CN111826397A - Method for producing recombinant target protein, overexpression vector and virus suspension - Google Patents

Method for producing recombinant target protein, overexpression vector and virus suspension Download PDF

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CN111826397A
CN111826397A CN202010977350.9A CN202010977350A CN111826397A CN 111826397 A CN111826397 A CN 111826397A CN 202010977350 A CN202010977350 A CN 202010977350A CN 111826397 A CN111826397 A CN 111826397A
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protein
virus
sequence
target protein
vector
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黄跃进
杨盼
粟子丹
张敬辉
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Nanjing Jenomed Biotech Co ltd
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Nanjing Jenomed Biotech Co ltd
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Abstract

The present invention provides a method for producing a recombinant protein of interest, comprising: after virus packaging is carried out on an overexpression vector containing a target protein gene and an adeno-associated virus vector, the obtained virus suspension is introduced into mammary tissues or mammary ducts of a non-human mammal in real time in an adeno-associated virus mediated transduction mode to complete virus introduction. The method for producing the recombinant target protein uses the overexpression vector containing the gene of the target protein and the adeno-associated virus vector, combines a transduction mode mediated by the animal mammary gland adeno-associated virus to complete virus introduction, does not need expensive instruments and equipment, is beneficial to realizing the rapid and high-level expression of the recombinant target protein in animal milk, and achieves unexpected technical effects. The invention also provides the overexpression vector and the virus suspension.

Description

Method for producing recombinant target protein, overexpression vector and virus suspension
Technical Field
The invention relates to the technical field of biological medicines, in particular to a method for producing recombinant target protein, an overexpression vector and a virus suspension.
Background
In recent years, animal mammary gland bioreactors have become a very active area of research and development due to the continuous improvement of genetic engineering techniques and the development of animal cloning techniques. The animal mammary gland bioreactor is used for introducing exogenous genes into an animal genome and positioning to express target proteins in animal mammary glands, and biological products with important values are produced in milk of animals by utilizing the capability of natural and efficient synthesis and secretion of the target proteins of the animal mammary glands.
Compared with a genetic engineering cell reactor, the biological product obtained by the animal mammary gland bioreactor has high activity, no pollution, easy separation and purification and is beneficial to reducing the cost. For example, chinese patent application publication No. CN1247899A discloses a method for constructing recombinant DNA for expressing interferon in the mammary gland of an animal, which comprises directly injecting an injection solution containing recombinant DNA into the duct of the mammary gland of the animal, so that the recombinant DNA enters the secretory mammary gland cell, and when the secretory mammary gland cell secretes milk, the protein encoded by the recombinant DNA is expressed. Chinese patent application publication nos. CN1744814A, CN101245351A and CN1869217A all disclose methods for expressing pharmaceutical proteins in animal milk using adenovirus vectors. U.S. patent publication No. US6743966B2 discloses a method for producing membrane proteins by expression in an animal mammary bioreactor, comprising inserting a mammary gland-specific expression construct into the genome of a non-human mammal, which causes the membrane proteins to be expressed in mammary epithelial cells and secreted into the milk.
However, the above-described cycles for expressing target proteins using animal mammary gland bioreactors or adenovirus vectors are generally long, the expression level of the target proteins in milk per ml cannot reach milligram level, and the search for mammary duct inlets in the prior art requires operation under a microscope or a magnifying glass, which easily damages the mammary duct once the operation is not proper.
Therefore, there is a need to provide a novel method for producing recombinant target proteins, which avoids the above-mentioned problems of the prior art.
Disclosure of Invention
The invention aims to provide a method for producing recombinant target protein, an overexpression vector applied to the method for producing the recombinant target protein and a virus suspension, so as to realize rapid and efficient production and high-level expression of the recombinant target protein.
To achieve the above object, the method for producing a recombinant target protein according to the present invention comprises: providing an overexpression vector comprising a gene for a target protein and an adeno-associated viral vector; viral packaging said overexpression vector to form a viral suspension; introducing the virus suspension into mammary tissue or mammary duct of a temporary non-human mammal by means of adeno-associated virus-mediated transduction to complete virus introduction, and allowing the non-human mammal to express and secrete the recombinant target protein through mammary cells in a lactation process; obtaining milk secreted by said non-human mammal during the lactation period, and obtaining said recombinant protein of interest from said milk.
The method for producing the recombinant target protein has the advantages that: the method uses an overexpression vector containing a gene of a target protein and an adeno-associated virus vector, performs virus packaging, and then uses the obtained virus suspension to be introduced into mammary tissues or mammary ducts of a non-human mammal in real time in an adeno-associated virus-mediated transduction mode to complete virus introduction, does not need expensive instruments and equipment, can realize rapid and efficient production and high-level expression of the recombinant target protein through simple operation, and achieves unexpected technical effects.
Preferably, the promoter sequence in the overexpression vector is any one of CMV, SV40, RSV, CAG and EF, Whey Acidic Protein (WAP), α S1-casein, α S2-casein, β -casein, κ -casein, β -lactoglobulin and α -lactalbumin.
Preferably, the enhancer sequence in the overexpression vector is a sequence of any one of CMV, SV40 and RSV.
Preferably, the signal peptide coding sequence for secretion of the expressed recombinant target protein provided in the overexpression vector is selected from the sequences of any one of Whey Acidic Protein (WAP), α S1-casein, α S2-casein, β -casein, κ -casein, β -lactoglobulin, α -lactalbumin, human alkaline phosphatase, melittin and immunoglobulin light chain protein Igk.
Further preferably, the signal peptide coding sequence secreted by the expressed recombinant target protein provided in the overexpression vector is derived from the sequence of goat beta-casein.
Preferably, the recombinant target protein is any one of cholinesterase, peroxidase, growth hormone, antiviral protein, immunoglobulin, a virus structural protein, a viroid, and a membrane protein, or a fragment or derivative of any one of the above.
Further preferably, the recombinant target protein is any one of human acetylcholinesterase and human butyrylcholinesterase.
Further preferably, the recombinant target protein is any one of peroxidase, oxidase and dehydrogenase.
Further preferably, the recombinant target protein is a membrane protein, and the membrane protein is any one of a GPCR protein, an ion channel protein and a transporter.
Further preferably, the recombinant target protein is any one of human ACE2 and ACE2-Fc fusion protein.
Preferably, the sequence of the overexpression vector further comprises a woodchuck hepatitis virus transcription regulatory element sequence (WPRE) and a SV40 polyadenylation signal sequence.
Preferably, the sequence of the overexpression vector is shown as any one of SEQ ID Nos. 1 to 7.
Further preferably, the sequence structure of the overexpression vector is pAAV-CAG promoter-target protein coding sequence-WPRE-SV 40 polyadenylation signal sequence, and the nucleotide sequence of the overexpression vector is shown in any one of SEQ ID Nos. 1-4.
Further preferably, the sequence structure of the overexpression vector is pAAV-goat beta-casein gene (BCN) promoter-target protein coding sequence-WPRE-SV 40 polyadenylation signal sequence, and the nucleotide sequence of the overexpression vector is shown in any one of SEQ ID Nos. 5-6.
Further preferably, the sequence structure of the over-expression vector is pAAV-CMV promoter-target protein coding sequence-WPRE-SV 40 polyadenylation signal sequence, and the nucleotide sequence of the over-expression vector is shown as SEQ ID No. 7.
Preferably, the adeno-associated viral vector serotype comprises any one of AAV1-AAV4 and AAV7-AAV 9.
Further preferably, the adeno-associated viral vector serotype is any one of AAV1 and AAV 9.
