阅读说明：本技术 奶牛溶菌酶基因乳腺特异性表达重组质粒及其构建方法和应用 (Dairy cow lysozyme gene mammary gland specificity expression recombinant plasmid and construction method and application thereof ) 是由 杨章平 张志鹏 杨奕 陈代杰 张慧敏 于 2019-10-25 设计创作，主要内容包括：本发明提供一种奶牛溶菌酶基因(<I>Lyz</I>)乳腺特异性表达质粒及其构建方法和应用。具体地,PCR扩增<I>Lyz</I>基因的编码序列,将其插入到携带增强的绿色荧光蛋白基因的通用表达载体pEGFP-N1的多克隆位点中。通用表达载体pEGFP-N1经<I>Ase</I> I+<I>Hin</I>d III双酶切,去除<I>CMV</I>启动子,克隆奶牛乳腺特异性表达β-乳球蛋白基因(<I>BLG</I>)启动子序列并取代<I>Ase</I> I和<I>Hin</I>dIII酶切位点,构建奶牛溶菌酶基因(<I>Lyz</I>)乳腺特异性表达质粒。采用上述重组质粒转染奶牛乳腺上皮细胞,得到的细胞系可用于分泌具有生物活性的奶牛溶菌酶,用于奶牛乳腺组织免疫机制研究；该重组质粒还可应用于基因治疗奶牛乳房炎制剂的研发。(The present invention provides a kind of cow lysozyme gene Lyz ) Mammary gland specificity expression plasmid and its construction method and application. Specifically, PCR amplification Lyz The coding sequence of the gene, which is inserted into the multiple cloning site of the universal expression vector pEGFP-N1 carrying the enhanced green fluorescent protein gene. The general expression vector pEGFP-N1 was obtained Ase I+ Hin d III double digestion, removal CMV Promoter, cloning of milk cow mammary gland-specific expression beta-lactoglobulin Gene: ( BLG ) Promoter sequence and substitution Ase I and Hin enzyme digestion site of dIII, construction of the milk cow lysozyme gene (II) Lyz ) Mammary gland specific expression plasmid. The recombinant plasmid is adopted to transfect milkThe obtained cell line can be used for secreting cow lysozyme with biological activity and is used for the research of the immune mechanism of the cow mammary tissue; the recombinant plasmid can also be applied to the research and development of gene therapy cow mastitis preparations.)

1. A mammary gland specificity expression recombinant plasmid of cow lysozyme gene is characterized in that,Lyzgene insertion into enhanced green fluorescent protein general expression vector pEGFP-N1 plasmidHindIII andSacII multiple cloning site, and of the pEGFP-N1 plasmidCMVReplacement of promoter sequence byBLGA gene promoter sequence; saidLyzThe nucleotide sequence of the gene is shown as SEQ ID NO. 2BLGThe nucleotide sequence of the gene promoter is shown as SEQ ID NO. 3.

2. The method for constructing the mammary gland specific expression recombinant plasmid of the cow lysozyme gene as claimed in claim 1, which comprises the following steps:

by usingHindIII and SacIICarrying out double digestion on the pEGFP-N1 plasmid, carrying out homologous recombination on the digested pEGFP-N1 plasmid, and carrying out homologous recombinationLyzConnecting the genes to obtain a recombined plasmid pEGFP-N1-Lyz;

the recombinant plasmid pEGFP-N1-Lyz is subjected toAseI+HindIIIDouble enzyme digestion, cutting pEGFP-N1 carrier originalCMVPromoter sequence obtained byBLGHomologous recombination of promoter gene sequence toAseIAndHindIIIand (3) constructing a mammary gland specific expression recombinant plasmid pBLG-EGFP-N1-Lyz of the dairy cow lysozyme gene at the enzyme restriction site.

3. The method of construction according to claim 2, wherein saidLyzGene preparation methodThe method comprises the following steps:

extracting total RNA of the mammary epithelial cells of the dairy cattle;

reverse transcription of total RNA of mammary epithelial cells of the milk cow into cDNA;

taking the milk cow mammary gland epithelial cell cDNA as a template, and taking P1 and P2 as milk cowsLyzPrimers, RT-PCR amplificationLyzA gene coding sequence;

wherein the P1 primer has a nucleotide sequence shown as SEQ ID No. 4 in a sequence table;

the P2 primer has a nucleotide sequence shown as SEQ ID No. 5 in the sequence table.

4. The construction method according to claim 3, wherein the RT-PCR amplification system is: the template cDNA is 1 mul, the primers P1 and P2 are 1 mul respectively, the Takara primeSTAR MIX is 10 mul, and ddH₂Supplementing O to 20 mu l; the concentrations of the P1 and P2 primers are both 0.1 nmol/. mu.l;

the RT-PCR amplification program comprises the following steps: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 1min for 35 cycles; extension at 72 ℃ for 7 min.

