CN107410206A - A kind of cad gene PR39 DNA murines - Google Patents

Abstract

The invention discloses a kind of cad gene PR39 DNA murines, antibacterial peptide PR39 gene integrations are on No. 13 chromosome of mouse, between GM36264 genes and GM8784 genes, and PR39 genes stabilization can be entailed offspring by the transgenic mice, and can in each tissue of whole body specifically expressing antibacterial peptide PR39 albumen, there is preferable fungistatic effect, and the disease-resistant effect of subjective antisepsis can be played, for being laid a good foundation for follow-up Cecropin-GM gene domestic animal, and a kind of solution of feasibility is provided to reduce the use of antibiotic.

Description

A kind of cad gene PR39 DNA murines

Technical field

The present invention relates to genetic engineering field, more particularly to a kind of cad gene PR39 DNA murines.

Background technology

Antibacterial peptide (Antimicrobial peptides, AMPs) is a kind of micromolecule polypeptide with antibacterial activity, it It is the important component of body natural immune defence system.Research shows that antibacterial peptide has broader spectrum of compared with antibiotic Antibacterial ability, but also with the effect such as antiviral, antimycotic, anticancer, it easily by proteolysis, is not easy residual in animal body Stay, be not easy to make bacterium produce drug resistance, thus by the extensive concern of researcher.

The classification of antibacterial peptide：

Antibacterial peptide can be classified according to the powered situation of molecule, structure and source.Antibacterial peptide is according to molecular band total electrical charge Situation, can be divided into cationic antibacterial peptide and Anionic Antimicrobial Peptides, and cationic antibacterial peptide accounts for the overwhelming majority；It can be divided according to structure For polytype, wherein cathelicidins and defensins are main two types；Source can be divided into according to source In the antibacterial peptide of the biology such as insect, mammal, amphibian, fish, mollusk, shellfish, plant and bacterium.

The molecular structure of antibacterial peptide：

Antibacterial peptide molecular weight is smaller, generally less than 40kD, is generally made up of 20-60 amino acid, and it has different points Minor structure, including one, two, three and quaternary structure, wherein secondary structure include linear α-helical structure, the foldable structures of β mono-, contained Stretch the antibacterial peptide of the type of lamellar structure and loop configuration etc. 4.The molecular structure of antibacterial peptide determines its molecular activity, its moderate resistance The charge number of bacterium peptide institute band, the conserved sequence in molecular structure, α-helical structure, disulfide bond etc. are influence its activity important Factor.

The mechanism of action of antibacterial peptide：

Antibacterial peptide has broad spectrum antibiotic activity, can effectively suppress bacterium (including gram-positive bacterium and gram-negative Property bacterium), do not damage eukaryotic.The antibacterial mechanisms of antibacterial peptide are mainly to act on based on bacterial cell membrane, a small number of antibacterials Peptide can also kill bacterium by suppressing the synthesis of DNA of bacteria, RNA and protein.Antibacterial peptide acts on bacterial cell membrane bag Include two ways：(1) film destructive mode (membrane disruptive) is the cell membrane of dissolution of bacteria；(2) non-film is destroyed Sexual norm (non-membrane disruptive) changes the permeability of bacterial cell membrane.

Antibacterial peptide is adsorbed onto on pathogen cell membrane by the electrostatic interaction between pathogen.Bacterial cell membrane contain compared with More phosphatide, so it is negatively charged, generally can more rapid and better it be adsorbed with cationic antibacterial peptide.Research shows, the suction of antibacterial peptide Attached effect is also closely related with molecular length itself, and peptide chain is longer in molecular structure, and the difficulty being attached on bacterial cell membrane is got over Greatly.In contrast, the antibacterial peptide of small molecule structure is more easy to play antibacterial activity.Therefore, there is researcher to attempt reduction long-chain to resist The peptide chain length of bacterium peptide, it is set to be more easy to be attached on bacterial cell membrane, but such a way may destroy the two level knot of antibacterial peptide Structure, so as to influence the function of its dissolution of bacteria cell membrane.

Application of the antibacterial peptide in biological medicine and animal husbandry：

With the progress of research, as part important in animal body immune system, it is cured antibacterial peptide in biology Medicine and animal husbandry show huge application prospect.At present, in human medical field, researcher is by animal sources antibacterial Peptide is used for clinical test.Colitis clinical model Therapy study proves that cathelicidins antibacterial peptides have fiber reparation work With the treatment available for IBD.In mouse tuberculosis clinical pattern Therapy study, people's derived antimicrobial peptide LL-37 passes through The mode of collunarium infects for mouse lung, the results showed that LL-37 can resist tuberculosis.

In livestock-raising, abuse of antibiotics for a long time causes medicine in the generation and livestock and poultry body of drug tolerant bacteria residual Stay, serious threat is caused to human health so that the research and development of antibacterial peptide substitute antibiotics medicine are highly valued.In recent years Come, application study of the animal derived antimicrobial peptide in livestock-raising mainly includes following two：(1) improved as feed addictive dynamic Thing immunity of organisms, promote animal body growth.Show to add antibacterial peptide in Piglet Diets as studied, feed conversion can be improved Rate, promote piglet growth, while immunity can also be improved.Antibacterial Peptide Extracted from Pig Small Intestine is expelled in SPF level chicken muscles, Ke Yixian Write and improve efficiency of feed utilization, improve small intestine immunocompetent cell quantity, strengthen the barrier function of enteron aisle.(2) as medicine to dynamic Thing disease is prevented and treated.Bacteriostatic activity experiment is done using the antibacterial peptide from sheep leucocyte, finds it to pig source Pathogenic bacteria have good inhibition.It is huge in the Marc-145 cell lines and pig alveolar of infection porcine reproductive and respiratory syndrome virus The antibacterial peptide PG-1 of continuous addition 36 hours, can substantially suppress the viral infection and duplication in the nutrient solution of phagocyte.

Antibacterial peptide PR-39 is to resist earliest from the small molecule in the isolated Cathelicidin families in the top of chitterlings Bacterium peptide, then show that PR-39 is also expressed in pig bone marrow primary cell by cDNA clone, and neutrality is stored in propeptide forms In granulocyte.