Preferably, 293T cells are co-transfected with the over-expression vector, the first helper plasmid and the second helper plasmid, and the resulting transfection solution is then subjected to a purification treatment to complete the viral packaging.
Further preferably, the first helper plasmid is a pAAV-RC plasmid and the second helper plasmid is a pHelper plasmid.
Preferably, the titer of the virus suspension is not less than 1X 1012Gene copy number/ml. The beneficial effects are that: facilitating rapid and high-level expression of the recombinant protein of interest.
Preferably, the temporary non-human mammal is a healthy non-human mammal 2 to 10 days from the expected date of labor, and the temporary non-human mammal is anesthetized before the virus introduction. The beneficial effects are that: ensuring that the relevant virus can infect mammary cells, and leading the recombinant target protein to be secreted into milk for expression through a lactation process.
Further preferably, the antenatal non-human mammal is a healthy non-human mammal 2-4 days from the expected date of labour.
Further preferably, the method further comprises electrical stimulation, and after the virus introduction is completed, the stimulation device is used for performing the electrical stimulation on the mammary gland part of the parturient non-human mammal for 1-2 minutes so as to facilitate rapid transfection.
Preferably, the non-human mammal is any one of a mouse, a rat, a rabbit, a goat, a sheep, a pig and a cow.
The overexpression vector provided by the invention comprises a gene of the target protein and the adeno-associated virus vector, and is used for forming virus suspension through virus packaging; the virus suspension is used for being introduced into mammary tissue or lactiferous ducts of a non-human mammal in labor through an adeno-associated virus-mediated transduction mode to complete virus introduction, the non-human mammal expresses and secretes the recombinant target protein through mammary cells in a lactation process, and the recombinant target protein is obtained from milk of the non-human mammal in a lactation period by obtaining the milk.
The virus suspension provided by the invention is formed by virus packaging of the overexpression vector.
The overexpression vector and the virus suspension provided by the invention have the beneficial effects that: the overexpression vector comprises the gene of the target protein and the adeno-associated virus vector, so that the overexpression vector is favorable for further forming a virus suspension through virus packaging and then introducing the virus suspension into mammary tissues or mammary ducts of a non-human mammal in real time through an adeno-associated virus-mediated transduction mode to complete virus introduction, expensive instruments and equipment are not needed, the rapid and efficient production and high-level expression of the recombinant target protein can be realized through simple operation, and an unexpected technical effect is achieved.
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FIG. 1 is a flowchart of a method for producing a recombinant target protein by transduction mediated by an animal mammary gland-associated virus according to an embodiment of the present invention;
FIG. 2 is a plasmid map of an overexpression vector of example 1 of the present invention;
FIG. 3 is a plasmid map of an overexpression vector of the present invention, abbreviated pAAV-BCN-BChE;
FIG. 4 is a plasmid map of an overexpression vector of the present invention, abbreviated pAAV-CMV-HRP;
FIG. 5a is a photograph showing the expression of recombinant human CB1 by SDS-PAGE silver staining of a sample of animal milk according to example 1 of the present invention;
FIG. 5b is a photograph showing the expression of recombinant human CB1 by Western blotting of three samples of MI6, MI7 and MI8 shown in FIG. 5 a;
FIG. 6a is a photograph showing the expression of recombinant human A2b by SDS-PAGE silver staining of a milk sample according to example 2 of the present invention;
FIG. 6b is a photograph showing the expression of recombinant human A2b by Western blotting of 12 samples shown in FIG. 6 a;
FIG. 6c is a graph showing the protein-ligand binding affinity of the animal milk sample of MI15 numbered as shown in FIGS. 6a and 6b positive for recombinant human A2b following a conventional isotopic label ligand binding assay after primary purification of casein;
FIG. 7a is a photograph showing the expression of recombinant human ACE2 by SDS-PAGE silver staining of a sample of animal milk according to example 3 of the present invention;
FIG. 7b is a photograph showing protein bands by SDS-PAGE silver staining of an eluate obtained after protein purification of the sample No. MI25 from example 3 using Anti-His resin;
FIG. 7c is a photograph showing a protein band in an eluate obtained after protein purification of the sample No. MI25 of example 3 using an Anti-His resin by Western blotting;
FIG. 8a is a photograph showing the expression of recombinant human CXCR4 by SDS-PAGE silver staining of a sample of animal milk according to example 4 of the present invention;
figure 8b is a photograph showing the expression of recombinant human CXCR4 by western blot of the sample numbered MI30 shown in figure 8 a;
FIG. 8c is a photograph showing protein bands by SDS-PAGE silver staining of an eluate obtained after protein purification of the sample No. MI30 of example 4 by Anti-Flag resin;
FIG. 8d is a photograph showing protein bands of an eluate obtained after protein purification of the sample No. MI30 of example 4 using an Anti-Flag resin by Western blotting.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The use of "including" and "comprising" and similar words, as used herein, is intended to mean that the element or item preceding the word comprises the element or item listed after the word and its equivalents, without excluding other elements or items.
One of the major limitations in the prior art for the production of transgenic protein pharmaceuticals by adenovirus vector-mediated expression in animal mammary glands is that adenovirus vectors are known to support only transient protein expression, which can last only about a week, and is generally thought to be caused by the induction of a strong immune response in the animal body to the adenovirus. This immune response may also prevent the re-expression of the same recombinant protein in the mammary gland of the animal.
Considering that the seasonal lactation period of large animals such as goats and cattle is 10 months or more, the lack of persistence of recombinant protein expression production is a major disadvantage of adenovirus vector-mediated production of transgenic protein drugs by animal mammary gland expression.
In view of the problems of the prior art, the embodiments of the present invention provide a method for producing a target protein by animal mammary gland-associated virus-mediated transduction to achieve rapid and high-level expression of the target protein.
FIG. 1 is a flowchart of a method for producing a recombinant target protein by transduction mediated by an animal mammary gland-associated virus according to an embodiment of the present invention. Referring to fig. 1, the method for producing a recombinant target protein by an animal mammary gland-associated virus-mediated transduction according to an embodiment of the present invention includes:
s1: providing an overexpression vector comprising a gene for a protein of interest and an adeno-associated viral vector, and viral packaging the overexpression vector to form a viral suspension;
s2: introducing the virus suspension into mammary tissue or mammary duct of a temporary non-human mammal by means of adeno-associated virus-mediated transduction to complete virus introduction, and allowing the non-human mammal to express and secrete the recombinant target protein through mammary cells in a lactation process;
s3: obtaining milk secreted by said non-human mammal during the lactation period, and obtaining said recombinant protein of interest from said milk.
In the prior art, the period of protein expression by an animal mammary gland bioreactor or an adenovirus vector is long, in the embodiment of the invention, an overexpression vector comprises a target protein gene and an adeno-associated virus vector, the overexpression vector is subjected to virus packaging, and then the obtained virus suspension is introduced into mammary gland tissues or mammary ducts of a non-human mammal in real time in an adeno-associated virus-mediated transduction mode to complete virus introduction, so that the applicant finds that the adeno-associated virus vector-mediated recombinant target protein is continuously expressed in animal milk for at least 10-30 days, and the expression amount of the recombinant target protein is 0.3-12 g/L. In addition, the parturient female rabbits infected locally with adeno-associated virus can recover quickly and produce milk normally like other healthy parturient female rabbits. It can be seen that the adeno-associated viral vector helps to achieve rapid and high-level expression of the recombinant target protein, and achieves unexpected technical effects.