5. The method of construction according to claim 2, wherein saidBLGThe preparation method of the promoter gene comprises the following steps:

extracting milk cow blood genome DNA;

taking milk cow blood genome DNA as a template, and taking P3 and P4 as milk cowsBLGPrimers, PCR amplificationBLGA promoter gene;

wherein the P3 primer has a nucleotide sequence shown as SEQ ID No. 6 in a sequence table;

the P4 primer has a nucleotide sequence shown as SEQ ID No. 7 in the sequence table.

6. The method of claim 5, wherein the PCR amplification system is: 1. mu.l of template DNA, 1. mu.l of each of primers P3 and P4, 10. mu.l of Takara primeSTAR MIX, and dH₂O is complemented to 20 mu l, and the concentrations of the P3 primer and the P4 primer are both 0.1 nmol/mu l;

the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 2min for 35 cycles; extension at 72 ℃ for 7 min.

7. The recombinant plasmid according to claim 1 or the recombinant plasmid obtained by the construction method according to any one of claims 2-6, for use in the study of the immune mechanism of the mammary tissue of a cow.

8. The use of claim 7, wherein the recombinant plasmid of claim 1 or the recombinant plasmid obtained by the construction method of any one of claims 2-6 is transfected into mammary gland cells, and the expression of the green fluorescent protein gene is observed by a green fluorescent microscope to detect the expression of the lysozyme gene in dairy cows.

9. The recombinant plasmid according to claim 1 or the recombinant plasmid obtained by the construction method according to any one of claims 2-6, which is applied to the development of a gene therapy cow mastitis preparation.

10. The use according to claim 9, wherein the recombinant plasmid according to claim 1 or the recombinant plasmid obtained by the construction method according to any one of claims 2 to 6 is transfected into a mammal, and the recombinant plasmid expresses a cow lysozyme gene in the mammal, and the expression product has an antibacterial effect.

Technical Field

The invention relates to a mammary gland specificity expression recombinant plasmid of a milk cow lysozyme gene, a construction method and application thereof, belonging to the technical field of gene recombination and molecular cloning.

Background

The mastitis of the dairy cow is a common disease and a frequently encountered disease of the dairy cow, and the milk yield of the dairy cow is greatly reduced along with the increase of the incidence rate of mastitis of the dairy cow, and the quality of raw milk is seriously influenced. Therefore, the treatment of cow mastitis is increasingly gaining importance. At present, the main treatment means is the drug treatment of antibiotics and Chinese medicinal preparations, and the use of antibiotics, the generation of drug-resistant strains and the drug residues in milk seriously harm the health of consumers, so the treatment of the cow mastitis becomes a world problem. The method has the advantages of effectively controlling drug residues by using little or no antibiotics, ensuring the safety of raw milk and the milk yield of the dairy cows, and is the main target of preventing and controlling the mastitis of the dairy cows.

Lysozyme (lysozyme, Lyz), also known as muramidase or N-acetylmuramyl polysaccharide hydrolase, is an alkaline enzyme that hydrolyzes mucopolysaccharides in pathogenic bacteria. The bacterial lysis is achieved by breaking the beta-1, 4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, breaking down the cell wall insoluble mucopolysaccharide into soluble glycopeptides, causing the contents of the broken cell wall to escape. Has the functions of resisting bacteria, diminishing inflammation, repairing tissue, raising immunity, etc. At present, negative effects brought by abuse of antibiotics are increasingly prominent, the drug resistance of bacteria and fungi to the antibiotics is gradually enhanced, and lysozyme only acts on bacterial cell walls, is a natural antibacterial substance in organisms and is non-toxic and harmless to animal organisms, so that the lysozyme is expected to become a substitute of novel antibiotics. The research shows that about 10 lysozyme similar genes exist in the genome of higher ruminants (such as cattle, sheep, deer and the like), and although the ruminants have a plurality of lysozyme genes, the expression level of the milk cow lysozyme in mammary gland is lower and is only 0.05-0.13 mg/L, and in contrast, the expression level of the human mammary gland lysozyme is 2000-4000 times higher than that of the human mammary gland lysozyme.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, construct a mammary gland specificity expression recombinant plasmid of a cow lysozyme gene, construct a mammary gland specificity expression regulation and control region at the 5' end of the plasmid, specifically express a target gene in mammary gland tissues, and provide an important material for preventing and treating cow mastitis by improving the expression level of natural antibacterial substances, namely lysozyme in the cow mammary gland.