Antibacterial peptide PR39 molecular structure feature：

The antibacterial peptide PR-39 assignment of genes gene mapping is in No. 13 chromosomes of pig, mrna length 1784bp, containing in 4 extrons and 3 Containing son, wherein the 4th exons coding antibacterial peptide PR-39 mature peptide.Antibacterial peptide PR39 molecules are made up of 39 amino acid, wherein There are 22 Pro and 11 Arg, its relative molecular mass is 4719Da.PR39 peptide chain structures order be： RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPREP, wherein proline tie with the Pro-Arg-Pro that arginine is formed Structure helps to act on bacterial phospholipid film.The molecular secondary structure of antibacterial peptide PR-39 is linearly changed, and forms α-helical structure.

Antibacterial peptide PR39 mechanism of action：

Antibacterial peptide is broadly divided into two kinds of cathelicidins and defensins according to configuration.Cathelicidins families Antibacterial peptide relies primarily on to be adsorbed between the positive charge and bacterial cell membrane of arginine residues institute band by the effect of electrostatic attraction On bacterial cell membrane, its bacteriostasis is played by non-pore-forming pattern formula.Its antibacterial process is not so good as defensins type antibacterial peptides Rapidly.Defensins directly destroys cell membrane, the synthesis that bacterial cell membrane suppresses DNA and protein is then penetrated, so as to quick Bacterium is killed, there is stronger killing ability to the bacterium in exponential phase.PR39 belongs to Cathelicidin antibacterial peptides, It mainly suppresses Gram-negative bacteria, but also has antibacterial effect to small part gram-positive bacteria, plays the machine of bacteriostasis System is related to its close structure.

A pancreas peptide enzyme recognition site between Pro propetides and PR39 mature peptides in PR39 structures be present, this with PR39 into The formation of ripe peptide is extracellular relevant with being discharged into.The antibacterial activity of N-terminal peptide chain is lived higher than C-terminal antibacterial in ripe PR39 peptide chain structures Property, multiple avtive spots be present in the structure of preceding 26 amino acid composition.PR39 different structures bacteriostatic experiment, which is studied, to be shown, PR-15 Fungistatic effect compared with PR39 effect it is even better, PR26 fungistatic effect is similar to PR39, but the peptide chain of different length It can all be influenceed when playing bacteriostasis by salinity.

Antibacterial peptide PR39 antibacterial action：

Antibacterial peptide PR39 has broad spectrum antibiotic activity, to gram-positive bacteria and Gram-negative bacteria (in addition to pseudomonad) It is respectively provided with preferable fungistatic effect.There is research by the PR39 gene clonings of synthesis to carrier pHIL-S1, construction recombination plasmid PHIL-S1-PR39, the plasmid of structure is transformed into saccharomycete, obtains recombinant protein.Restructuring PR39 albumen will be obtained and be used for large intestine The bacteriostatic experiment of bacillus, Salmonella typhimurtum and staphylococcus aureus, the results showed that pass through weight caused by Pichia anomala expression Group PR39 albumen, fungistatic effect is respectively provided with to Escherichia coli, Salmonella typhimurtum and staphylococcus aureus.By the PR39 of synthesis After gene is connected with expression vector pCold, it is transferred in Escherichia coli, obtains restructuring PR39 albumen.By the restructuring PR39 eggs of acquisition The white bacteriostatic experiment for Escherichia coli and Salmonella typhimurtum, the results showed that by recombinating PR39 caused by Bacillus coli expression Albumen is similarly obtained has fungistatic effect to Escherichia coli and Salmonella typhimurtum.There is research to be carried by building PR39 eukaryotic expressions Body pIRES2-PR39, after transfected macrophage, the energy of intracellular bacterium is killed and removed to expression antibacterial peptide PR39 macrophage Power is remarkably reinforced, and shows that antibacterial peptide PR39 can be expressed in macrophage and be had fungistatic effect.

Antibacterial peptide has a variety of advantages compared with antibiotic, therefore turns into the study hotspot in herding disease prevention and cure at present. Antibacterial peptide is applied to the disease prevention and cure of farming animals mainly as feed addictive or veterinary drug at present.But use artificial synthesized or table The antibacterial peptide reached not only increases aquaculture cost, and because the activity of antibacterial peptide is easy in the production, transportation and storage process It is affected, its antibacterial effect reduces.Therefore derived antimicrobial peptide in animal body oneself expression is made using transgenic technology, so as to improve Its premunition, it can solve the above problems.

The content of the invention

It is an object of the invention to provide a kind of cad gene PR39 DNA murines, the transgenic mice oneself expression PR39 eggs In vain, and to bacterium fungistatic effect is produced, to obtain the livestock and poultry of cad gene PR39 genes, especially cad gene PR39 gene pigs Laid the foundation.

According to an aspect of the invention, there is provided a kind of cad gene PR39 DNA murines, antibacterial peptide PR39 genes It is incorporated on No. 13 chromosome of mouse, what thus the transgenic mice can stablize PR39 genes entails offspring, and Energy specifically expressing PR39 albumen, and there is preferably resistance effect to bacterium.

In some embodiments, antibacterial peptide PR39 genes are located on No. 13 chromosome of transgenic mice Between GM36264 genes and GM8784 genes, what thus the transgenic mice can stablize PR39 genes entails offspring, and Energy specifically expressing PR39 albumen, and there is preferably resistance effect to bacterium.

In some embodiments, cad gene PR39 DNA murines obtain as follows：

(1) according to PR39 gene C DS sequences, restriction enzyme site, artificial synthesized corresponding DNA sequences are increased at gene order both ends Row；

(2) DNA sequence dna of synthesis is connected on pCMS-EGFP empty carriers, obtains antibacterial peptide PR39 expression vectors pCMS-EGFP-PR39；

(3) functional verification is carried out to antibacterial peptide PR39 expression vectors pCMS-EGFP-PR39；

(4) it is the expression vector pCMS-EGFP-PR39 microinjections mouse fertilized egg of energy effective expression PR39 genes is male former Core, and by the zygote transplation after injection to replace-conceive dams intrauterine, F0 is obtained for transgenic mice by gestation；

(5) F0 of acquisition is detected for transgenic mice, chooses PR39 gene expression amounts height, and integration site is single Individual carry out follow-up study；

(6) F0 filtered out generations individual is bred with wild-type mice breeding, obtains F1 generation transgenic mice；

(7) the F1 generation transgenic mice of acquisition is detected, filtering out can stable heredity and high efficient expression PR39 gene Individual carry out follow-up study；

(8) the F1 generation individual filtered out is bred, obtains corresponding family, and it is real to carry out antibacterium to the family Test, obtain being highly resistant to the cad gene PR39 DNA murines of bacterium.

