CN109735519A - A method of birds, beasts and eggs bacteriolyze production of enzyme is improved using gene editing technology - Google Patents

Abstract

The invention discloses a kind of methods for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology, method includes the following steps: will include that the targeting sgRNA for carrying out fixed point editor for fowl lysozyme promoter region is cloned into the V2 plasmid of the Cas9 gene comprising DNA double chain shearing, the plasmid edited for fowl lysozyme promoter be obtained；Gene editing template ssDNA is constructed, template ssDNA is the linear ssDNA comprising homologous sequence, 2000bp ovalbumin transcripting starting complementary series and target spot complementary strand right side homologous sequence on the left of target spot complementary strand；Said gene editor plasmid, gene editing template ssDNA are mixed and made into plasmid vector group mixed liquor, and mixed liquor is mixed with liposome and is fostered, is injected into blastaea hatching nearby, obtains the female poultry individual of the lysozyme gene containing efficient promoter.The method of the present invention has the characteristics of technically reliable, permanent and safe and efficient expression, and the translation amount of lysozyme improves nearly a hundred times.

Description

A method of birds, beasts and eggs bacteriolyze production of enzyme is improved using gene editing technology

Technical field

The invention belongs to poultry kind molecules accurately to improve breeding technical field more particularly to a kind of utilization gene editing skill The method of art raising birds, beasts and eggs bacteriolyze production of enzyme.

Background technique

Lysozyme (lysozyme) is also known as muramidase or N-acetylmuramide lycanohydrlase, is a kind of from fowls egg protein Middle separation and Extraction can hydrolyze the alkaline enzyme that polysaccharide is sticked in pathogenic bacteria.It can pass through the N- acetyl born of the same parents in destruction bacteria cell wall β-Isosorbide-5-Nitrae glycosidic bond between Teichaic acid and n-acetylglucosamine makes the insoluble glutinous polysaccharide of cell wall resolve into soluble glycopeptide, leads It causes the evolution of cell wall rupture content and bacterolysis is made to play sterilization effect.Lysozyme can also be with negatively charged virus protein It binds directly, forms double salt with DNA, RNA, apoprotein, breed virally inactivated inhibition virus amplification.Therefore, which has Have antibacterial, anti-inflammatory, it is antiviral the effects of.Because of its extensive antibacterial and antiviral effect, but after being claimed the antibiotic epoch it is new Type immune factor, the novel immune factor is used widely and effect in medical domain, food production field at present Verifying.Since animal body fluid that it mainly comes is a kind of native enzyme, the additive of safe green, no drug resistance, and to not having The human body cell of cell wall will not have an adverse effect, the anti-corrosion particularly suitable for various food.In addition, the enzyme can also kill intestines Road corruption coccus increases enteron aisle resistance infection, while baby intestinal bifidobacterium can also be promoted to be proliferated, and promotes milk casein Curdled milk is conducive to digestion, so being the good additive of baby food, beverage again.Just because of lysozyme is to human body totally nontoxic, nothing Side effect has effects that antibacterial, antiviral and antitumor, is a kind of safe natural antiseptic agent, therefore have wide application Prospect, but the lysozyme due to being produced using biotechnology is deposited in the various aspects such as bioactivity and natural lysozyme activity In larger difference, therefore at present based on the extraction of the body fluid such as the main source of lysozyme or egg white, the animal blood of poultry, wherein The abundantest with the content of poultry egg white, activity is best.The lysozyme content that the previous egg of mesh can extract is only the left side 3mg The right side is far from satisfying the ever-expanding lysozyme market demand according to the current such extracted amount of market demand, this also makes It holds at high price (4~5 yuan every gram or so) at lysozyme, although the lysozyme content in egg white and other animal body liquid phases Compare that content is very abundant, but there is also very big differences for the content in egg white is up to tens of grams of ovalbumin It is different.Therefore how to improve lysozyme content in birds, beasts and eggs and extracted amount using biotechnology is current limitation lysozyme industry development Critical bottleneck, this is also the direction of a great development prospect in poultry industry transition and upgrade, for poultry industry, food, The development of medical industry will all have tremendous influence and contribution.

Summary of the invention

The purpose of the present invention is to provide a kind of methods for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology, more Body is to provide one kind the controlling element of the ovalbumin of high efficient expression in birds, beasts and eggs can be inserted into avian lysozyme gene expression Regulatory region come the method that improves birds, beasts and eggs lysozyme high efficient expression in birds, beasts and eggs, this is intended to improve birds, beasts and eggs from genomic level molten Bacterium enzyme content, to obtain the laying poultry individual with production lysozyme.