In order to realize the aim, the invention provides a mammary gland specific expression recombinant plasmid of a dairy cow lysozyme gene, wherein Lyz gene is inserted into HindIII and SacII multiple cloning sites of an enhanced green fluorescent protein general expression vector pEGFP-N1 plasmid, and a CMV promoter sequence of the pEGFP-N1 plasmid is replaced by a BLG gene promoter sequence; the nucleotide sequence of the Lyz gene is shown as SEQ ID NO. 2, and the nucleotide sequence of the BLG gene promoter is shown as SEQ ID NO. 3.

The invention also provides a construction method of the milk cow lysozyme gene mammary gland specificity expression recombinant plasmid, which comprises the following steps:

carrying out double enzyme digestion on the pEGFP-N1 plasmid by adopting HindIII and SacII, and connecting the enzyme digested pEGFP-N1 plasmid with Lyz genes through homologous recombination to obtain a recombined plasmid pEGFP-N1-Lyz;

carrying out AseI + HindIII double enzyme digestion on the recombinant plasmid pEGFP-N1-Lyz, cutting the original CMV promoter sequence of the pEGFP-N1 vector, taking the gene sequence of the BLG promoter to carry out homologous recombination and replace the gene sequence to the enzyme digestion sites of AseI and HindIII, and constructing the mammary gland specific expression recombinant plasmid pBLG-EGFP-N1-Lyz of the lysozyme gene of the dairy cow.

Further, the preparation method of the Lyz gene comprises the following steps:

extracting total RNA of the mammary epithelial cells of the dairy cattle;

reverse transcription of total RNA of mammary epithelial cells of the milk cow into cDNA;

taking the cDNA of the mammary epithelial cells of the dairy cattle as a template, taking P1 and P2 as primers of Lyz of the dairy cattle, and carrying out RT-PCR amplification on Lyz gene coding sequences;

wherein the P1 primer has a nucleotide sequence shown as SEQ ID No. 4 in a sequence table;

the P2 primer has a nucleotide sequence shown as SEQ ID No. 5 in the sequence table.

Further, the RT-PCR amplification system is as follows: 1. mu.l of template cDNA, 1. mu.l of each of primers P1 and P2, 10. mu.l of Takara primeSTAR MIX, and ddH₂O is complemented to 20 mu l; the concentrations of the P1 and P2 primers are both 0.1 nmol/. mu.l;

the RT-PCR amplification program comprises the following steps: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 20s, and extension at 72 ℃ for 1min for 35 cycles; extension at 72 ℃ for 7 min.

Further, the method for preparing the BLG promoter gene includes:

extracting milk cow blood genome DNA;

PCR amplification is carried out on BLG promoter genes by taking milk cow blood genome DNA as a template and P3 and P4 as milk cow BLG primers;

wherein the P3 primer has a nucleotide sequence shown as SEQ ID No. 6 in a sequence table;

the P4 primer has a nucleotide sequence shown as SEQ ID No. 7 in the sequence table.

Further, the PCR amplification system is as follows: 1. mu.l of template DNA, 1. mu.l of each of primers P3 and P4, 10. mu.l of Takara primeSTAR MIX, and dH₂O is complemented to 20 mu l, and the concentrations of the P3 primer and the P4 primer are both 0.1 nmol/mu l;

the PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 2min for 35 cycles; extension at 72 ℃ for 7 min.

The invention also provides an application of the recombinant plasmid in the research of the breast tissue immune mechanism of the milk cow.

Further, the recombinant plasmid or the recombinant plasmid obtained by the construction method is transfected into mammary cells, the expression condition of the green fluorescent protein gene is observed through a green fluorescent microscope, and the expression condition of the lysozyme gene of the dairy cow is detected.

The invention also provides application of the recombinant plasmid or the recombinant plasmid obtained by the construction method in the research and development of a preparation for treating the cow mastitis through genes.

Further, the recombinant plasmid or the recombinant plasmid obtained by the construction method is transfected into a mammal body, the recombinant plasmid expresses the cow lysozyme gene in the mammal body, and the expression product has an antibacterial effect.

The invention achieves the following beneficial effects:

1. the invention constructs a recombinant plasmid containing a BLG gene promoter, a cow lysozyme gene (Lyz) and an enhanced green fluorescent protein gene for mammary gland specific expression of the cow lysozyme gene, and can be used for the research of the mammary tissue immune mechanism of a cow and the research and development of a preparation for gene therapy of cow mastitis.

2. The recombinant plasmid constructed based on the method has high specificity, high sensitivity and high accuracy, and can ensure that the lysozyme gene of the dairy cow is specifically and highly expressed in mammary cells.

