexpression in mammary epithelial cells. The goat mammary epithelial cells with overexpression of Ltf gene obtained by the method of the present invention can effectively protect mammary epithelial cells from LPS-induced inflammatory response. The present method provides a method for constructing Ltf overexpression, providing theoretical support for the field of gene therapy for mastitis.

Description

Construction method and application of goat mammary epithelial cells overexpressing Ltf gene

Technical Field

The invention relates to the technical field of genetic engineering, and in particular to a construction method and application of goat mammary epithelial cells overexpressing Ltf genes.

Background Art

Lactoferrin (Ltf) is an iron-binding glycoprotein with a molecular weight of 80 kDa. It is mainly found in breast milk and has multiple biological functions, such as participation in iron metabolism, anti-inflammation, broad-spectrum antibacterial, and antiviral. Ltf's antibacterial effect is mainly manifested in antibacterial and bactericidal effects; its antiviral effect is manifested in not preventing the replication of the virus after infection, but preventing the virus from infecting cells to achieve its antiviral activity; in addition, Ltf is produced by mammary cells and white blood cells, and has a regulatory effect on blood coagulation, antibody production, interleukin and tumor necrosis factor production, T cell maturation, prostaglandin production, and natural killer cell (NK cell) activation. In addition, studies have shown that Ltf can enhance the phagocytosis of exogenous microorganisms by macrophages and monocytes, the killing of intracellular parasites, and the promotion of leukocyte adhesion on vascular endothelial cells, thereby facilitating the infiltration and translocation of leukocytes to the inflammatory site outside the blood vessel.

Ltf has important biological functions and has become a hot topic in international research. Studies have shown that the concentration of Ltf in milk varies greatly, mainly affected by physiological conditions, lactation stage, parity and breed, but the research results in this regard are not completely consistent, so it is worth further study. In addition, most current studies focus on the biological activity of Ltf, and there are few studies on the expression and regulation mechanism of Ltf, which still needs further in-depth analysis.

Summary of the invention

In view of the deficiencies in the prior art, the present invention aims to provide a method for constructing goat mammary epithelial cells overexpressing the Ltf gene and its application. Using goat primary mammary epithelial cells as materials, CRISPR/Cas9 gene editing technology is used to replace the promoter encoding the Ltf gene with an endogenous strong promoter to achieve high expression. After obtaining the positive clone cell line, the recombination efficiency is evaluated by quantitative and protein immunoblotting, and the function of the Ltf gene in the mastitis model is evaluated by LPS-induced inflammatory response, so as to obtain mammary epithelial cells that stably overexpress the goat Ltf gene and are anti-inflammatory.

In order to solve the above technical problems, the technical solution provided by the present invention is:

Method for constructing goat mammary epithelial cells overexpressing Ltf gene,

Using gene editing technology, the promoter encoding the Ltf gene was replaced with the promoter of the endogenous b-actin gene, which strongly expressed the Ltf gene, to overexpress the Ltf gene and obtain primary mammary epithelial cells that stably overexpressed the goat Ltf gene;

The specific steps include:

S1. Amplification of the repair fragment b-actin promoter: Using goat genomic DNA as a template, the b-actin promoter was amplified by PCR and purified; the nucleotide sequence of the b-actin promoter is shown in SEQ ID No.1;

S2. Amplification of the left homology arm and the right homology arm of the Ltf gene: Using the genomic DNA of goat Ltf as a template, 1 kb before and after the 5'UTR of the Ltf gene were used as the left and right homology arms, and PCR amplification and purification were performed; the nucleotide sequence of the left homology arm of the Ltf gene is shown in SEQ ID No.2; the nucleotide sequence of the right homology arm of the Ltf gene is shown in SEQ ID No.3;

S3. Preparation of Donor linear backbone: pZ-Tomato plasmid was double-digested with Sal I endonuclease and Hind III endonuclease, purified, and Donor linear backbone was obtained;

S4. Construction of pZ-Tomato-Ltf plasmid: digest the b-actin promoter obtained in S1 and the left homology arm and right homology arm fragments of the Ltf gene obtained in S2, and connect them to obtain homology arm fragments: left homology arm-promoter-right homology arm; connect the homology arm fragments with the Donor linear skeleton obtained in S3 to construct pZ-Tomato-Ltf plasmid;