Thus, cad gene PR39 DNA murines are obtained by the above method, hereditary PR39 genes can be stablized, and turning Specifically expressing PR39 albumen in DNA murine different tissues, is advantageous to the performance of the antibacterial and bacteriostasis of each tissue, plays and turn base Because of the effect of mouse systemic tissue synthesis antibacterial.

In some embodiments, antibacterial peptide PR39 expression vectors pCMS-EGFP-PR39 gene orders are SEQ ID NO:1, transgenic mice is obtained from there through the expression vector, there can be preferably suppression with the expression PR39 albumen of differential high efficient Bacterium effect.

In some embodiments, expression vector pCMS-EGFP-PR39 functional verification, including expression vector is transfected After 293 cells, carry out qPCR detections and fungistatic effect detection, it is possible thereby to judge expression vector pCMS-EGFP-PR39 whether structure Work(is built up, and can PR39 genes correctly express in 293 cells.

In some embodiments, qPCR is detected in expression vector pCMS-EGFP-PR39 functional verification, including as follows Step：

(1) pCMS-EGFP-PR39 expression vectors rotaring redyeing 293 cell, in fluorescence microscopy Microscopic observation green fluorescence after 24h Protein expression situation；

(2) after 72h, cell extraction RNA is collected；

(3) qPCR detections are carried out to the RNA of extraction, qRCP response procedures are 42 DEG C of 15min, 95 DEG C of 3min.

In some embodiments, fungistatic effect detection is included such as in expression vector pCMS-EGFP-PR39 functional verification Lower step：

(1) 293 cell culture of pCMS-EGFP-PR39 expression vectors rotaring redyeing 293 cell and blank control group untransfected After 48h, with serum free medium culture cell 24h, culture medium is collected, by culture medium elastin laminin ferment treatment；

(2) preparation of Escherichia coli：The picking single bacterium colony in Escherichia coli plate, with 5mlLB fluid nutrient mediums, it is placed in 37 DEG C, 18-24h is cultivated in 220r/min shaking tables, 50ul bacterium solutions is drawn and expands in new 5mlLB fluid nutrient mediums and cultivates 3h, Draw 1ml bacterium solutions and centrifuge 5min in the centrifuge that rotating speed is 2000g, outwell supernatant, be resuspended with PBS, draw 200ul in 96 In orifice plate, carry out OD values using ELIASA and detect, be from bacterial concentration：2×10⁶CFU/ml；

(3) bacteriostatic experiment step：With 96 orifice plates, it is 2 × 10 that 25ul concentration is added per hole⁶CFU/ml Escherichia coli bacterium Liquid, then add culture mediums of the 25ul after step (1) processing and mix, be positioned in 37 DEG C of incubators and cultivate 3h, added per hole 200ulLB fluid nutrient mediums, it is incubated overnight in 37 DEG C of incubators, its OD value is detected with ELIASA within second day, according to the big of OD values It is small, to judge the fungistatic effect of pCMS-EGFP-PR39 expression vectors.

In some embodiments, F0 for transgenic mice detection, including PCR detection and PR39 gene mRNA expression amounts Detection.

In some embodiments, the antibacterium of transgenic mice family is tested, comprised the following steps:

(1) it is 6x10 with concentration to the transgenic lines and nontransgenic mice of acquisition⁵CFU/, 3x10⁶CFU/, 6x10⁶CFU/, 6x10⁷The actinobacillus pleuropneumoniaes of CFU/ only are by being injected intraperitoneally transgenic mice group and non-transgenic Mouse group；

(2) mouse treated to step (1), is cultivated, in different time node, the death rate of mouse is united Meter analysis.

In some embodiments, actinobacillus pleuropneumoniae concentration is in the detection of transgenic mice antibacterial 3x10⁶CFU/ is only.

The cad gene PR39 DNA murines obtained through the above way, there is beneficial effect：

1st, the PR39 gene integrations of cad gene PR39 DNA murines are located on No. 13 chromosome of mouse Between GM36264 genes and GM8784 genes, what can be stablized entail offspring, and can specifically expressing PR39 albumen, have compared with Good fungistatic effect.

2nd, cad gene PR39 DNA murines show that PR39 genes can express in different tissues of mice, and can send out Wave bacteriostasis.

3rd, acquisition being provided for the domestic animal of other cad genes PR39 genes of cad gene PR39 DNA murines Research foundation has been established, resolving ideas is provided to solve the use of antibiotic during cattle breeding.

Brief description of the drawings

Fig. 1 is the digestion qualification result figure of expression vector pCMS-EGFP-PR39 in embodiment 1：Wherein 1 is plasmid pCMS- EGFP-PR39,2 be BamHI and XhoI double digestions pCMS-EGFP-PR39, and 3 be DNALadder；

Fig. 2 is the sequencing result figure of expression vector pCMS-EGFP-PR39 in embodiment 1；

Fig. 3 is the fluoroscopic examination result figure after expression vector pCMS-EGFP-PR39 rotaring redyeing 293 cells 24h in embodiment 2；

Fig. 4 is PR39 gene mRNA relative expressions after expression vector pCMS-EGFP-PR39 rotaring redyeing 293 cells in embodiment 2 Measure result figure：Wherein ND is that PR39 expression is not detected by mRNA level in-site, (1) and (2) culture that to be respectively two different Hole；

PCR testing result figures of the Fig. 5 for F0 in embodiment 3 for transgenic mice：Wherein P is positive control, and Neg is feminine gender Control, W are water, and 426-433 is to turn PR39 DNA murines numbering in F0 generations, and 165 be PR39 genetic fragment sizes, and 110 be GAPDH pieces Duan great little；