3. The recombinant plasmid is transfected into an ICR mouse body in a lactation peak period, the recombinant plasmid specifically expresses the lysozyme gene of the dairy cow in mammary tissue in the body, and an expression product has an antibacterial effect. The lysozyme expression quantity in mammary tissue and cow milk can be greatly improved after the lysozyme gene-modified bovine mastitis gene is used, pathogenic bacteria can be killed by directly reaching the focus, the effect of treating and treating cow mastitis is achieved, a passage for pathogenic bacteria to enter breasts from a nipple duct can be cut off, the infection way of the mastitis is reduced, the purpose of treating and preventing the mastitis is achieved, meanwhile, the problem of drug resistance is avoided because no antibiotic is used, the cow milk is not easy to deteriorate due to the increase of the content of the lysozyme in the cow milk, and the cow mastitis gene-modified bovine mastitis gene is healthier.

4. The invention has wide application prospect in the aspect of researching and developing novel replaceable antibiotic products.

Drawings

FIG. 1 shows PCR amplification of Lyz gene in example 1. M: 100bp DNA Ladder; 1-5: lyz amplifying the fragment; 6: ddH 2O. Note: 488bp is 444bpLyz +22bp upstream homology arm +22bp downstream homology arm.

FIG. 2 is a schematic structural diagram of the enhanced green fluorescent protein general expression vector pEGFP-N1 in example 1.

FIG. 3 shows PCR detection of Lyz recombinant vector bacterial liquid in example 1. M: 100bp DNA Ladder; 1-5: and (5) carrying out PCR detection on the bacterial liquid. Note: wherein No. 1, 2 and 4 detect the target stripe.

FIG. 4 is a PCR amplification diagram of the BLG promoter in example 1. M: DL5000, DNAMarker; 1-5: and (3) amplifying the promoter fragment. Note: 2358bp is 2318bp promoter +20bp upstream homology arm +20bp downstream homology arm.

FIG. 5 is a diagram of the analysis of the BLG promoter element in example 1. Note: promoter elements of GC-box, CAAT-box and TATA-box exist at the positions of 887bp-892bp, 1188bp-1191bp and 2080-2085 bp.

FIG. 6 construction of mammary gland-specific expression plasmid for Lyz gene in example 1. Note: the pEGFP-N1 vector was inserted into the Lyz gene and replaced the CMV promoter.

FIG. 7. expression pattern of pBLG-EGFP-N1-Lyz in primary mammary epithelial cells in example 1. Bpmecc cells (× 100); pBLG-EGFP-N1-Lyz transfected bPMEC cells (x 100);

hek293t cells (× 100); pBLG-EGFP-N1-Lyz transfected HEK293T cells (. times.100).

FIG. 8 is a graph showing the relative expression levels of the recombinant plasmid pBLG-EGFP-N1-Lyz in various tissues of mice in example 2. 1. The blank group was not injected with any reagents; 2. injecting a transfection reagent into mice in a control group; 3. the expression level of the breast tissue Lyz gene after injection of pBLG-EGFP-N1-Lyz and mouse transfection reagent; 4-8, after the injection of pBLG-EGFP-N1-Lyz and mouse in vivo transfection reagent, the expression level of heart, liver, spleen, lung and kidney tissues Lyz genes is increased. Note: the 3 is very different from 1, 2; 4-8 were not significantly different relative to 1, 2.

FIG. 9 is a graph showing the results of western blot detection in example 2. Note: the left three bands are the results of western blot detection of the mammary tissue of the mouse transfected with the recombinant plasmid pBLG-EGFP-N1-Lyz, and the right three bands are the results of western blot detection of the mammary tissue of the untransfected control group mouse.

Detailed Description

The invention designs a primer according to a cDNA sequence (GenBank number: NM-180999.1) of a milk cow Lyz gene in GenBank, amplifies a Lyz gene coding sequence from a milk cow mammary gland epithelial cell by RT-PCR, and clones the sequence into a general expression vector pEGFP-N1 carrying enhanced green fluorescent protein for sequencing. The recombinant plasmid pEGFP-N1-Lyz is obtained by excising a CMV promoter through AseI + HindIII double enzymes, and the mammary gland specific expression beta-lactoglobulin gene (BLG) promoter of the dairy cow is homologous recombined and replaced to AseI and HindIII enzyme cutting sites to construct a mammary gland specific expression plasmid pBLG-EGFP-N1-Lyz of a dairy cow lysozyme gene (Lyz). The method is characterized in that primary dairy cow mammary gland epithelial cells (bMECs) and 293T cells are transfected by a liposome method, and the expression condition of green fluorescent protein in the transfected cells is detected by a fluorescence microscope, so that the specific expression condition of the lysozyme gene of the dairy cow in the primary dairy cow mammary gland epithelial cells is detected, and the method is further applied to the research field of the immune mechanism of the dairy cow mammary gland tissue.

The following detailed description of the present invention will be made with reference to the accompanying drawings.