S5. Construction of pX459-Ltf plasmid: determine the PAM site of the Ltf gene promoter and construct a double-stranded gRNA fragment; after the pX459 vector is digested with BbsI, the double-stranded gRNA fragment is connected to the pX459 plasmid vector to obtain the pX459-Ltf plasmid; transfer into Escherichia coli competent cells, screen and verify positive single clones;

S6. The pX459-Ltf plasmid obtained in S5 and the pZ-Tomato-Ltf plasmid obtained in S4 were electroporated into goat mammary epithelial cells at a mass ratio of 1:1, and resistance screening was performed to obtain mammary epithelial cells that stably overexpressed the goat Ltf gene.

Preferably,

The sequence of the gRNA is shown in SEQ ID No.10 and SEQ ID No.11.

Preferably,

The sequences of the primers used for amplifying the repair fragment b-actin promoter in S1 are shown in SEQ ID No.4 and SEQ ID No.5.

Preferably,

The sequences of the primers used to amplify the left homologous arm of the Ltf gene in S2 are shown in SEQ ID No.6 and SEQ ID No.7; the sequences of the primers used to amplify the right homologous arm of the Ltf gene are shown in SEQ ID No.8 and SEQ ID No.9.

Preferably,

The verification primer sequences of the pX459-Ltf plasmid in S5 are shown in SEQ ID No.11 and SEQ ID No.12.

Preferably,

The homology arm fragments in the S4 were connected to the Donor linear backbone by Gibson connection, and the mass ratio of the homology arm fragments to the Donor linear backbone was 2:1.

The above method constructs goat mammary epithelial cells overexpressing the Ltf gene.

The application of the above-mentioned Ltf gene overexpressing goat mammary epithelial cells in constructing an inflammatory response animal model.

Preferably,

The steps to construct an animal model of inflammatory response are:

After wild-type goat mammary epithelial cells and gene-overexpressing mammary epithelial cells were revived, when the cell confluence reached 70% to 80%, 10 μg/mL LPS was added for 12 hours, and total cell mRNA was extracted. The expression levels of inflammatory factor genes were detected by real-time fluorescence quantitative PCR.

Preferably,

The sequences of the primers for PCR detection of inflammatory factors are shown in SEQ ID No.13 to SEQ ID No.24.

The beneficial effects of the present invention are:

The present invention utilizes CRISPR/Cas9 gene editing technology, uses goat primary mammary epithelial cells as materials, replaces the promoter encoding the Ltf gene with the promoter of the endogenous b-actin gene that strongly expresses, and overexpresses the Ltf gene to obtain primary mammary epithelial cells that stably overexpress the goat Ltf gene. The present method provides a method for constructing Ltf gene overexpression, and the goat mammary epithelial cells overexpressing the Ltf gene obtained by the method can effectively protect the mammary epithelial cells from LPS-induced inflammatory response, providing theoretical support for the field of mastitis gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

Figure 1 shows the Ltf gene site-directed overexpression strategy based on the CRISPR/Cas9 system

Figure 2 shows the pX459 plasmid backbone and pZ-Tomato plasmid backbone targeting the Ltf gene

Figure 3 shows the homology arm fragment of Ltf gene and the amplified fragment of b-actin promoter

Figure 4 is the pZ-Tomato-Ltf plasmid restriction PCR verification

Figure 5 is a picture of stably transfected goat mammary epithelial cells after Ltf gene overexpression

Figure 6 is the expression results of Ltf mRNA and protein levels in goat mammary epithelial cells with overexpression of Ltf gene

Figure 7 is a graph showing the expression of inflammatory gene mRNA in goat mammary epithelial cells overexpressing Ltf gene after infection