The invention is realized in this way a method of birds, beasts and eggs bacteriolyze production of enzyme, the party are improved using gene editing technology Method the following steps are included:

It (1) will include that the targeting sgRNA for carrying out fixed point editor for fowl lysozyme promoter region is cloned into comprising DNA In the V2 plasmid of the Cas9 gene of double-strand shearing, the plasmid edited for fowl lysozyme promoter is obtained；

(2) construct gene editing template ssDNA, template ssDNA be comprising homologous sequence on the left of target spot complementary strand, The linear ssDNA of homologous sequence on the right side of 2000bp ovalbumin transcripting starting complementary series and target spot complementary strand；

(3) said gene editor plasmid, gene editing template ssDNA are mixed and made into plasmid vector group mixed liquor, and will Mixed liquor is mixed with liposome and is fostered；

(4) after addition liposome is fostered near the blastodisc of hatching fowl embryo egg in vitro, the mixing after being fostered in step (3) Liquid is injected near the blastaea to germinate, and entire embryo egg is moved to external container, dual anti-rear sealing, hatching is added, in shape Verifying screening is carried out to the segment of insertion at after individual, obtains the female poultry of the lysozyme gene containing efficient promoter Body.

Preferably, the birds, beasts and eggs are egg.Preferably, in step (1), the targeting sgRNA of chicken genome such as SEQ ID Shown in NO:1；In step (2), homologous sequence is as shown in SEQ ID NO:2 on the left of the target spot complementary strand of chicken genome, chicken gene The 2000bp ovalbumin transcripting starting complementary series of group is as shown in SEQ ID NO:3, on the right side of the target spot complementary strand of chicken genome Homologous sequence is as shown in SEQ ID NO:4.

Preferably, the birds, beasts and eggs are duck's egg.Preferably, in step (1), the targeting sgRNA of Duck genome such as SEQ ID Shown in NO:5；In step (2), homologous sequence is as shown in SEQ ID NO:6 on the left of the target spot complementary strand of Duck genome, duck gene The 2000bp ovalbumin transcripting starting complementary series of group is as shown in SEQ ID NO:7, on the right side of the target spot complementary strand of Duck genome Homologous sequence is as shown in SEQ ID NO:8.

Preferably, in step (3), the molecular weight of gene editing plasmid, gene editing template ssDNA in the mixed liquor The ratio between be 1:9；The liposome is liposome 2000 or liposome 3000；The volume ratio of the mixed liquor and liposome is 1: 1.5；The temperature fostered is 37 DEG C, time 10min.

Preferably, in step (4), the liposome is liposome 2000 or liposome 3000；The liposome is in embryo The amount that disk is nearby added is 15 microlitres；The temperature fostered is 37 DEG C, time 10min.

Preferably, described dual anti-for ampicillin and streptomysin in step (4).

The present invention, which overcomes the deficiencies of the prior art and provide, a kind of improves birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology Method, the present invention utilizes precise target editor's Insertion action of gene editing technology, accurately by the efficient table of the ovalbumin of birds The lysozyme gene expression regulation area of birds is properly inserted up to controlling element sequence, generating has ovalbumin high efficient expression tune The new laying poultry individual of element and the combination of lysozyme gene translated region is controlled, and then improves the content of lysozyme from egg white.

Compared with the prior art the shortcomings that and deficiency, the invention has the following advantages:

(1) the method for the present invention has the characteristics of technically reliable, permanent and safe and efficient expression；

(2) present invention is accurately inserted by the method for gene editing by efficient ovalbumin gene expression regulation element Enter the control region to lysozyme gene, effectively raises the expression quantity of lysozyme gene, and then improve the translation amount of lysozyme Nearly a hundred times；

(3) present invention changes the bacteriolyze of birds due to the method using gene editing on the basis of genetic expression Expression of enzymes efficiency, therefore the heredity that the change can be stable form the group that efficiently production is translated, and be highly advantageous to lysozyme The batch production industrialization of product is extracted and application；

(4) present invention inserts existing genetic fragment in avian subject body using gene editing means, therefore will not make Uncertain biohazardous caused by being imported at alien gene；

(5) segment be inserted into of the present invention only for be lysozyme gene expression efficiency, for original fowls egg protein Expression regulation, which does not make, to be changed, therefore the hatching egg that the change generates can also hatch to form new individual, ensure that individual change Stablize heredity.