In step S1 of some embodiments of the present invention, the target protein is any one of cholinesterase, peroxidase, growth hormone, antiviral protein, virus structural protein, viroid, and membrane protein, or a fragment or derivative of any one of the above.
In embodiments 1 to 4 of the present invention, the viral vector is an adeno-associated viral vector, and the target proteins are a human CB1 receptor, a human adenosine A2b receptor, a human angiotensin-converting enzyme II receptor, and a human CXCR4 receptor, respectively. For convenience of description, abbreviated as human CB1, human A2b, human ACE2 and human CXCR4, respectively. Wherein, human CB1, human A2b and human CXCR4 all belong to membrane protein, and human ACE2 belongs to membrane protein and antiviral protein.
Specifically, a cDNA plasmid of human CB1, a cDNA plasmid of human A2b, a cDNA plasmid of human ACE2 and a cDNA plasmid of human CXCR4 are respectively cloned to the adeno-associated virus vector, and the formed overexpression vectors are respectively marked as pAAV-CAG-CB1, pAAV-CAG-A2b, pAAV-CAG-ACE2 and pAAV-CAG-CXCR 4. pAAV-CAG-CB1, pAAV-CAG-A2b, pAAV-CAG-ACE2 and pAAV-CAG-CXCR4, and the corresponding sequences are shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, respectively.
The sequence structure of the overexpression vector of the embodiment 1-4 is pAAV-CAG promoter-target protein coding sequence-WPRE-SV 40 polyadenylation signal sequence.
FIG. 2 is a plasmid map of the overexpression vector of example 1 of the present invention.
Referring to FIG. 2, the overexpression vector having the sequence structure pAAV-CAG-CB1 has a promoter which is a CAG promoter. More specifically, two ITRs at the 5 'end and the 3' end of the adeno-associated viral vector represent adeno-associated viral inverted terminal repeat sequences, CMVenh represents a CMV enhancer sequence, CAG pro represents a CAG promoter sequence, CB1 represents a coding sequence of a target protein CB1, WPRE represents a posttranscriptional regulatory sequence of the woodchuck hepatitis virus, and SV40 pA represents an SV40 polyadenylation signal sequence.
As is common knowledge in the art, the CAG promoter comprises the enhancer sequence of the CMV viral promoter and the promoter sequence of chicken beta-actin. The CAG promoter selected by the embodiment of the invention reserves the enhancer of the CMV virus, so the transcription capability is comparable to that of the CMV promoter, and the chicken beta-actin promoter provides a wider expression spectrum for the CMV promoter, thereby being beneficial to driving the high-level expression of the gene of the target protein in different mammalian vectors.
Specifically, the CAG promoter sequence is common knowledge of those skilled in the art and will not be described herein.
The sequence structures of the CAG promoters respectively possessed by pAAV-CAG-CB1, pAAV-CAG-A2b, pAAV-CAG-ACE2 and pAAV-CAG-CXCR4 in examples 2 to 4 of the present invention are consistent with the sequence structure of the CAG promoter in pAAV-CAG-CB1 in example 1, and are not repeated herein.
In some embodiments of the invention, the sequence structure of the overexpression vector is pAAV-goat beta-casein gene (BCN) promoter-target protein coding sequence-WPRE-SV 40 polyadenylation signal sequence.
In some embodiments of the present invention, the viral vector is an adeno-associated viral vector, and the target protein is human butyrylcholinesterase, abbreviated as human BChE. Human BChE belongs to cholinesterase.
Specifically, a cDNA plasmid of human BChE is cloned to the adeno-associated virus vector, the formed overexpression vector is marked as pAAV-BCN-BChE, the sequence is shown as SEQ ID No.5, and the corresponding plasmid map is shown as figure 3.
Referring to FIG. 3, pAAV-BCN-BChE has a promoter sequence of BCN promoter. Specifically, two ITRs at the 5 'end and the 3' end of the adeno-associated virus vector represent adeno-associated virus inverted terminal repeat sequences, CMV enh represents a CMV enhancer sequence, BCN pro represents a goat beta-casein gene promoter sequence, BChE represents a coding sequence of a target protein BChE, WPRE represents a woodchuck hepatitis virus post-transcriptional regulatory sequence, and SV40 pA represents an SV40 polyadenylation signal sequence.
The BCN promoter in pAAV-BCN-BChE contains enhancer sequences of CMV virus promoter, complex elements such as BCE-1, TATA box, STAT5 and NFKB and action sites of transcription and translation regulatory factors. Wherein BCE-1 is one of the most characteristic elements in the goat beta-casein gene promoter and is responsible for hormone-dependent regulation of gene expression.
As the BCN promoter sequence selected by the embodiment of the invention reserves the enhancer of the CMV virus, the transcription capability is comparable to that of the CMV promoter, and the BCN promoter provides a promoter sequence for specific expression of animal mammary glands, thereby being beneficial to driving high-level expression and secretion of target proteins in different mammary glands of mammals.
More specifically, the sequence of the BCN promoter corresponds to a sequence segment from position 2646 to position 4591 in the sequence of SEQ ID No.5, wherein the sequence of the BCN promoter comprises a CMV enhancer sequence from position 2646 to position 2895 in the sequence of SEQ ID No. 5.
Referring to FIG. 3, the base pair length of the sequence segment located between two ITRs is no more than 5 Kb. In some embodiments of the invention, the sequence segment between the two ITRs corresponds to the sequence segment from position 2457 to position 7457 in the sequence of SEQ ID No. 5.
In some embodiments of the invention, the viral vector is an adeno-associated viral vector, and the target protein is horseradish peroxidase (HRP). HRP belongs to peroxidase.
Specifically, the cDNA plasmid of HRP is cloned to the adeno-associated virus vector, and the formed overexpression vector is marked as pAAV-BCN-HRP, and the sequence is shown as SEQ ID No. 6. The sequence structure of the BCN promoter contained in the pAAV-BCN-HRP is consistent with that of the BCN promoter contained in an overexpression vector with the sequence structure of pAAV-BCN-BChE and the sequence of the promoter shown in SEQ ID No.5, and the description is omitted here. In some embodiments of the invention, the sequence structure of the overexpression vector is the pAAV-CMV promoter-target protein coding sequence-WPRE-SV 40 polyadenylation signal sequence.
In some embodiments of the invention, the viral vector is an adeno-associated viral vector and the protein of interest is HRP. The cDNA plasmid of HRP is cloned to the adeno-associated virus vector, the formed over-expression vector is marked as pAAV-CMV-HRP, the sequence is shown as SEQ ID No.7, and the plasmid map is shown as figure 4.
Referring to FIG. 4, pAAV-CMV-HRP has a CMV promoter sequence. Specifically, two ITRs at the 5 'end and the 3' end of the adeno-associated virus vector represent adeno-associated virus inverted terminal repeat sequences, CMV pro represents a CMV promoter sequence, HRP represents a coding sequence of a target protein HRP, WPRE represents a posttranscriptional regulatory sequence of woodchuck hepatitis virus, and SV40 pA represents an SV40 polyadenylation signal sequence.
As is well known in the art, the CMV promoter contains the enhancer sequence of the CMV viral promoter, and is a powerful promoter for promoting expression of eukaryotic genes, which is advantageous for driving high-level expression of genes in different mammalian vectors.
Specifically, the sequence of the CMV promoter is common knowledge of those skilled in the art and will not be described in detail herein.
More specifically, the signal peptide coding sequence secreted by the expressed recombinant target protein in the overexpression vector sequence shown as SEQ ID number 1-7 is selected from goat beta-casein gene, and can sufficiently guide the newly synthesized target protein to transfer to an animal milk secretion pathway.