Fig. 6 is transgenic mice EGFP gene copy number results figure in embodiment 3；

Fig. 7 is transgenic mice PR39 gene mRNA relative expression quantity result figures in embodiment 3：Wherein 426-433 is to turn DNA murine is numbered, and is organized as transgenic mice tail in figure used in quantitative PCR, and ND is to be not detected by PR39's in mRNA level in-site Expression；

Fig. 8 is the PCR testing result figures of F1 generation transgenic mice in embodiment 4：Wherein M is 500bp marker, numbering It is respectively F1 generation birth mouse for 1-11, P is positive control, and W is water；

Fig. 9 is that F1-9 transgenic mices respectively organize PR39 gene mRNA relative expression quantity result figures in embodiment 4；

Figure 10 is F1-9 transgenic mice portion of tissue PR39 protein concentration expression of results figures in embodiment 4；

Figure 11 is F1-9 transgenic mice integration site analysis result figures in embodiment 5；

Figure 12 is that F1-9 transgenic mices family attacks death rate testing result after poison with nontransgenic mice in embodiment 6 Figure.

Embodiment

Invention is described in further detail below in conjunction with the accompanying drawings.

Unless otherwise specified, the reagent used in following examples derives from commercially available.

Embodiment 1：Antibacterial peptide PR39 expression vectors pCMS-EGFP-PR39 structure

(1) synthesis of antibacterial peptide PR39 genes

PR-39 gene C DS region sequences (the GenBank accession number announced according to NCBI：：L23825.1), The artificial synthesized of gene is carried out, and XhoI and NotI restriction enzyme sites are provided with gene order both ends.

(2) carrier for expression of eukaryon pCMS-EGFP digestion

1) carrier for expression of eukaryon pCMS-EGFP linearisation

Using two enzyme digestion reporter gene EGFP of Xhol, Notl carrier for expression of eukaryon pCMS-EGFP (purchased in market), Make vector linearization, while there is cohesive terminus,cohesive termini, digestion system is with reference to Fermentas companies restriction enzyme XhoI and NotI Specification, as shown in table 1.

The double digestion system of the carrier pCMS-EGFP sequences of table 1

Above-mentioned reaction system is fully mixed, 37 DEG C of digestion 15-30min, with reference to the purification kit of Transgen companies EasyTMPCRPurification specifications carry out purifying recovery to digestion products.

2) digestion of PR39 genes

The double digestion of two enzymes of Xhol, Notl is carried out to the PR39 gene DNA fragments of synthesis, makes DNA fragmentation that there is stickiness End, digestion system reference Fermentas companies restriction enzyme XhoI and NotI specification, as shown in table 2：

The double digestion system of the PR39 gene DNA sequences of table 2

Above-mentioned reaction system is fully mixed, 37 DEG C of digestion 15-30min, with reference to the purification kit of Transgen companies EasyTM PCR Purification specifications carry out purifying recovery to digestion products.

(3) after digestion carrier pCMS-EGFP and-PR39 connection and conversion

The digestion products of above-mentioned recovery are attached by system in table 3, linked system is with reference to TaKaRa company DNA Ligation Kit Ver.2.0 connections kit illustrates that concrete operations are as follows：

The linked system of the PR39 genes of table 3 and carrier pCMS-EGFP

The competent cell DH5 α of preservation are melted in ice, take 30 μ L competent cells gently to be mixed with the μ L of connection product 1 It is even, ice bath 30min, 42 DEG C of heat shock 45s, it is quickly transferred to cool down 2min in ice bath, adds the LB Liquid Cultures of 1mL antibiotic-frees Base, 37 DEG C are placed in, 1h is cultivated in 220r/min shaking tables, take 200 μ L bacterium solutions to be coated in LA flat boards, 37 DEG C of overnight incubations.

(4) PCR and sequencing identification

The picking single bacterium colony in the flat board of above-mentioned culture, it is suspended from 10 μ L aqua sterilisas, takes 2 μ L to carry out bacterium colony as template PCR, remaining 4 DEG C preservations.Positive bacterium solution PCR identification primers (being shown in Table 4) are designed, directly take bacterium solution to enter performing PCR identification as template, Reaction system and program are carried out with reference to GenestarMix Kit specifications, and PCR primer is examined with 1% agarose gel electrophoresis Survey.

The positive bacterium solution PCR identifications design of primers of table 4

The bacterium solution of test positive is re-seeded into LB culture mediums of the 5mL containing Amp, 37 DEG C of 220r/min shaking table cultures 12-16h, operated with reference to OMEGA companies E.Z.N.A.TM Plasmid Mini Kit II kit specifications.

The plasmid for taking 1 μ L to extract, the restriction enzyme site XhoI and NotI that both sides are inserted from exogenous sequences carry out double digestion mirror It is fixed, electrophoresis detection digestion result, the results showed that plasmid, which is cut open, is divided into 4683bp and 1382bp two parts, with expected size phase Symbol, it was demonstrated that carrier pCMS-EGFP-PR39 structures are qualified, and testing result refers to accompanying drawing 1.

When digestion result with expection be consistent, select 2-3 clone be sent to lifetechnologies companies progress positive and negative two Individual direction sequencing, sequencing result and aim sequence is compared, testing result such as accompanying drawing 2, sequencing result is correct, enters one Step shows that expression vector pCMS-EGFP-PR39 structures are qualified, and gene order number is SEQ ID NO:1.

Embodiment 2：Antibacterial peptide PR39 expression vectors pCMS-EGFP-PR39 functional verification

(1) cell of plasmid transfection 293, and carry out fluorescence microscope detection

Correct transgene carrier pCMS-EGFP-PR39 and control group carrier pCMS-EGFP bacterium solutions will be sequenced in 37 DEG C Expand culture 12-16h in 220r/min shaking tables, collect bacterium solution and carry out endotoxin plasmid extraction, plasmid extraction process reference OMEGA companies E.Z.N.A.TM EndoFree Plasmid Mini Kit II kit specifications are operated.Take 1 μ L matter Grain DNA carries out quality testing using nucleic acid concentration instrument, and DNA of 260/280 value between 1.8~2.0 can be used for cell Transfection experiment.