Specific embodiment

In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to Limit the present invention.

Embodiment 1

(1) by include for fowl lysozyme promoter region carry out fixed point editor targeting sgRNA (such as SEQ ID NO: Shown in 1) being cloned into the V2 plasmid of Cas9 gene comprising DNA double chain shearing, (V2 plasmid is commercially available for market, the enzyme of V2 plasmid Cut operation, restriction enzyme site is recorded according to product description and carried out) in, obtain the matter edited for fowl lysozyme promoter Grain；

(2) gene editing template DNA is constructed, which is comprising homologous sequence (such as SEQ ID on the left of target spot complementary strand Shown in NO:2), on the right side of 2000bp ovalbumin transcripting starting complementary series (as shown in SEQ ID NO:3) and target spot complementary strand The linear ssDNA of homologous sequence (as shown in SEQ ID NO:4)；

It (3) is 1:9 according to the ratio between molecular weight by said gene editor plasmid, gene editing template ssDNA；Mixing；Later It is again that 1:1.5 is mixed according to volume ratio with liposome, fosters for 37 DEG C after mixing；

(4) blastodisc for hatching fowl embryo egg in vitro is nearby added 15 microlitres of the liposome containing plasmid mixed liquor, and 37 DEG C Foster after five minutes, be injected near the blastaea to germinate, entire embryo egg is moved into external container, be added it is dual anti-after with sealing, Hatching carries out verifying screening to the segment of insertion after forming individual, obtains the female of the lysozyme gene containing efficient promoter Property poultry individual.

(5) 100 pieces of embryo egg of conversion is completed, hatching forms individual 65 chickens, wherein hen 29, by target sequence Detection discovery is successively inserted into female individuals 3 of ovalbumin promoter, survives 2 after sexal maturity of growing up, and lay eggs.Utilize survey Fixed two individual egg product contents are shown in Table 1 compared with lysozyme content in control group egg white:

1 egg lysozyme content of table and active contrast table

Group	Lysozyme content (mg/mL)	Active (U/mg)
			Gene editing group	645.2±83.85	15587.3±1343.27
Control group	5.7±0.15	16479.2±1023.71

Embodiment 2

(1) by include for fowl lysozyme promoter region carry out fixed point editor targeting sgRNA (such as SEQ ID NO: Shown in 5) it is cloned into the V2 plasmid of the Cas9 gene comprising DNA double chain shearing, it obtains being compiled for fowl lysozyme promoter The plasmid collected；

(2) gene editing template DNA is constructed, which is comprising homologous sequence (such as SEQ ID on the left of target spot complementary strand Shown in NO:6), on the right side of 2000bp ovalbumin transcripting starting complementary series (as shown in SEQ ID NO:7) and target spot complementary strand The linear ssDNA of homologous sequence (as shown in SEQ ID NO:8)；

It (3) is 1:9 according to the ratio between molecular weight by said gene editor plasmid, gene editing template ssDNA；Mixing；Later It is again that 1:1.5 is mixed according to volume ratio with liposome, fosters for 37 DEG C after mixing；

(4) blastodisc for hatching duck egg in vitro is nearby added 15 microlitres of the liposome containing plasmid mixed liquor, and 37 DEG C Foster after five minutes, be injected near the blastaea to germinate, entire embryo egg is moved into external container, be added it is dual anti-after with sealing, Hatching carries out verifying screening to the segment of insertion after forming individual, obtains the female of the lysozyme gene containing efficient promoter Property poultry individual.

(5) 100 pieces of duck egg of conversion is completed, hatching forms individual 65 ducklings, wherein duck 29, by target spot sequence Column detection discovery is successively inserted into female individuals 4 of ovalbumin promoter, survives 4 after sexal maturity of growing up, and lay eggs.It utilizes It measures two individual egg product contents and is shown in Table 2 compared with lysozyme content in control group Ovum Anas domestica album:

2 duck's egg lysozyme content of table and active contrast table

Group	Lysozyme content (mg/mL)	Active (U/mg)
			Gene editing group	213.5±34.16	2571.5±205.4
Control group	3.5±0.12	2571.3±529.3

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.