The cDNA plasmid of the human CB1 receptor and the cDNA plasmid of the human adenosine A2b receptor of the embodiment of the invention are from Shanghai medicine Mingkude new medicine development company Limited; the cDNA plasmid of human ACE2 was derived from Qincheng Haizheng Biotech, Inc., Shanghai; the cDNA plasmid of the human CXCR4 receptor is from Nanjing Kinshire Biotech, Inc.; the cDNA plasmids of human BChE and HRP are from Shanghai name of the last ruler of the Xia Dynasty Mongolian biotechnology, Inc.; adeno-associated virus vectors are derived from the national Biotechnology (Shanghai) Co., Ltd.
Specifically, the process of cloning cDNA plasmids of various receptors into the adeno-associated viral vector is a routine technique for those skilled in the art, and the detailed procedures are described in Cold spring harbor Laboratory (Cold spring harbor Laboratory) in New York, USA, published in 1989 as molecular cloning: laboratory Manual, second edition, is not repeated herein.
In some embodiments of the invention, the overexpression vector comprises any one of a CAG promoter, a BCN promoter or a CMV promoter, a goat β -casein signal peptide coding sequence, a WPRE and a SV40 polyadenylation signal.
In some embodiments of the invention, 293T cells are co-transfected with the overexpression vector, the first helper plasmid and the second helper plasmid for not less than 48 hours, and the resulting transfection solution is then purified to complete the viral packaging.
In examples 1-4 of the present invention, the first helper plasmid was pAAV-RC plasmid, the second helper plasmid was pHelper plasmid, and the cotransfection time was not less than 48 hours.
Specifically, the specific methods of the co-transfection and the purification treatment are conventional technical means of those skilled in the art, and the specific operations are performed by the company yohimoto (shanghai) gmbh, which are not described herein again.
In some embodiments of the invention, the titer of the virus suspension is detected by a fluorescent quantitative PCR method, and the titer is not lower than 1 × 1012Gene copy number/ml to facilitate rapid and high level expression of the recombinant protein of interest.
In examples 1 to 4 of the present invention, the titers were all 1X 1013Gene copy number/ml.
In step S2 of some embodiments of the present invention, the parturient non-human mammal is a healthy non-human mammal 2-10 days away from the expected delivery stage to ensure that the adeno-associated virus can infect mammary cells, so that the recombinant target protein is secreted into milk for expression through the lactation process.
In some embodiments of the invention, the recombinant target protein is expressed in the milk of the animal for 10-30 days in an amount of 0.3-12 g/L.
In some embodiments of the invention, the recombinant target protein is expressed in the milk of the animal for a period of 20-30 days, and the recombinant target protein is expressed in an amount of 1-10 g/L.
In some embodiments of the invention, the non-human mammal is any one of a mouse, rat, rabbit, goat, sheep, pig and cow.
The non-human mammals used in examples 1 to 4 of the present invention are all healthy parturient female rabbits 2 to 4 days from the expected date of delivery. The healthy antenatal female rabbit comes from Shanghai Fengxian rabbit industry professional cooperative.
Specifically, after the body hair of the mammary gland part of each healthy parturient female rabbit is removed and cleaned, the nipple and the surrounding skin are disinfected by alcohol, and the virus suspension is injected into the mammary gland tissue or the mammary gland duct of the parturient non-human mammal by a medical injector to complete virus introduction. Injecting 2-4 nipples into each healthy parturient female rabbit, and disinfecting the injection part with alcohol after the injection is finished.
In some embodiments of the invention, after the introduction of the virus is completed, the stimulation device is used to electrically stimulate the mammary gland of the parturient non-human mammal for 1-2 minutes, which facilitates rapid transfection.
In some embodiments of the invention, the stimulation device is a conventional electrical pulse stimulator.
In some embodiments of the invention, after the introduction of the virus is completed, milk secreted by the non-human mammal in the lactation period is obtained, and the expression of the recombinant target protein is detected.
In embodiments 1 to 4 of the present invention, after the introduction of the virus is completed, after each healthy parturient rabbit completes the production of the baby rabbit, milk secreted by each healthy parturient rabbit is collected by a milker, and the expression of recombinant human CB1, recombinant human A2b, recombinant human ACE2, recombinant human CXCR4 and recombinant human BChE in the milk of the animal is detected by at least one of Western Blot (Western Blot) and SDS-PAGE silver staining. In examples 1-4 of the invention, all animal milk samples were initially purified by conventional casein removal after collection.
Wherein, embodiment 2 of the present invention further includes: and carrying out isotope labeling ligand combination experiments on the obtained animal milk sample containing the recombinant human A2b after conventional casein removal primary purification. The conventional casein-removing initial purity and isotope labeled ligand binding experiments are the conventional technical means of the technicians in the field, and the isotope labeled ligand binding experiments are carried out by Shanghai medicine Mingkude new drug development Limited company, which is not described herein.
Embodiment 4 of the present invention further includes: the obtained animal milk sample containing the recombinant human CXCR4 is subjected to conventional casein removal and SMA primary purification, and then is subjected to protein purification by Anti-Flag resin. Conventional casein removal, primary SMA purification and protein purification are conventional technical means of technicians in the field, and protein purification is performed by Shanghai Medicine GmbH, which is not described herein.
In example 1 of the present invention, the animal milk was subjected to 1:1000 dilution. SDS-PAGE silver staining is a routine technique used by those skilled in the art, and the detailed procedures are described in Cold spring Harbor Laboratory (Cold spring Harbor Laboratory) in New York, USA, molecular cloning: laboratory Manual, second edition, is not repeated herein.
FIG. 5a is a photograph showing the expression of recombinant human CB1 by SDS-PAGE silver staining of a sample of animal milk according to example 1 of the present invention.
Referring to fig. 5a, the milk sample from the first animal secreted by the healthy parturient female rabbit of example 1 was numbered MI1, the milk was collected 4 days apart and numbered MI2, and then every 4 days apart and numbered MI3, MI4 and MI5 in that order.
The milk samples from the animals collected for the first time, the milk samples collected for the second time and the milk samples collected for the third time were subjected to casein precipitation and WGA kit purification, respectively, and the formed milk samples were numbered MI6, MI7 and MI8 in this order. The WGA kit was from Vector Labs, USA.
M1 is the protein size-labeled sample of example 1 (from san assist, Biotech, Inc. in Shanghai), and N1 and N2 are negative controls. Specifically, the negative control is a sample of milk from an animal positive for rBChE.
As can be seen from the white asterisk marks in FIG. 5a, the five samples MI3, MI4, MI5, MI7, and MI8 showed about 130K dimer-positive faint bands, while the 65K monomer band may be hidden in the 50-90K strong protein band. As can be seen from the black asterisk markers in FIG. 5a, both N1 and N2 showed approximately 90K rBChE bands, with expression levels of approximately 10-12 g/l. Since MI5 was a sample collected on day 20 after the first lactation of the animal from a healthy pre-parturient female rabbit, it was found that recombinant human CB1 of example 1 was expressed in the animal's milk for a sustained period of up to 20 days.
In example 1 of the present invention, before detection by western blotting, the animal milk was subjected to 1: 300, respectively. Specifically, the indirect detection is performed by using a mouse anti-CB 1 monoclonal antibody as a primary antibody and using a goat anti-mouse-HRP as a secondary antibody, and the specific procedures of the indirect detection are routine technical means of those skilled in the art, and refer to the molecular cloning published in 1989 by Cold spring harbor Laboratory (Cold spring harbor Laboratory) in New York, USA: laboratory Manual, second edition, is not repeated herein.