Passage to 6 orifice plates is cultivated, when cell grow to degree of converging be 80% when, the plasmid that will build PCMS-EGFP-PR39 and carrier pCMS-EGFP is in liposome-mediated lower rotaring redyeing 293 cell.Experimental procedure is with reference to life Technologies companies Lipofectamine TM LTX and PLUSTM Reagent kit specifications are carried out.

When observation cell confluency degree reaches 80%, liposome-mediated transfection can be carried out.

4 kinds of interference plasmids are diluted to 500 μIn basic culture solution, mix.Matter are disturbed by with 4 kinds Grain volume ratio 1：1 ratio adds Lipofectamine^TMPLUS Reagent (gently being shaken up using preceding) arrive above-mentioned mixed solution In, gently shake up, be incubated at room temperature 5min.By Lipofectamine^TMLTX (gently being shaken up using preceding) is added directly into dilute In the 4 kinds of interference plasmid solution released, gently shake up, be incubated at room temperature 30min.

In the process, after washing the cell of mouse 293 with serum-free medium, fresh 10% hyclone training is added Nutrient solution.

DNA- liposome mixed liquors are added dropwise in each hole, cell is put back into 37 DEG C of 5%CO2 after gently shaking up and satisfied Continue to cultivate with the incubator of humidity.

After 4-6h, renew the fresh nutrient solution containing 10% hyclone.After cultivating 24h, seen under fluorescence microscope Examine.As a result as shown in Figure 3：Display eukaryon expression plasmid pCMS-EGFP-PR39 and pCMS-EGFP empty carrier can detect To specificity fluorescent, illustrate two plasmid Successful transfections into 293 cells, and transfection efficiency is up to more than 60%.

(2) after the cell of plasmid transfection 293 PR39 genes the detection of expression in mRNA level in-site

It is rough to judge respectively with the fluorescence power situation in each hole of fluorescence microscope after the cell 24h of plasmid transfection 293 The transfection efficiency of plasmid.72h collects cell, and cell RNA is extracted using conventional method, by the RNA of extracting, reference TAKARA companies Reverse transcription reagent box specification carries out reverse transcription, and RT-PCR relative quantifications are carried out with the cDNA after reverse transcription.Reverse transcription step is such as Under, genomic DNA removes system such as table 5, and reverse transcription reaction system is as shown in table 6.PR39 genes, EGFP gene and reference gene Primer, it is as shown in table 8 that primer sequence is shown in Table 7, RT-PCR reaction systems.

Cell RNA extraction steps are as follows：

1) RNA extraction steps

Cell is collected, cell is blown and beaten after addition 1mLTRIzol repeatedly and is stored at room temperature 5min 200 μ L chloroforms of addition, vibration is mixed It is even, 5min12000g is stored at room temperature, supernatant is transferred to the body such as addition and supernatant in new centrifuge tube by 4 DEG C of centrifugation 15min Long-pending isopropanol, 10min, 12000g, 4 DEG C of centrifugation 10min are stored at room temperature, abandoning supernatant, the 75% of 1mL are added into precipitation Ethanol 12000g, 4 DEG C of centrifugation 5min abandoning supernatants, 10min is dried at room temperature and adds 36 μ LDEPC processing water, dissolves RNA.

2) reverse transcription step

1. organize RNA digestion：Purpose is in order to remove the genomic DNA in RNA, to prevent influenceing RT-PCR results.Make It is as shown in table 5 with the gDNAEraser that reagent is Takara companies, reaction system.

The genomic DNA of table 5 removes system

After sample has added mixing, 2min is incubated in 42 DEG C, removes genomic DNA；Then in 4 DEG C of preservations.

2. mixed liquor is placed in the mixing for carrying out reaction system according to table 6 on ice, Suo Youcao by above-mentioned reaction immediately after terminating Work is carried out on ice.

The reverse transcription reaction system of table 6

Above-mentioned system is mixed, 15min is incubated in 37 DEG C, then heats 5seconds terminating reactions in 85 DEG C, synthesis CDNA is placed in -20 DEG C of preservations.

It is 3. enterprising in PCR instrument according to 42 DEG C of 15min of response procedures, 95 DEG C of 3min after above-mentioned reactive component is fully mixed Row reaction.React -20 DEG C of preservations of reverse transcription product or progress quantitative RT-PCR detection after terminating.The testing result such as institute of accompanying drawing 4 Show, 293 cell lines of transfection pCMS-EGFP-PR39 plasmids have the expression of PR39 genes, and (pCMS-EGFP plasmids turn NC control groups Dye) and blank control group (no processing) be not detected by the expression of PR39 genes.

The detection primer of the PR39 genes of table 7, EGFP gene and reference gene (GAPDH)

The RT-PCR reaction systems of table 8

(3) detection after the cell of plasmid transfection 293 to Escherichia coli inhibition

With serum free medium culture cell after 293 cell 48h of the cell of plasmid transfection 293 and blank control group untransfected 24h, culture medium is collected, by culture medium elastin laminin ferment treatment, the transgene carrier pCMS- after elastin laminin ferment treatment EGFP-PR39 can obtain the PR39 of maturation.Elastoser application method reference SIGMA companies, product article No. are：E-8140 Operation instruction carries out digestion and (300ul physiology salt aqueous fusions is used, with 1:10 system carries out digestion).

The preparation of Escherichia coli, the picking single bacterium colony in Escherichia coli plate, with 5mlLB fluid nutrient mediums, it is placed in 37 DEG C, 18-24h is cultivated in 220r/min shaking tables, 50ul bacterium solutions is drawn and expands culture 3h in new 5ml LB fluid nutrient mediums, inhale Take 1ml bacterium solutions to centrifuge 5min in the centrifuge that rotating speed is 2000g, outwell supernatant, be resuspended with PBS, draw 200ul in 96 holes In plate, carry out OD values using ELIASA and detect, with reference to OD value corresponding concentration formula：Bacterial concentration (CFU/ml)=OD (620nm) X2.5x108 calculates its concentration, is from bacterial concentration：2×10⁶CFU/ml。

Bacteriostatic experiment step, with 96 orifice plates, 25ul concentration is added per hole is：2×10⁶CFU/ml Escherichia coli bacteria liquid, Then 25ul culture mediums are added to mix, is positioned in 37 DEG C of incubators and cultivates 3h, the addition 200ulLB fluid nutrient mediums per hole, 37 It is incubated overnight in DEG C incubator, detects its OD value with ELIASA within second day.