Sequence table

<110>Taizhou University

Zhejiang Academy of Agricultural Science

Xibei Univ. of Agricultural & Forest Science & Technology

<120>a kind of method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology

<141> 2019-01-03

<160> 8

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Gallus domesticus

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gcgctcagct cccgccccgg cccggcccgg ccccgccgca gcgctcagcc ccacggcccc 240

ggcgcccgca ccgcgtcacg ggggcggggc ggtgccgtca cggggcgacg gttggccgcg 300

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<212> DNA

<213> Gallus domesticus

<400> 3

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tggaaattca aagagcaaat ttcagtaaac tttaggtttg gacagatata tgagaaagca 360

gaggcttgct gactatttta tttcttattt ttattcccta aaaataaatg tagagaaata 420

tctgtttgtt gcacactact tgctatgagt agatcttcaa aagtattttt acctttgttt 480

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gacacgcatg taattcatat tctgctctat tgcactttca ggtatggttt gctttgctca 600

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agcttttgga gccagcatga atctagttgc tttcctgaga tttatataat ctgtgaaagt 900

cagaagtcct tcagagccca gccctttata tgcgtactga gtgctggggc ctcaggattg 960

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agaataagcc tcctgaaaac aaacacaaag cttttacaca taataaaata gtgcagaatg 1380

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tgacccaccc acaggttccc ttcagaagtt tgacctttga cgccatagaa atcatttaat 1680

gggattgggt tagattttag tttcaatagg tccattttgg attgaatgga gagcaaatat 1740

tagtttttaa ttctgggtaa caatgtgttt tctgcctgtt ctgctaatcc atcaggactg 1800

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ttcatttgat ccttcttgta tgtacaacgt gcatctgagc catgctaggc aggacataag 2040

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<212> DNA

<213> Gallus domesticus

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gctctcctgg gaaccccccg ccacctacca cgcgttcatg ccgtttccat cgctgacgat 300

cttcttcgcg cagttcacgc tcgctgttat gtctgagctc agcagggctt aagaaaggaa 360

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<213> Anatinae

<400> 5

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<212> DNA

<213> Anatinae

<400> 6

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gtgtgagggc tgttggctgt tgagaacggg ccccgctgag cacctgcgcc tcatagcctg 360

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<210> 7

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<212> DNA

<213> Anatinae

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tgtgaaatcc ctgttgtcta gagcaaacag cagaagcaca atgacaatgt aatgatggaa 60

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agtagttttt aggccattaa ataaaacaac aaatagtttt aaagtcattc attgttaaca 180

tgcctattgc cataggcatc tgtcctgcat gttcaaaact gtattcataa atctatttct 240

ttttgaattt gcattcagtt gcctttttag cagtaaattt tctcagaagt cttagttcca 300

tatacagatt ctgtttacct tttaaaagca gtcaaataac aattgtttgt acatattttc 360

attcagtgaa agatagaatt cctctgatca aaacaagaaa atatgacctg tttgatagac 420

ttgcaaagct ggaacttcaa agtgcaaatt tcagtaggtt tggacagata aatgtgaaag 480

cagaggctcg ctgtttttat attctaaaaa taaatgagtg cagaaaaata tctacttgtt 540

ccattgtacc tactgccagt ggacttttaa aggcttgcat ttctactctt gcttcagtaa 600

tggaagaata gatagggaac atagtttgtc taaggtcatc tagtctagga gagagactga 660

tgcacataat tcgtatttat gaatattcaa ttccacccta tcatgctttc agacatactt 720

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ttagcgacat tcagacagaa gaggaatgca gacatgataa attagcaact ataatacagc 840

actgtcagat taatatttgt gtaaagcagt aggatcttta tatttgtttc tgtcttgaac 900

tactataaag tgccagggtg tacaagtcat gtatagccag tatgcttata taaagcacag 960

cttttgaggt cagcatgaac ctagttttct tcctgtaatt tatagaaatt gttaaagtta 1020

tttgtctttc agaatccagc cttttatata ggtgctaatg ctggagcctg aagtttgtct 1080

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ccacatactt ctcccacttt catttctgta tacaaattaa catttataga ttattgtaag 1200

aatataagtt tatacaccta cactaaaatg accctaaaaa ttcactgaat gccacagtgt 1260

tgctctctgt gcattctgtt gaactggaag tcaatcaagc aaactgcaac aaataaaatt 1320

taaaagatac tgaacaatac aacaaggtgc tctgaaatat aaacatattt taagcgcaaa 1380

gaataactgt tttagaaatc tcctgaaaac caacacatag tttttacaca taataaaaca 1440

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agagcaaata tagtggaaag gtaaattttc ctagagaagc acttaccgtt tgagcttcag 1620