DETAILED DESCRIPTION

The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

definition

As used herein, the terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides) of any length or their analogs. Examples of polynucleotides include, but are not limited to, coding or non-coding regions of genes or gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes and primers. One or more nucleotides in a polynucleotide may be further modified. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may also be modified after polymerization, for example by coupling with a labeling agent. "CRISPR/Cas9" is an adaptive immune defense formed by bacteria and archaea during long-term evolution, which can be used to fight invading viruses and foreign DNA. CRISPR/Cas9 gene editing technology is a technology for specific DNA modification of targeted genes. Gene editing technology based on CRISPR/Cas9 has shown great application prospects in a series of gene therapy application fields, such as blood diseases, tumors and other genetic diseases. This technical achievement has been applied to the precise modification of the genomes of human cells, zebrafish, mice and bacteria. The terms "gRNA", "guide RNA" and "CRISPR guide sequence" used in this article can be used interchangeably throughout and refer to RNA containing specific sequences that determine the Cas protein in the CRISPR/Cas system. The gRNA hybridizes with the target nucleic acid sequence in the host cell genome (partially or completely complementary). The length of the gRNA or part thereof hybridized with the target nucleic acid sequence may be between 15-25 nucleotides, 18-22 nucleotides or 19-21 nucleotides. In some embodiments, the length of the gRNA sequence hybridized with the target nucleic acid sequence may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In some embodiments, the length of the gRNA sequence hybridized to the target nucleic acid is between 10-30 or 15-25 nucleotides. "sgRNA" refers to a single-molecule guide RNA or a single-stranded guide RNA in the artificial CRISPR/Cas9 system, which refers to the RNA that guides the Cas protein to specifically bind to the target DNA sequence, and is an important component of the CRISPR gene knockout knock-in system. The sgRNA comprises a guide sequence that targets the target sequence. In a preferred embodiment, the sgRNA further comprises a tracr sequence and a tracr partner sequence.

"Guide sequence" refers to a sequence of about 17-20bp that specifies a targeting site, and can be used interchangeably with "guide sequence" or "spacer". In the context of forming a CRISPR complex, the "target sequence" is a sequence that the guide sequence is designed to have complementarity with, wherein hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex, and hybridization requires that the "target sequence" and the "guide sequence" or "guide sequence" have sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex, and complete complementarity is not required. "Complementary" means that the "guide sequence" or "guide sequence" and the target nucleotide sequence (in the case of the present invention, the target nucleotide sequence in the third exon region of the FTO gene of the cell line) can hybridize through the nucleotide pairing principle discovered by Watson and Crick. Those skilled in the art will understand that as long as there is sufficient complementarity, the "guide sequence" can hybridize with the target nucleotide sequence without the need for 100% complete complementarity between them. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence can be about or greater than about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more when optimally aligned using an appropriate alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, including the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, an algorithm based on the Burrows-Wheeler Transform, and the like.

Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (including hybridization of a guide sequence to a target sequence and complexing with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). The tracr sequence may comprise all or a portion of a wild-type tracr sequence, e.g., about or greater than about 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 70, 75, 80, 85 or more nucleotides of a wild-type tracr sequence) or consist of the above tracr sequence can also form part of a CRISPR complex, e.g., by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr partner sequence operably linked to a guide sequence.

In some embodiments, the tracr sequence has sufficient complementarity with the tracr partner sequence to hybridize and participate in the formation of the CRISPR complex. Similar to the hybridization of the "target sequence" and the "guide sequence" or "guide sequence", complete complementarity is not required, as long as it is sufficient to perform its function. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complementarity with the length of the tracr partner sequence under optimal alignment.

"Gene overexpression" or "overexpression" refers to editing a gene in a mammary epithelial cell (e.g., inserting, replacing, and/or deleting the gene) so that the gene acquires function (e.g., overexpressing a functional protein). Genes in the cell genome can be edited using various known molecular biology techniques (e.g., using zinc finger nuclease-based gene editing technology, TALEN gene editing technology, and CRISPR/Cas (e.g., CRISPR/Cas9) gene editing technology).

"Vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors include, but are not limited to: plasmids; phagemids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription start sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

The present invention provides a method for constructing goat mammary epithelial cells that overexpress the Ltf gene based on the CRISPR/Cas9 system and an application thereof, comprising: utilizing gene editing technology, taking goat primary mammary epithelial cells as materials, and overexpressing the Ltf gene by a promoter replacement method to obtain mammary epithelial cells that stably overexpress the goat Ltf gene and are anti-inflammatory.