Testing result is as shown in table 8, the results showed that the cell training collected after the cell of pCMS-EGFP-PR39 plasmid transfections 293 The bacterial concentration value for supporting the Escherichia coli of base processing is substantially less than the cell culture medium collected after pCMS-EGFP rotaring redyeing 293 cells The bacterial concentration value of the Escherichia coli of processing, illustrate that the PR39 albumen that secretion is expressed from HEK293 cells can significantly inhibit large intestine The growth of bacillus, expression vector pCMS-EGFP-PR39 are successfully constructed.

Inhibitory action of the cell conditioned medium to Escherichia coli after the cell of 8 plasmid transfection of table 293

Embodiment 3：In F0 generations, turn the preparation and identification of PR39 DNA murines

By the transgene expression vector pCMS-EGFP-PR39 of linearisation, after empirical tests are qualified, using pronuclear microinjection Method prepare turn PR39 DNA murines.

(1) in F0 generations, turn the PCR identifications of PR39 DNA murines

Clip birth mouse tail is organized on a small quantity, extracts mouse tail tissue DNA, operating procedure is according to OMEGA companies DNA Extraction agent box specification is carried out.

PCR is detected：The both ends of PR39 genes insertion position separately design primer in carrier sequence, using clone with plasmid as Positive control, foreign gene is expanded, while the internal control primer GAPDH for designing mouse comes the integrality of validating DNA, PCR reactants System is shown in Table 9.As a result see accompanying drawing 5, the results showed that have 8 mouse for transgenic mice (i.e. F0 for mouse, be respectively labeled as 426, 427th, 428,429,430,431,432 and No. 433), wherein 6 transgenosis public affairs mouse (marked as：426、427、428、429、430、 431), 2 transgenosis dams (marked as：432 and 433).

The PCR reaction systems of table 9

(2) in F0 generations, turn the detection of PR39 DNA murine EGFP luciferase expressions

Using EGFP albumen blue light excite it is lower it is observed that green fluorescence come the preliminary mouse for judging to be born whether be Transgenic mice.Taken pictures respectively under white light background and blue light background, observe the expression of green fluorescence.Camera is adjusted to light Mode of priority is enclosed, parameter is respectively：White light aperture F2.8, ISO100；Blue light aperture F2.8, ISO4000.

As a result see accompanying drawing 6, the results showed that the EGFP gene copy number of 8 positive transgenic mices of PCR detections 1-8 it Between, wherein having the copy numbers of 4 transgenic mices (be respectively 426,429,430 and 431) close to single copy.Because EGFP is marked Remember gene and target gene PR39 amalgamation and expressions, thus, thus EGFP gene is also single copy if single copy, target gene.

(3) in F0 generations, turn the qPCR detections of PR39 DNA murines

To detect PR39 gene mRNA expression situations, using the mouse tail tissue of extracting, pass through real-time fluorescence quantitative PCR Method, the detection of PR39 gene mRNAs relative expression quantity is carried out, testing result is as shown in Figure 7.In 8 transgenic mices only 426th, detect that PR39mRNA is expressed in the tail tissue of 427 and 430 3 transgenic mices, and transgenic mice 427 PR39mRNA relative expression quantity highests.

Embodiment 4：F1 generation turns the preparation identification and detection of PR39 DNA murines

It is high for PR39mRNA expression quantity in mouse, is selected in F0 according to testing result, and the F0 generations 427 that integration site is single With 430 mouse with carrying out breeding breeding with kind wild-type mice, F1 generation mouse is obtained, and F1 generation mouse is identified.

(1) F1 generation turns the PCR identifications of PR39 DNA murines

Clip birth mouse tail is organized on a small quantity, extracts mouse tail tissue DNA, operating procedure is according to kit specification Carry out.

PCR is detected：The both ends of PR39 genes insertion position separately design primer in carrier sequence, using clone with plasmid as Positive control, foreign gene is expanded, while the internal control primer GAPDH for designing mouse carrys out the integrality of validating DNA.As a result such as accompanying drawing Shown in 8, the results showed that have 8 F1 generation transgenic mices can stablize expression PR39 genes, respectively F1-2, F1-3, F1-5, F1-7, F1-8, F1-9, F1-10, F1-11, PR39 gene expression amount highests in F1-9 individuals can be seen that by accompanying drawing 8, thus select Select F1-9 individuals and carry out follow-up study.

(2) detection of F1-9 transgenic mices different tissues PR39mRNA relative expression quantities

The primer of PR39 genes can be expanded according to PR39 genes design in plasmid, detection plasmid turns PR39 genes in difference Relative expression's situation in mouse.Select the F1-9 transgenic mices that can stablize expression PR39 genes in F1 generation individual, extracting group Knit RNA and PR39 gene mRNA expression amount situations in tissue are detected by real-time fluorescence quantitative PCR.As a result as shown in Figure 9, F1-9 Totally 7 tissues can express PR39 gene mRNAs for the transgenic mice heart, liver, spleen, lung, kidney, muscle, lymph node.

(3) detection of the ELISA method to F1-9 transgenic mice PR39 protein concentrations is used

1) antigenic content detects：

Collection F1-9 turns PR39 DNA murine blood, is collected with EDTA anticoagulant tubes, blood plasma is obtained, according to EIAab companies The content of PR39 antigens in ELISAKitforpigPR39 kits operating procedure detection blood.F1 is detected using identical method In generation, turns the content of PR39 antigens in the tissue such as the PR39 DNA murines heart, liver, spleen, lung, kidney, muscle.

2) ELISA operating procedures：

Before detection, each reagent all should be balanced to room temperature (reagent directly can not dissolve at 37 DEG C)；Reagent or sample dilution When, it is both needed to mix, avoids bubbling as far as possible during mixing.Sample size should be predicted before experiment, as sample concentration it is too high when, tackle sample Be diluted so that after dilution samples met kit detection range, multiplied by with corresponding extension rate during calculating.