FIG. 5b shows the expression of recombinant human CB1 by Western blotting of three samples MI6, MI7 and MI8 shown in FIG. 5 a. As can be seen from the white asterisk marks shown in fig. 5b, MI7 and MI8 showed about 130K dimer-positive faint bands, while all three samples showed about 65K protein monomer bands.
In example 2 of the present invention, the animal milk was subjected to 1:1000 dilution. For a specific procedure of SDS-PAGE silver staining, see example 1.
FIG. 6a is a photograph showing the expression of recombinant human A2b by SDS-PAGE silver staining of a sample of animal milk according to example 2 of the present invention.
Referring to fig. 6a, the milk sample of the first time expressed animal of a healthy parturient female rabbit of example 2 was numbered MI9, the milk was collected every 4 days and numbered MI10, and then the milk was collected every 4 days and numbered MI11, MI12 and MI13 in that order; meanwhile, the milk of another healthy parturient female rabbit is respectively collected at corresponding time and numbered as MI14, MI15, MI16, MI17 and MI18 in sequence.
M2 is a negative control for 10 samples of the protein size marker sample of example 2 (from san assist, Biotech, Inc. in Shanghai), N3 from MI9 to MI13 and MI14 to MI 18. The negative control was a sample of milk from an animal positive for rBChE.
From the black asterisk markers in FIG. 6a, MI9, MI14, and MI15 showed about 40K positive bands, and from the white asterisk markers in FIG. 6a, N2 showed about 90K rBChE bands with expression levels of about 10-12 g/L.
In example 2 of the present invention, the animal milk was subjected to 1: dilution of 500. Specifically, indirect detection was performed using rabbit anti-A2 b polyclonal antibody as the primary antibody and goat anti-rabbit-HRP as the secondary antibody. For a specific detection method, see example 1.
FIG. 6b is a photograph showing the expression of recombinant human A2b by Western blotting of 12 samples shown in FIG. 6 a. As can be seen from the black asterisk markers shown in fig. 6b, MI9, MI14, and MI15 showed about 40K positive bands.
In example 2, fig. 6c is a graph of protein-ligand binding affinity (radioelement radioactivity determination intensity) of the animal milk sample numbered MI15 shown in fig. 6a and 6b as positive for recombinant human A2b expression, for an isotopically labeled ligand binding experiment following conventional casein removal. The negative sample is a milk sample N4 collected from female rabbits after the same period of healthy production, the positive control is a recombinant human A2b overexpression cell lineage membrane structure provided by Shanghai medicine Mingkude New drug development Limited company, and the isotope labeled ligand is3H-CGS 15943. Referring to fig. 6c, the affinity of protein-isotope labeled ligand of MI15 is equivalent to that of the positive control, and has good activity, thus proving that the recombinant human A2b receptor membrane structure derived from animal milk can effectively replace the recombinant human A2b overexpression cell lineage membrane structure which is expensive in market at present. It is worth noting that the experimental result is shown for the first time at home and abroad.
In example 3 of the present invention, the animal milk was subjected to 1:1000 dilution. For SDS-PAGE silver staining see example 1.
FIG. 7a is a photograph showing the expression of recombinant human ACE2 by SDS-PAGE silver staining of a sample of animal milk according to example 3 of the present invention.
Referring to fig. 7a, the milk sample of the first time expressed animal of a healthy parturient female rabbit of example 3 was numbered MI19, the milk was collected every 4 days and numbered MI20, and then the milk was collected every 4 days and numbered MI21, MI22 and MI23 in that order; meanwhile, the milk sample secreted for the first time by another healthy parturient female rabbit was numbered MI24, the milk was collected at 4-day intervals and numbered MI25, and then the milk was collected at 4-day intervals and numbered MI26, MI27 and MI28 in this order.
M3 is a negative control for 10 samples of the protein size marker sample of example 3 (from san assist, Biotech, Inc. in Shanghai), N5 is MI19 to MI23 and MI24 to MI 28. The negative control was a sample of milk from an animal positive for rBChE.
As can be seen from the white asterisk markers in FIG. 7a, 7 samples of MI19, MI20, and MI24 to MI28 showed about 95K positive bands, and N3 showed about 90K rBChE bands with expression levels of about 10-12 g/L, as seen from the black asterisk markers in FIG. 7 a. Using the rBChE band of N3 as a reference, it was confirmed that recombinant human ACE2 was expressed in 1-10g/L for 20-30 days in 7 samples of MI19, MI20 and MI 24-MI 28.
FIG. 7b is a photograph showing protein bands by SDS-PAGE silver staining of eluates obtained by protein-purifying the MI25 sample and the negative MI5 sample N5 with Anti-Flag resin shown in FIG. 7 a. The MI25 eluate showed an approximately 95K positive band, which was not present in the N5 eluate.
FIG. 7c is a photograph showing the expression of recombinant human ACE2 by Western blotting of eluates obtained by protein-purifying the sample No. MI25 of example 3 and the negative sample N5 with Anti-Flag resin. The MI25 eluate showed an approximately 95K positive band, which was not present in the N5 eluate. In the experiment, a mouse anti-ACE 2 monoclonal antibody is used as a primary antibody, and goat anti-mouse-HRP is used as a secondary antibody for indirect detection. For a specific detection method, see example 1.
In example 4 of the present invention, the animal milk was diluted with the same conventional PBS buffer as in example 1 before detection by SDS-PAGE silver staining. For SDS-PAGE silver staining, see example 1.
FIG. 8a is a photograph showing the expression of recombinant human CXCR4 by SDS-PAGE silver staining of a sample of animal milk according to example 4 of the present invention.
Referring to fig. 8a, the milk of the first-time lactating animal of a healthy parturient female rabbit of example 4 was treated with conventional PBS buffer 1: the sample obtained after 500 dilutions was assigned the number MI29, and the sample obtained after 1:1000 dilutions was assigned the number MI 30. N6 and N7 are negative controls for MI29 and MI30, respectively. The negative control was a sample of milk from an animal positive for rBChE.
As can be seen from the black asterisk markers in fig. 8a, MI29 and MI30 showed about 37K positive bands, which were not shown in the N6 and N7 negative control samples.
In example 4 of the present invention, the animal milk was subjected to 1:1000 dilution. Specifically, rabbit anti-CXCR 4 polyclonal antibody was used as the primary antibody, and goat anti-rabbit-HRP was used as the secondary antibody for indirect detection. For a specific detection method, see example 1.
Figure 8b is a photograph showing the expression of recombinant human CXCR4 by western blot of the sample numbered MI30 shown in figure 8 a. As shown in fig. 8b, MI30 showed an approximately 37K positive band, which was not shown in the N7 negative control sample; the band of about 60K protein is background protein, which indicates that the amount of protein loaded in each sample is the same.
FIG. 8c is a photograph showing protein bands by SDS-PAGE silver staining of an eluate obtained after protein purification of the sample No. MI30 from example 4 by Anti-Flag resin. From the black asterisk markers in fig. 8c, the three eluents numbered MI30a, MI30b, and MI30c, all showed about 37K positive bands, while the about 80K band was probably recombinant human CXCR4 dimer.
FIG. 8d is a photograph showing a protein band in an eluate obtained by protein-purifying the sample No. MI30 shown in FIG. 8c using Anti-Flag resin, which was subjected to Western blotting. As shown in fig. 8d, the three eluates numbered respectively for MI30a, MI30b, and MI30c of fig. 8c all showed about 37K positive bands, while the about 80K band was likely to be recombinant human CXCR4 dimer. In the experiment, rabbit anti-CXCR 4 polyclonal antibody is used as a primary antibody, and goat anti-rabbit-HRP is used as a secondary antibody for indirect detection. For a specific detection method, see example 1.