After knowing the efficient gene editing region (the promoter region of the Ltf gene in the present invention), those skilled in the art can use any gene editing method, such as zinc finger nuclease-based gene editing technology, TALEN gene editing technology, CRISPR/Cas (such as CRISPR/Cas9) gene editing technology, and other gene editing methods discovered in the future, to edit the known efficient gene editing region, optimize the gene editing conditions, and achieve the purpose of efficient editing. Therefore, the present invention covers the technical solution of knocking out the promoter region target sequence of the Ltf gene identified by the present invention by any available gene editing method.

In a specific embodiment, the present invention overexpresses the Ltf gene of mammary epithelial cells through CRISPR/Cas9 technology, wherein the gRNA used to target the Ltf gene of mammary epithelial cells in the CRISPR/Cas9 technology comprises a nucleotide sequence complementary to the promoter region of the Ltf gene of mammary epithelial cells.

FIG1 is a strategy for site-directed overexpression of the Ltf gene based on the CRISPR/Cas9 system, and FIG2 is a map of the pX459-Ltf plasmid and pZ-Tomato-Ltf plasmid targeting the Ltf gene.

Example 1

Instruments used in the present invention:

96-well plate (centrifuge Beijing Dinghaoyuan Technology Co., Ltd.), electronic balance (METTLER TOLEDO, Switzerland), inverted fluorescence microscope (AMG), constant temperature water bath (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.), agarose horizontal electrophoresis instrument (Beijing Liuyi Biotechnology Co., Ltd.), protein transfer instrument (Bio-Rad, USA), real-time fluorescence quantitative PCR instrument (ABI, USA), _CO2 constant temperature cell culture incubator (Thermo Fisher), cell electrofection instrument (NEPAGENE).

Reagents used in the present invention:

Blood/cell/tissue genomic DNA extraction kit (TIANGEN, DP304-02), endotoxin-free plasmid extraction kit (Magen, P1156-02), KOD FX NEO high-efficiency PCR enzyme (TOYOBO, KFX-201), DH5α (Kangwei Century, CW0808B), ampicillin storage solution (Coolaber, SL3810), SalI restriction endonuclease (NEB, R3138V), BbsI restriction endonuclease (NEB, R3539S), HindIII restriction endonuclease (R0104S, NEB), T4 DNA ligase (M0201S, NEB), nucleic acid dye (CooLaber, SL2140), 1kb DNA ladder (Zhongke Ruitai, RTM417), CloneExpressIIOne Step Clonging Kit (Vazyme, C112), DMEM-F12 medium (Gibco, A4192001), FBS (Gibco, 16140071), DMSO (Sigma, D2650), penicillin-streptomycin double antibody (Invitrogen, 15140122), recombinant human insulin (Solarbio, I8830-20), recombinant human EGF (Sangong, c610033-0100), dexamethasone (Sangong, A601187-0005), 0.25% Trypsin-EDTA (1×) (Gibco, 25200-072), Trizol (Mei5, C0121), StarScript III one-tube de-genomic reverse transcription premix (GenStar, A230-10), HieffqPCR SYBRGreen Master Mix (High Rox Plus) (YEASEN, 11203ES08), Prestained Protein Ladder (Life, 26619), Recombinant Anti-LTF Antibody (abcam, ab109216), RIPA Cell Lysis Buffer (Strong) (Beyotime, P0013B), RNasin Inhibitor (Promega, N2112S), SDS-PAGE Gel Preparation Kit (Beyotime, P0012A), TEMED (Beyotime, ST728), 30% Arc-Bis (Beyotime, ST003), 1.0 M Tris-HCl (pH 6.8) (Beyotime, ST768), PhosSTOP (Roche, 04906845001), 1.5 M Tris-HCl (pH 8.8) (Beyotime, ST789), Western primary antibody diluent (Beyotime, P0256), Western secondary antibody diluent (Beyotime, P0258), 0.25% Trypsin-EDTA (Gibco, 25200056), PVDF membrane (Beyotime, FFP24), skimmed milk powder (Yili Group, 422535), PMSF (100 mM) (Beyotime, ST506).