1. it is loaded：Blank well, gauge orifice, testing sample hole are set respectively.In addition to blank well, remaining hole add respectively standard liquid or Testing sample 100ul, is gently mixed, and ELISA Plate is plus lid, 37 DEG C of incubation 120min.(it is loaded and sample is added on ELISA Plate bottom, Bubble has been careful not to, has not touched hole wall as far as possible)

2. discarding liquid, dry, without washing.Do not have directly hole to add detection solution A working solution 100ul (small using first half When interior preparation), gently rock mixing, ELISA Plate adds overlay film, 37 DEG C of incubation 60min.

3. discarding liquid, dry, board-washing 3 times.Immersion 1-2min every time, about 400ul/ holes, dry (gently patting dry).

4. adding detection liquid B working solution 100ul per hole, ELISA Plate adds overlay film, 37 DEG C of incubation 60min, board-washing 5 times.

5. substrate solution 90ul, 37 DEG C of preceding 3-4 for being incubated 15-30min, now naked eyes visual standard product are added per plate successively There are obvious gradient blueness, rear 3-4 gradient pores unobvious in hole.

6. stop bath 50ul is added per plate successively, terminating reaction (now blueness switchs to yellow immediately).

7. measure the optical density (OD values) in each hole successively in 450nm wavelength with enzyme-linked instrument.

8. liquid A and detection liquid B are detected to detect liquid：Liquid=1 after dilution:100 dilution proportion

It is 9. same according to concentration value corresponding to the extension rate provided on standard curve difference extension rate OD values and specification When use CurveExpert1.4 analysis softwares.

ELISA testing results as shown in Figure 10, antibacterial peptide are detected in F1-9 transgenic mices kidney, spleen and blood plasma PR39 is present, wherein PR39 protein concentration values are in nephridial tissue：1430pg/mg；PR39 protein concentration values are in tissue spleen： 835pg/mg；PR39 protein concentration values are in blood plasma：58446pg/ml.

Embodiment 5：Inverse PCR determines integration site of the antibacterial peptide PR39 genes in F1-9 transgenic mice individuals

DNA using F1-9 transgenic mices individual is template, and design direction PCR draws based on antibacterial peptide PR39 genes Thing, inverse PCR is carried out, and PCR primer is sequenced, sequencing result is compared with pig whole genome sequence, as a result such as Shown in accompanying drawing 11, antibacterial peptide gene is incorporated on No. 13 chromosome of transgenic mice, positioned at GM36264 genes and GM8784 Between two functional genes of gene, the expression of the F1-9 transgenic mice individual normal genes, while antibacterial peptide are not interfered with PR39 gene integrations have higher expression effect in the site.

Embodiment 6：F1-9 transgenic mice family antibacterium (APP) infection ability is analyzed

Bred to obtaining F1-9 transgenic mices, obtain F1-9 transgenic mice familys, and to the transgenic mice The analysis of anti-actinobacillus pleuropneumoniae (App) infection ability is carried out, it is specific as follows：

It is 6x10 with toxic agent amount is attacked⁶The actinobacillus pleuropneumoniaes of CFU/ only are injected simultaneously by way of intraperitoneal injection Transgenic mice 20, nontransgenic mice 20.Attack 5h mouse after poison start to occur it is dead, to attacking 17h dead mouses after poison Rate is not further added by.

As a result see accompanying drawing 12, show that 5h mouse start death occur after attacking poison, are raising always from the death rate between 5-17h. The non-transgenic group death rate reaches 64.7% wherein in 9h, is extremely significantly significantly higher than the 33.3% of transgenosis group；In 13h The non-transgenic group death rate reaches 82.4%, is significantly higher than the 66.7% of transgenosis group.This shows antibacterial in transgenic mice body Peptide PR39 may strengthen the antibacterial ability of mouse, delay mouse diing time, reduce mouse death rate.

In other embodiments, the toxic agent amount of attacking of actinobacillus pleuropneumoniae (App) can also be 6x10⁵CFU/, 3x10⁶CFU/, 6x10⁷CFU/ isoconcentration.

Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, not On the premise of departing from the invention design, various modifications and improvements can be made, these belong to the protection domain of invention.

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>A kind of cad gene PR39 DNA murines