Although the embodiments of the present invention have been described in detail hereinabove, it is apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it is to be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention as described herein is capable of other embodiments and of being practiced or of being carried out in various ways.
Sequence listing
<110> Shanghai name of the last ruler of the Xia Dynasty Mongolian Biotechnology Co., Ltd
<120> method for producing recombinant target protein, overexpression vector and virus suspension
<130>2020.09.14
<150>HRCN20L31041-Y
<151>2020-09-14
<160>7
<170>SIPOSequenceListing 1.0
<210>1
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acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180
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gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
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gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600
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tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900
aatcaatcta aagtatatatgagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1020
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260
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acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1920
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atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2100
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caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2460
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gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 3120
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ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 3540
aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgctagc ataacgcgta 3600
ctggtaccgc caccatgaag gtcctcatcc ttgcctgtct ggtggctctg gcccttgcaa 3660
agtcgatcct agatggcctt gcagatacca ccttccgcac catcaccact gacctcctgt 3720
acgtgggctc aaatgacatt cagtacgaag acatcaaagg tgacatggca tccaaattag 3780
ggtacttccc acagaaattc cctttaactt cctttagggg aagtcccttc caagagaaga 3840
tgactgcggg agacaacccc cagctagtcc cagcagacca ggtgaacatt acagaatttt 3900
acaacaagtc tctctcgtcc ttcaaggaga atgaggagaa catccagtgt ggggagaact 3960
tcatggacat agagtgtttc atggtcctga accccagcca gcagctggcc attgcagtcc 4020
tgtccctcac gctgggcacc ttcacggtcc tggagaacct cctggtgctg tgcgtcatcc 4080
tccactcccg cagcctccgc tgcaggcctt cctaccactt catcggcagc ctggcggtgg 4140
cagacctcct ggggagtgtc atttttgtct acagcttcat tgacttccac gtgttccacc 4200
gcaaagatag ccgcaacgtg tttctgttca aactgggtgg ggtcacggcc tccttcactg 4260
cctccgtggg cagcctgttc ctcacagcca tcgacaggta catatccatt cacaggcccc 4320
tggcctataa gaggattgtc accaggccca aggccgtggt ggcgttttgc ctgatgtgga 4380
ccatagccat tgtgatcgcc gtgctgcctc tcctgggctg gaactgcgag aaactgcaat 4440
ctgtttgctc agacattttc ccacacattg atgaaaccta cctgatgttc tggatcgggg 4500
tcaccagcgt actgcttctg ttcatcgtgt atgcgtacat gtatattctc tggaaggctc 4560
acagccacgc cgtccgcatg attcagcgtg gcacccagaa gagcatcatc atccacacgt 4620
ctgaggatgg gaaggtacag gtgacccggc cagaccaagc ccgcatggac attaggttag 4680
ccaagaccct ggtcctgatc ctggtggtgt tgatcatctg ctggggccct ctgcttgcaa 4740
tcatggtgta tgatgtcttt gggaagatga acaagctcat taagacggtg tttgcattct 4800
gcagtatgct ctgcctgctg aactccaccg tgaaccccat catctatgct ctgaggagta 4860
aggacctgcg acacgctttc cggagcatgt ttccctcttg tgaaggcact gcgcagcctc 4920
tggataacag catgggggac tcggactgcc tgcacaaaca cgcaaacaat gcagccagtg 4980
ttcacagggc cgcagaaagc tgcatcaaga gcacggtcaa gattgccaag gtaaccatgt 5040
ctgtgtccac agacacgtct gccgaggctc tgtgagaatt cgatatatca agcttatcga 5100
taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 5160
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 5220
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 5280
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 5340
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 5400
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 5460
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 5520
cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 5580
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 5640
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 5700
tcgacccggg cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac 5760
cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 5820
atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 5880
gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 5940
tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt 6000
taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 6060
gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 6120
cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg cggggagagg 6180
cggtttgcgt attgggcgct6200
<210>2
<211>5780
<212>DNA
<213>Artificial Sequence
<400>2
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 540
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaatgaagtttta 900
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1020
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1860
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1920
aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1980
aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2040
atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2100
cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2160
accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2220
taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2280
agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2340
cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2400
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2460
gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2520
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2580
gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2640
gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 2700
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 2760
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 2820
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 2880
tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 2940
ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 3000
gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg 3060
gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 3120
ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 3180
tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 3240
ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 3300
gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 3360
ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg 3420
gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 3480
ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 3540
aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgctagc ataacgcgta 3600
ctggtaccgc caccatgaag gtcctcatcc ttgcctgtct ggtggctctg gcccttgcac 3660
tgctggagac acaggacgcg ctgtacgtgg cgctggagct ggtcatcgcc gcgctttcgg 3720
tggcgggcaa cgtgctggtg tgcgccgcgg tgggcacggc gaacactctg cagacgccca 3780
ccaactactt cctggtgtcc ctggctgcgg ccgacgtggc cgtggggctc ttcgccatcc 3840
cctttgccat caccatcagc ctgggcttct gcactgactt ctacggctgc ctcttcctcg 3900
cctgcttcgt gctggtgctc acgcagagct ccatcttcag ccttctggcc gtggcagtcg 3960
acagatacct ggccatctgt gtcccgctca ggtataaaag tttggtcacg gggacccgag 4020
caagaggggt cattgctgtc ctctgggtcc ttgcctttgg catcggattg actccattcc 4080
tggggtggaa cagtaaagac agtgccacca acaactgcac agaaccctgg gatggaacca 4140
cgaatgaaag ctgctgcctt gtgaagtgtc tctttgagaa tgtggtcccc atgagctaca 4200
tggtatattt caatttcttt gggtgtgttc tgcccccact gcttataatg ctggtgatct 4260
acattaagat cttcctggtg gcctgcaggc agcttcagcg cactgagctg atggaccact 4320
cgaggaccac cctccagcgg gagatccatg cagccaagtc actggccatg attgtgggga 4380
tttttgccct gtgctggtta cctgtgcatg ctgttaactg tgtcactctt ttccagccag 4440
ctcagggtaa aaataagccc aagtgggcaa tgaatatggc cattcttctg tcacatgcca 4500
attcagttgt caatcccatt gtctatgctt accggaaccg agacttccgc tacacttttc 4560
acaaaattat ctccaggtat cttctctgcc aagcagatgt caagagtggg aatggtcagg 4620
ctggggtaca gcctgctctc ggtgtgggcc tatgagaatt cgatatatca agcttatcga 4680
taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 4740
tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 4800
tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 4860
gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 4920
tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 4980
tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 5040
gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 5100
cgcctgtgttgccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 5160
caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 5220
tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg 5280
tcgacccggg cggccgcttc gagcagacat gataagatac attgatgagt ttggacaaac 5340
cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt 5400
atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat 5460
gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg 5520
tggtaaaatc gataaggatc ttcctagagc atggctacgt agataagtag catggcgggt 5580
taatcattaa ctacaaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc 5640
gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg 5700
cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc ggccaacgcg cggggagagg 5760
cggtttgcgt attgggcgct 5780
<210>3
<211>7190
<212>DNA
<213>Artificial Sequence
<400>3
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180
ttttccataggctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 540
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1020
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1860
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1920
aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1980
aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2040
atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2100
cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2160
accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2220
taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2280
agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2340
cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2400
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2460
gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2520
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2580
gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2640
gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 2700
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 2760
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 2820
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 2880
tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 2940
ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 3000
gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg 3060
gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 3120
ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 3180
tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 3240
ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 3300
gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 3360
ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg 3420
gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 3480
ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 3540
aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgctagc ataacgcgta 3600
ctggtaccgc caccatgaag gtcctcatcc ttgcctgtct ggtggctctg gcccttgcac 3660
accatcacca ccatcacgag aacctgtact tccaaggcca gtccaccatt gaggaacagg 3720
ccaagacatt tttggacaag tttaaccacg aagccgaaga cctgttctat caaagttcac 3780
ttgcttcttg gaattataac accaatatta ctgaagagaa tgtccaaaac atgaataatg 3840
ctggggacaa atggtctgcc tttttaaagg aacagtccac acttgcccaa atgtatccac 3900
tacaagaaat tcagaatctc acagtcaagc ttcagctgca ggctcttcag caaaatgggt 3960
cttcagtgct ctcagaagac aagagcaaac ggttgaacac aattctaaat acaatgagca 4020
ccatctacag tactggaaaa gtttgtaacc cagataatcc acaagaatgc ttattacttg 4080
aaccaggttt gaatgaaata atggcaaaca gtttagacta caatgagagg ctctgggctt 4140
gggaaagctg gagatctgag gtcggcaagc agctgaggcc attatatgaa gagtatgtgg 4200
tcttgaaaaa tgagatggca agagcaaatc attatgagga ctatggggat tattggagag 4260
gagactatga agtaaatggg gtagatggct atgactacag ccgcggccag ttgattgaag 4320
atgtggaaca tacctttgaa gagattaaac cattatatga acatcttcat gcctatgtga 4380
gggcaaagtt gatgaatgcc tatccttcct atatcagtcc aattggatgc ctccctgctc 4440
atttgcttgg tgatatgtgg ggtagatttt ggacaaatct gtactctttg acagttccct 4500
ttggacagaa accaaacata gatgttactg atgcaatggt ggaccaggcc tgggatgcac 4560
agagaatatt caaggaggcc gagaagttct ttgtatctgt tggtcttcct aatatgactc 4620
aaggattctg ggaaaattcc atgctaacgg acccaggaaa tgttcagaaa gcagtctgcc 4680
atcccacagc ttgggacctg gggaagggcg acttcaggat ccttatgtgc acaaaggtga 4740
caatggacga cttcctgaca gctcatcatg agatggggca tatccagtat gatatggcat 4800
atgctgcaca accttttctg ctaagaaatg gagctaatga aggattccat gaagctgttg 4860
gggaaatcat gtcactttct gcagccacac ctaagcattt aaaatccatt ggtcttctgt 4920
cacccgattt tcaagaagac aatgaaacag aaataaactt cctgctcaaa caagcactca 4980
cgattgttgg gactctgcca tttacttaca tgttagagaa gtggaggtgg atggtcttta 5040
aaggggaaat tcccaaagac cagtggatga aaaagtggtg ggagatgaag cgagagatag 5100
ttggggtggt ggaacctgtg ccccatgatg aaacatactg tgaccccgca tctctgttcc 5160
atgtttctaa tgattactca ttcattcgat attacacaag gaccctttac caattccagt 5220
ttcaagaagc actttgtcaa gcagctaaac atgaaggccc tctgcacaaa tgtgacatct 5280
caaactctac agaagctgga cagaaactgt tcaatatgct gaggcttgga aaatcagaac 5340
cctggaccct agcattggaa aatgttgtag gagcaaagaa catgaatgta aggccactgc 5400
tcaactactt tgagccctta tttacctggc tgaaagacca gaacaagaat tcttttgtgg 5460
gatggagtac cgactggagt ccatatgcag accaaagcat caaagtgagg ataagcctaa 5520
aatcagctct tggagataaa gcatatgaat ggaacgacaa tgaaatgtac ctgttccgat 5580
catctgttgc atatgctatg aggcagtact ttttaaaagt aaaaaatcag atgattcttt 5640
ttggggagga ggatgtgcga gtggctaatt tgaaaccaag aatctccttt aatttctttg 5700
tcactgcacc taaaaatgtg tctgatatca ttcctagaac tgaagttgaa aaggccatca 5760
ggatgtcccg gagccgtatc aatgatgctt tccgtctgaa tgacaacagc ctagagtttc 5820
tggggataca gccaacactt ggacctccta accagccccc tgtttccata tggctgattg 5880
tttttggagt tgtgatggga gtgatagtgg ttggcattgt catcctgatc ttcactggga 5940
tcagagatcg gaagaagaaa aataaagcaa gaagtggaga aaatccttat gcctccatcg 6000
atattagcaa aggagaaaat aatccaggat tccaaaacac tgatgatgtt cagacctcct 6060
tttaggaatt cgatatatca agcttatcga taatcaacct ctggattaca aaatttgtga 6120
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 6180
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 6240
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 6300
gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 6360
cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 6420
ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 6480
ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg 6540
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 6600
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 6660
cctttgggcc gcctccccgc atcgataccg tcgacccggg cggccgcttc gagcagacat 6720
gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt 6780
tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 6840
agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt 6900
tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc ttcctagagc 6960
atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga 7020
tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg 7080
tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca 7140
ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 7190
<210>4
<211>5864
<212>DNA
<213>Artificial Sequence
<400>4
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 540
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1020
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1860
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1920
aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1980
aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2040
atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2100
cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2160
accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2220
taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2280
agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2340
cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2400
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2460
gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2520
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2580
gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2640
gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 2700
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 2760
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 2820
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 2880
tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc 2940
ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg 3000