This embodiment provides a method and application of constructing goat mammary epithelial cells for site-directed overexpression of the Ltf gene based on the CRISPR/Cas9 system, comprising the following steps:

Step 1: Amplification of the b-actin promoter (repair fragment)

Using goat genomic DNA as a template, the b-actin promoter (i.e., the repair fragment) was amplified by PCR and purified, and the nucleotide sequence is shown in SEQ ID No.1 in Table 1. The PCR reaction system and reaction conditions are shown in Table 2. RF F1/RF R1 are b-actin primers containing homology arms, and the nucleotide sequences are shown in SEQ ID No.4 and SEQ ID No.5 in Table 3. The PCR product was electrophoresed on a 1% agarose gel (100V, 15min), stained in EB for 30min, and the target band was verified under ultraviolet light. The size of the target band was 2kb, and the results are shown in Figure 3. The obtained PCR product was stored in a -20°C refrigerator for later use.

Step 2: Amplification of the homology arms of the Ltf gene

Using goat genomic DNA as a template, 1 kb before and after the 5'UTR of the Ltf gene was used as the left and right homology arms, and the nucleotide sequences were shown in SEQ ID No. 2 and SEQ ID No. 3 in Table 1, and PCR amplification and purification were performed. The reaction system and reaction conditions are shown in Table 2, RF F2/RF R2 and RF F3/RF R3 are the left and right homology arm primers containing b-actin, and the nucleotide sequences are shown in SEQ ID No. 6, SEQ ID No. 7 (left homology arm); SEQ ID No. 8, SEQ ID No. 9 (right homology arm) in Table 3. The PCR product was electrophoresed (100V, 15min) in EB for 30min using 1% agarose gel, and the target band was verified under ultraviolet light. The target band size was 1kb, and the results are shown in Figure 3. The obtained PCR product was stored in a -20℃ refrigerator for standby use.

Table 1b-actin promoter (repair fragment) and Ltf gene left and right homologous arm sequence list

Table 2 PCR amplification reaction system and reaction conditions Reaction system

Reaction conditions

Table 3 b-actin primers containing homology arms and left and right homology arm primers

Step 3: Preparation of Donor Linear Skeleton

The Donor linear backbone digestion system and reaction conditions are shown in Table 4. Double digestion was performed with Hind III endonuclease and Sal I endonuclease. The amount of Sal I and Hind III added to the reaction system was determined according to the template amount of the pZ-Tomato plasmid. The Donor linear backbone was obtained after purification.

Table 4 Donor linear backbone template enzyme digestion system

Step 4: Construction of pZ-Tomato-Ltf plasmid

The promoter sequence obtained in step 1 and step 2 and the left and right homology arm sequences were connected by left homology arm-promoter-right homology arm to obtain homology arm fragments, and connected to the Donor linear backbone to construct pZ-Tomato-Ltf plasmid;

(1) The amplified left and right homology arms and the promoter were added to the connection system at a mass ratio of 1:1:2 to obtain homology arm fragments. The homology arm fragments and the Donor linear backbone were added to the connection system at a mass ratio of 2:1. The promoter, homology arm fragments and the Donor linear backbone were connected by Gibson to obtain the pZ-Tomato-Ltf plasmid. The connection system is shown in Table 5 and Table 6. 10 μL of each reaction system was subjected to PCR reaction at 37°C for 30 min to obtain a reaction solution.

(2) Take 10 μL of the reaction solution for E. coli transformation.

(3) After transformation, the cells are first spread on LB-Amp plates. After the strain grows, it is streaked on the LB-Amp plate and cultured at 37°C for 12 hours. A small amount of cells is transferred to a test tube containing 5 mL of LB+Amp liquid culture medium and cultured for 12 to 16 hours (120 rpm, 37°C).

(4) Collect the bacteria and use a plasmid DNA large-scale extraction kit to extract the pZ-Tomato-Ltf plasmid from Escherichia coli.

(5) After restriction digestion, the pZ-Tomato-Ltf plasmid and the digestion product were subjected to electrophoresis (100 V, 15 min) using 0.7% agarose gel, stained in EB for 30 min, and the target band was verified under ultraviolet light. The results are shown in FIG4 . The left figure is a schematic diagram of the construction of the pZ-Tomato-Ltf plasmid, and its fragment size is 11,578 bp. After single digestion with EcoR I endonuclease, it is expected to produce 9306 bp, 7567 bp, 6283 bp, 5295 bp, 4011 bp and 2272 bp bands. The right figure is a single clone extracted from the PZ-Tomato-Ltf

Agarose gel image after plasmid digestion. After single digestion with 8# and 10#, bands larger than 2kbp, 5kbp and 6k bp appeared. It was preliminarily determined to be the expected plasmid and the subsequent DNA purification step could be carried out. The DNA ladder used in the agarose gel was 1kb plus marker. The obtained pZ-tomato-Ltf plasmid was sequenced and confirmed to obtain the pZ-tomato-Ltf plasmid with the correct sequence.