<130> 2017

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 6065

<212> DNA

<213>Artificial sequence

<400> 1

tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60

ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120

aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 300

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360

ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg 480

gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540

caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600

caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg 660

cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata 720

agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac 780

agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt 840

gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa 900

ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact 960

cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac 1020

aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact 1080

ataggctagc ctcgagaggc ctgccaccat ggagacccag agggccagcc tgtgcctggg 1140

gcgctggtca ctgtggcttc tgctgctggg actcgtggtg ccctcggcca gcacccaggc 1200

cctcagctac agggaggccg tgcttcgtgc tgtggatcgc ctcaacgagc agtcctcgga 1260

agctaatctc taccgcctcc tggagctgga ccaaccgccc aaggccgacg aggacccggg 1320

caccccaaaa cctgtgagct tcacggtgaa ggagactgtg tgtcccaggc cgacccagcg 1380

gcccccggag ctgtgtgact tcaaggagaa cgggcgagtg aagcagtgtg tggggacagt 1440

caccttgaac ccatccaatg acccactgga catctcctgt aatgagattc agagtgtcag 1500

gagacgtccc cgacccccat atttgccaag gccaaggcca cctccgtttt tcccaccaag 1560

gctcccacca aggatcccac cagggttccc accaaggttc ccaccacggt tccccggaaa 1620

acggcaccac caccaccacc actgaggcgc gccgcggccg cttcccttta gtgagggtta 1680

atgcttcgca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca 1740

gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat 1800

aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg 1860

ggagatgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta aaatccgata 1920

aggatcgatc cgggctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 1980

ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg 2040

tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 2100

ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 2160

ggctcccttt agggttccga tttagagctt tacggcacct cgaccgcaaa aaacttgatt 2220

tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 2280

tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 2340

tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 2400

atgagctgat ttaacaaata tttaacgcga attttaacaa aatattaacg tttacaattt 2460

cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgcgga 2520

tctgcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct 2580

gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 2640

ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa 2700

agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 2760

ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt 2820

ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct 2880

ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc 2940

ttgccacaac ccgggatcca ccggtcgcca ccatggtgag caagggcgag gagctgttca 3000

ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 3060

tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 3120

ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 3180

agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 3240

ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 3300

gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 3360

acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 3420

acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 3480

acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 3540

gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 3600

aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 3660

tcactctcgg catggacgag ctgtacaagt aaagcggccc tagagctcgc tgatcagcct 3720

cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 3780

ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 3840

gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 3900

attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg 3960

aaagaaccag ctggggcaga tccgcgtatg gtgcactctc agtacaatct gctctgatgc 4020

cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 4080

tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 4140

gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt 4200

tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 4260

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 4320

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 4380

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 4440

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 4500

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 4560

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 4620

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 4680

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 4740

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 4800

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 4860

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 4920

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 4980

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 5040

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 5100

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 5160

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 5220

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 5280

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 5340

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 5400

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 5460

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 5520

cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 5580

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 5640

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 5700

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 5760

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 5820

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 5880

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 5940

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 6000

caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tggctcgaca 6060

gatct 6065

Claims (10)

1. a kind of cad gene PR39 DNA murines, it is characterised in that the antibacterial peptide PR39 gene integrations turn antibacterial described On No. 13 chromosome of peptide PR39 DNA murines.

2. cad gene PR39 DNA murines according to claim 1, it is characterised in that the antibacterial peptide PR39 gene positions Between the GM36264 genes and GM8784 genes on the 13rd chromosome of transgenic mice.

3. cad gene PR39 DNA murines according to claim 2, it is characterised in that described cad gene PR39 bases Because mouse obtains as follows：

(1) according to PR39 gene C DS sequences, restriction enzyme site, artificial synthesized corresponding DNA sequence dna are increased at gene order both ends；

(2) DNA sequence dna of synthesis is connected on pCMS-EGFP empty carriers, obtains antibacterial peptide PR39 expression vectors pCMS- EGFP-PR39；

(3) functional verification is carried out to antibacterial peptide PR39 expression vectors pCMS-EGFP-PR39；

(4) by can effective expression PR39 genes expression vector pCMS-EGFP-PR39 microinjection mouse fertilized egg male pronucleus, And by the zygote transplation after injection to replace-conceive dams intrauterine, F0 is obtained for transgenic mice by gestation；

(5) F0 of acquisition is detected for transgenic mice, chooses PR39 gene expression amounts height, and that integration site is single Body carries out follow-up study；

(6) F0 filtered out generations individual is bred with wild-type mice breeding, obtains F1 generation transgenic mice；

(7) the F1 generation transgenic mice of acquisition is detected, filtering out can be stable hereditary and high efficient expression PR39 genes Body carries out integration site analysis, and is used for follow-up study；

(8) the F1 generation individual filtered out is bred, obtains corresponding family, and antibacterium experiment is carried out to the family, obtained To the cad gene PR39 DNA murines for being highly resistant to bacterium.

4. cad gene PR39 DNA murines according to claim 3, it is characterised in that the antibacterial peptide PR39 gene tables It is SEQ ID NO up to carrier pCMS-EGFP-PR39 gene orders:1.

5. cad gene PR39 DNA murines according to claim 3, it is characterised in that the step (3) is included table Up to after carrier rotaring redyeing 293 cell, qPCR detections and fungistatic effect detection are carried out.

6. cad gene PR39 DNA murines according to claim 5, it is characterised in that described qPCR detections, including Following steps：

(1) pCMS-EGFP-PR39 expression vectors rotaring redyeing 293 cell, in fluorescence microscopy Microscopic observation green fluorescent protein after 24h Expression；

(2) after 72h, cell extraction RNA is collected；

(3) qPCR detections are carried out to the RNA of extraction, qRCP response procedures are 42 DEG C of 15min, 95 DEG C of 3min.

7. cad gene PR39 DNA murines according to claim 5, it is characterised in that described fungistatic effect detection bag Include following steps：

(1) after 293 cell culture 48h of pCMS-EGFP-PR39 expression vectors rotaring redyeing 293 cell and blank control group untransfected, With serum free medium culture cell 24h, culture medium is collected, by culture medium elastin laminin ferment treatment；

(2) preparation of Escherichia coli：The picking single bacterium colony in Escherichia coli plate, it is inoculated in 5ml LB fluid nutrient mediums, puts 18-24h is cultivated in 37 DEG C, 220r/min shaking tables, 50ul bacterium solutions is drawn and expands culture in new 5ml LB fluid nutrient mediums 3h, draw 1ml bacterium solutions and centrifuge 5min in the centrifuge that rotating speed is 2000g, outwell supernatant, be resuspended with PBS, draw 200ul In 96 orifice plates, carry out OD values using ELIASA and detect, be from bacterial concentration：2×10⁶CFU/ml；

(3) bacteriostatic experiment step：With 96 orifice plates, it is 2 × 10 that 25ul concentration is added per hole⁶CFU/ml Escherichia coli bacteria liquid, then Add culture mediums of the 25ul after step (1) processing to mix, be positioned in 37 DEG C of incubators and cultivate 3h, 200ul LB are added per hole Fluid nutrient medium, it is incubated overnight in 37 DEG C of incubators, its OD value is detected with ELIASA within second day, according to the size of OD values, to sentence Determine the fungistatic effect of pCMS-EGFP-PR39 expression vectors.

8. cad gene PR39 DNA murines according to claim 3, it is characterised in that the step (5) is examined including PCR Survey and PR39 gene mRNA expressions amount detects.

9. cad gene PR39 DNA murines according to claim 3, it is characterised in that the step (8) includes as follows Step:

(1) it is 6x10 with concentration to the transgenic lines and nontransgenic mice of acquisition⁵CFU/, 3x10⁶CFU/, 6x10⁶CFU/, 6x10⁷The actinobacillus pleuropneumoniaes of CFU/ only are by being injected intraperitoneally transgenic mice group and non-transgenic Mouse group；

(2) mouse treated to step (1), is cultivated, and in different time node, statistical is carried out to the death rate of mouse Analysis.

10. cad gene PR39 DNA murines according to claim 9, it is characterised in that described pig pleuropneumonia is put Line bar bacteria concentration is 6x10⁶CFU/ is only.