caccaaaggt aatacagctt tccagctgta tgttgagaga ctggctgagc cctggagaat 1680

ttatacagtc tgaatttgga cccacccgct ggtgcctttc agaagtttga cctttgacac 1740

cataaaatca tttgatggaa ttgggtcaga tttactttca atgggtccat tctgggttga 1800

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cctaatccat caggactgtt gagtaggaca gaagactggg aaatactggt aaccttcctt 1920

taagtcttag ttacaactag gtacctggat gttaatgaaa acaagaaaaa gaaaaaaaat 1980

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<210> 8

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<212> DNA

<213> Anatinae

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aaaaggccag gctatatatc aagtactttg ctgcttcccc aaagagagag gcacaaccaa 180

gcattcctga tttagaaatc acagcacacc taaggccata ggttggactg gtctatgcgc 240

tctgttcttt gagagcgtca attctaagta gagcacgtgg gctttgtaaa agcaggtcaa 300

gatgactcga cccttcgcct gcagccagca aaatcaaagc cagtcacatg tacattgtag 360

gtcatttcgt ttgcacttca atactaaaag tttggggaat 400

Claims (8)

1. a kind of method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology, which is characterized in that this method includes following Step:

It (1) will include that the targeting sgRNA for carrying out fixed point editor for fowl lysozyme promoter region is cloned into comprising DNA double chain In the V2 plasmid of the Cas9 gene of shearing, the plasmid edited for fowl lysozyme promoter is obtained；

(2) gene editing template ssDNA is constructed, template ssDNA is comprising homologous sequence, 2000bp ovum on the left of target spot complementary strand The linear ssDNA of homologous sequence on the right side of albumin transcripting starting complementary series and target spot complementary strand；

(3) said gene editor plasmid, gene editing template ssDNA are mixed and made into plasmid vector group mixed liquor, and will mixing Liquid is mixed with liposome and is fostered；

(4) after addition liposome is fostered near the blastodisc of hatching fowl embryo egg in vitro, the mixed liquor after fostering in step (3) is infused It is mapped near the blastaea to germinate, entire embryo egg is moved into external container, dual anti-rear sealing, hatching is added, it is a being formed Verifying screening is carried out to the segment of insertion after body, obtains the female poultry individual of the lysozyme gene containing efficient promoter.

2. the method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology as described in claim 1, which is characterized in that institute Stating birds, beasts and eggs is egg.

3. the method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology as claimed in claim 2, which is characterized in that In step (1), the targeting sgRNA of chicken genome is as shown in SEQ ID NO:1；

In step (2), on the left of the target spot complementary strand of chicken genome homologous sequence as shown in SEQ ID NO:2, chicken genome 2000bp ovalbumin transcripting starting complementary series is homologous on the right side of the target spot complementary strand of chicken genome as shown in SEQ ID NO:3 Sequence is as shown in SEQ ID NO:4.

4. the method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology as described in claim 1, which is characterized in that institute Stating birds, beasts and eggs is duck's egg.

5. the method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology as claimed in claim 4, which is characterized in that In step (1), the targeting sgRNA of Duck genome is as shown in SEQ ID NO:5；

In step (2), on the left of the target spot complementary strand of Duck genome homologous sequence as shown in SEQ ID NO:6, Duck genome 2000bp ovalbumin transcripting starting complementary series is homologous on the right side of the target spot complementary strand of Duck genome as shown in SEQ ID NO:7 Sequence is as shown in SEQ ID NO:8.

6. the method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology as described in claim 1, which is characterized in that In step (3), the ratio between molecular weight of gene editing plasmid, gene editing template ssDNA is 1:9 in the mixed liquor；The rouge Plastid is liposome 2000 or liposome 3000；The volume ratio of the mixed liquor and liposome is 1:1.5；The temperature fostered For 37 DEG C, time 10min.

7. the method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology as described in claim 1, which is characterized in that In step (4), the liposome is liposome 2000 or liposome 3000；The amount that the liposome is added near blastodisc is 15 Microlitre；The temperature fostered is 37 DEG C, time 10min.

8. the method for improving birds, beasts and eggs bacteriolyze production of enzyme using gene editing technology as described in claim 1, which is characterized in that It is described dual anti-for ampicillin and streptomysin in step (4).