Table 5 Reaction system of Gibson ligation of promoter and homology arm

Table 6 Reaction system of Gibson connection of Donor linear skeleton

Step 5: Construction of pX459-Ltf plasmid

(1) Determine the PAM site of the Ltf gene promoter, construct a double-stranded gRNA fragment, the sequence is shown in SEQ ID No. 10 and SEQ ID No. 11 in Table 7, use pX459 plasmid as the vector, after Bbs I digestion, the double-stranded gRNA fragment is ligated to the pX459 vector to obtain the pX459-Ltf plasmid.

(2) The pX459-Ltf plasmid was transformed into competent E. coli cells, plated and cultured, screened, and single clones were verified by PCR reaction. The identification primers are shown in Table 7 for SEQ ID No. 11 and SEQ ID No. 12, and the PCR system and reaction conditions are shown in Table 8.

(3) The positive single clones were screened and cultured in a test tube containing 5 mL of LB+Amp liquid culture medium for 12 to 16 h (120 rpm, 37°C).

(4) Collect the bacteria, use a plasmid DNA large-scale extraction kit to extract the pX459-Ltf plasmid from Escherichia coli, and perform sequencing to confirm that the pX459-Ltf plasmid with the correct sequence is obtained.

Table 7pX459-Ltf plasmid verification primers

Table 8 pX459-Ltf plasmid verification PCR reaction system and conditions

Step 6: Cell transfection and positive screening

10 μg of each pX459-Ltf plasmid and pZ-Tomato-Ltf plasmid were mixed with 100 μL Opti-MEM solution and electroporated into 10*6 mammary epithelial cells. The electroporation conditions are shown in Table 9. The results are shown in Figure 5. The expression of red fluorescence (tdTomato protein) can be detected in mammary epithelial cells, which preliminarily indicates that the overexpression plasmid has been successfully transferred into the cells. After 24 hours, flow sorting was performed. When the cell confluence reached 60-70%, 10 μg/mL puromycin was used for resistance screening to obtain mammary epithelial cells that stably overexpressed the goat Ltf gene. The collected mammary epithelial cells were then cultured for monoclonal cloning, and the single cells grew into cell clusters after two weeks.

Table 9 Electroporation conditions

Step 7: Identification of Ltf gene overexpression

When the single cells grew into cell clusters, they were digested and lysed, and RNA and protein were extracted for real-time fluorescence quantification and protein immunoblotting to detect Ltf expression. The results are shown in Figure 6. The results show that compared with the wild type (NC) mammary epithelial cells, the transfected Ltf gene RNA and protein were significantly increased. This shows that gRNA successfully overexpressed the Ltf gene, and a positive clone cell line was obtained, which was named GMEC-Ltf-OE-Cell line.

Step 8: LPS-induced mammary inflammation model

After the wild-type mammary epithelial cells and the gene-overexpressing mammary epithelial cell GMEC-Ltf-OE-Cell line were revived, when the cell confluence reached 70% to 80%, 10 μg/mL LPS was added for 12 hours, and the total cell mRNA was extracted using the Trizol method. The expression levels of inflammatory factor genes were detected by real-time fluorescence quantitative PCR. The results showed that in the LPS-induced mastitis model, overexpression of the Ltf gene significantly reduced the expression of inflammatory factors (IL-6, IL-1B, IL-8, NF-kB, TNF-a), indicating that in the process of mammary epithelial inflammation, overexpression of the Ltf gene can significantly inhibit the expression of inflammatory factors, thereby alleviating the inflammatory state of the cells.

Table 10 Primers for Real-Time PCR Detection of Inflammatory Factors

The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (10)

1. A method for constructing goat mammary epithelial cells overexpressing Ltf gene, characterized in that: Using gene editing technology, the promoter encoding the Ltf gene was replaced with the promoter of the endogenous b-actin gene, which strongly expressed the Ltf gene, to overexpress the Ltf gene and obtain primary mammary epithelial cells that stably overexpressed the goat Ltf gene; The specific steps include: S1. Amplification of the repair fragment b-actin promoter: Using goat genomic DNA as a template, the b-actin promoter was amplified by PCR and purified; the nucleotide sequence of the b-actin promoter is shown in SEQ ID No.1; S2. Amplification of the left homology arm and the right homology arm of the Ltf gene: Using the genomic DNA of goat Ltf as a template, 1 kb before and after the 5'UTR of the Ltf gene were used as the left and right homology arms, and PCR amplification and purification were performed; the nucleotide sequence of the left homology arm of the Ltf gene is shown in SEQ ID No.2; the nucleotide sequence of the right homology arm of the Ltf gene is shown in SEQ ID No.3; S3. Preparation of Donor linear backbone: pZ-Tomato plasmid was double-digested with Sal I endonuclease and Hind III endonuclease, purified, and Donor linear backbone was obtained; S4. Construction of pZ-Tomato-Ltf plasmid: digest the b-actin promoter obtained in S1 and the left homology arm and right homology arm fragments of the Ltf gene obtained in S2, and connect them to obtain homology arm fragments: left homology arm-promoter-right homology arm; connect the homology arm fragments with the Donor linear skeleton obtained in S3 to construct pZ-Tomato-Ltf plasmid; S5. Construction of pX459-Ltf plasmid: determine the PAM site of the Ltf gene promoter and construct a double-stranded gRNA fragment; after the pX459 vector is digested with BbsI, the double-stranded gRNA fragment is connected to the pX459 plasmid vector to obtain the pX459-Ltf plasmid; transfer into Escherichia coli competent cells, screen and verify positive single clones; S6. The pX459-Ltf plasmid obtained in S5 and the pZ-Tomato-Ltf plasmid obtained in S4 were electroporated into goat mammary epithelial cells at a mass ratio of 1:1, and resistance screening was performed to obtain mammary epithelial cells that stably overexpressed the goat Ltf gene. 2. The method for constructing goat mammary epithelial cells with overexpression of Ltf gene according to claim 1, characterized in that: The sequence of the gRNA is shown in SEQ ID No.10 and SEQ ID No.11. 3. The method for constructing goat mammary epithelial cells with overexpression of Ltf gene according to claim 1, characterized in that: The sequences of the primers used for amplifying the repair fragment b-actin promoter in S1 are shown in SEQ ID No.4 and SEQ ID No.5. 4. The method for constructing goat mammary epithelial cells with overexpression of Ltf gene according to claim 1, characterized in that: The sequences of the primers used to amplify the left homologous arm of the Ltf gene in S2 are shown in SEQ ID No.6 and SEQ ID No.7; the sequences of the primers used to amplify the right homologous arm of the Ltf gene are shown in SEQ ID No.8 and SEQ ID No.9. 5. The method for constructing goat mammary epithelial cells with overexpression of Ltf gene according to claim 1, characterized in that: The verification primer sequences of the pX459-Ltf plasmid in S5 are shown in SEQ ID No.11 and SEQ ID No.12. 6. The method for constructing goat mammary epithelial cells overexpressing Ltf gene according to claim 1, characterized in that: The homology arm fragments in the S4 were connected to the Donor linear backbone by Gibson connection, and the mass ratio of the homology arm fragments to the Donor linear backbone was 2:1. 7. Goat mammary epithelial cells overexpressing Ltf gene constructed by the method according to any one of claims 1 to 6. 8. Use of the goat mammary epithelial cells overexpressing the Ltf gene according to any one of claims 1 to 6 in constructing an inflammatory response animal model. 9. The use according to claim 8, characterized in that: The steps to construct an animal model of inflammatory response are: After wild-type goat mammary epithelial cells and gene-overexpressing mammary epithelial cells were revived, when the cell confluence reached 70% to 80%, 10 μg/mL LPS was added for 12 hours, and total cell mRNA was extracted. The expression levels of inflammatory factor genes were detected by real-time fluorescence quantitative PCR. 10. The use according to claim 9, characterized in that: The sequences of the primers for PCR detection of inflammatory factors are shown in SEQ ID No.13 to SEQ ID No.24.