gggcgggggg gggggggggg cgcgcgccag gcggggcggg gcggggcgag gggcggggcg 3060
gggcgaggcg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt 3120
ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag 3180
tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc 3240
ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg 3300
gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc 3360
ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg 3420
gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 3480
ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 3540
aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgctagc ataacgcgta 3600
ctggtaccgc caccatgaag gtcctcatcc ttgcctgtct ggtggctctg gcccttgcag 3660
aaggaatctc tatctacacc tcagacaact acaccgaaga aatggggagc ggcgactacg 3720
actcaatgaa agaaccctgt tttcgcgagg aaaacgccaa tttcaacaag atctttctgc 3780
ccacaatcta cagcatcatt ttcctcactg ggatcgtggg caatggactg gtcatcctgg 3840
tcatgggtta ccagaagaaa ctgcgatcta tgactgacaa gtataggctc cacctgagtg 3900
tcgcagatct gctcttcgtg atcaccctgc ctttttgggc cgttgacgcc gtggctaatt 3960
ggtatttcgg gaactttctg tgcaaggccg tccacgtgat ctacacagtc aatctgtata 4020
gctccgtgct catcctggct ttcatttcac tcgatagata cctggcaatt gtgcatgcca 4080
ctaacagcca gaggccaaga aagctgctcg ctgagaaagt ggtctatgtc ggcgtgtgga 4140
tccccgcact gctcctgacc atccctgact tcatttttgc caatgtgagt gaagctgacg 4200
atcggtacat ctgtgaccgc ttttatccaa acgatctgtg ggtggtcgtg ttccagtttc 4260
agcacatcat ggtcgggctc attctgcccg gtattgtgat cctgagctgc tactgtatca 4320
ttatctccaa gctgtcacac agcaaaggcc atcagaagag aaaagctctg aagaccacag 4380
tgattctcat cctggctttc tttgcatgct ggctgcctta ctacatcggc atcagtatcg 4440
attcattcat cctcctggag attatcaagc agggctgtga gtttgaaaat accgtgcaca 4500
aatggatcag cattacagaa gcactggcct tctttcattg ctgtctcaac ccaatcctgt 4560
acgctttcct cggagcaaag tttaaaactt ccgcccagca tgctctgacc tccgtgtctc 4620
ggggctctag tctcaagatc ctgtctaaag gaaagagagg tggacattct agtgtgagta 4680
ccgaaagtga gagtagtagt tttcacagta gcgactacaa ggatgacgac gacaagtagg 4740
aattcgatat atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt 4800
gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 4860
tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 4920
gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 4980
tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 5040
cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 5100
ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 5160
atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc 5220
cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 5280
ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 5340
ggccgcctcc ccgcatcgat accgtcgacc cgggcggccg cttcgagcag acatgataag 5400
atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg 5460
tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa 5520
caacaacaat tgcattcatt ttatgtttca ggttcagggg gagatgtggg aggtttttta 5580
aagcaagtaa aacctctaca aatgtggtaa aatcgataag gatcttccta gagcatggct 5640
acgtagataa gtagcatggc gggttaatca ttaactacaa ggaaccccta gtgatggagt 5700
tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc 5760
gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcattaatg 5820
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgct 5864
<210>5
<211>7511
<212>DNA
<213>Artificial Sequence
<400>5
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 540
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1020
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1860
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1920
aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1980
aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2040
atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2100
cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2160
accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2220
taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2280
agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2340
cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2400
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2460
gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2520
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2580
gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2640
gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt2700
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 2760
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 2820
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 2880
tcatcgctat taccatggta attggtgaga gacagtcata aggaaatgct gtgtttattg 2940
cacaatatgt aaagcatctt cctgagaaaa taaaagggaa atgttgaatg ggaaggatat 3000
gctttctttt gtattccttt tctgagaaat caaacttttt cacctgtggc cttggccacc 3060
aaaagctaac aaataaaggc atatgaagta gccaaggcct tttctagtta tatctataac 3120
actgagttca tttcatcatt tattttcctg acttcctcct gggtccatat gagcagtctt 3180
agaatgaata ttagctgaat aatccaaata catagtagat gttgatttgg gttttctaag 3240
caatccaaga cttgtatgac agtaagatgt attaccatcc aacacacatc tcagcatgat 3300
ataaatgcaa ggtatattgt gaagaaaaat ttttaattat gtcaaagtgc ttactttaga 3360
aggtcatcta tctgtcccaa agctgtgaat atatatattg aaggtaatga atagatgaag 3420
ctaaccttgt aaaaatgagt agtgtgaaat acaactacaa ttatgaacat ctgtcactaa 3480
agaggcaaag aaacttgaag attgcttttg caaatgggct cctattaata aaaagtactt 3540
ttgaggtctg gctcagactc tattgtagta cttagggtaa gaccctcctc ctgtatgggc 3600
tttcattttc tttcttgctt ccctcatttg cccttccatg aatactagct gataaacatt 3660
gactataaaa gatatgaggc caaacttgag ctgtcccatt ttaataaatc tgtataaata 3720
atatttgttc tacaaaagta ttatctaaat aaatgttact ttctgtctta aaatccctca 3780
acaaatcccc actatctaga gaataagatt gacattccct ggaatcacag catgctttgt 3840
ctgccattat ctgacccctt tctctttctc tcttctcacc tccatctact cctttttcct 3900
tgcaattcat gacccagatt cactgtttga tttggcttgc atgtgtgtgt gctgagttgc 3960
gtctgactgt tatcaacccc atgaatgata gtccaccagg ctctactgtc catgaaattt 4020
tccagtcaag aatactggag tggattgcat ttcctactcc atttgattaa tttagtgact 4080
tttaaatttc tttttccata ttcgggagcc tattcttcct ttttagtcta tactctcttc 4140
actcttcagg tctaaggtat catcgtgtgc ttgttagctt gttactttct ccattatagc 4200
ttaagcacta acaactgttc aggttggcat gaaattgtgt tctttgtgtg gcctgtatat 4260
ttctgttgtg tattagaatt taccccaaga tctcaaagac ccactgaata ctaaagagac 4320
ctcattgtgg ttacaataat ttggggactg ggccaaaact accgtgcatc ccagccaaga 4380
tctgtagcta ctggacaatt tcatttcctt tatcagattg tgagttattc ctgttaaaat 4440
gctccccaga atttctgggg acagaaaaat aggaagaatt catttcctaa tcatgcagat 4500
ttctaggaat tcaaatccac tgttggtttt atttcaaacc acaaaattag catgccatta 4560
aatactatat ataaacagcc actaaatcag atcattctcg aggagagcac cggtagccat 4620
gaaggtcctc atccttgcct gtctggtggc tctggccctt gcagaagatg acatcataat 4680
tgcaacaaag aatggaaaag tcagagggat gaacttgaca gtttttggtg gcacggtaac 4740
agcctttctt ggaattccct atgcacagcc acctcttggt agacttcgat tcaaaaagcc 4800
acagtctctg accaagtggt ctgatatttg gaatgccaca aaatatgcaa attcttgctg 4860
tcagaacata gatcaaagtt ttccaggctt ccatggatca gagatgtgga acccaaacac 4920
tgacctcagt gaagactgtt tatatctaaa tgtatggatt ccagcaccta aaccaaaaaa 4980
tgccactgta ttgatatgga tttatggtgg tggttttcaa actggaacat catctttaca 5040
tgtttatgat ggcaagtttc tggctcgggt tgaaagagtt attgtagtgt caatgaacta 5100
tagggtgggt gccctaggat tcttagcttt gccaggaaat cctgaggctc cagggaacat 5160
gggtttattt gatcaacagt tggctcttca gtgggttcaa aaaaatatag cagcctttgg 5220
tggaaatcct aaaagtgtaa ctctctttgg agaaagtgca ggagcagctt cagttagcct 5280
gcatttgctt tctcctggaa gccattcatt gttcaccaga gccattctgc aaagtggatc 5340
ctttaatgct ccttgggcgg taacatctct ttatgaagct aggaacagaa cgttgaactt 5400
agctaaattg actggttgct ctagagagaa tgagactgaa ataatcaagt gtcttagaaa 5460
taaagatccc caagaaattc ttctgaatga agcatttgtt gtcccctatg ggactccttt 5520
gtcagtaaac tttggtccga ccgtggatgg tgattttctc actgacatgc cagacatatt 5580
acttgaactt ggacaattta aaaaaaccca gattttggtg ggtgttaata aagatgaagg 5640
gacagctttt ttagtctatg gtgctcctgg cttcagcaaa gataacaata gtatcataac 5700
tagaaaagaa tttcaggaag gtttaaaaat attttttcca ggagtgagtg agtttggaaa 5760
ggaatccatc ctttttcatt acacagactg ggtagatgat cagagacctg aaaactaccg 5820
tgaggccttg ggtgatgttg ttggggatta taatttcata tgccctgcct tggagttcac 5880
caagaagttc tcagaatggg gaaataatgc ctttttctac tattttgaac accgatcctc 5940
caaacttccg tggccagaat ggatgggagt gatgcatggc tatgaaattg aatttgtctt 6000
tggtttacct ctggaaagaa gagataatta cacaaaagcc gaggaaattt tgagtagatc 6060
catagtgaaa cggtgggcaa attttgcaaa atatgggaat ccaaatgaga ctcagaacaa 6120
tagcacaagc tggcctgtct tcaaaagcac tgaacaaaaa tatctaacct tgaatacaga 6180
gtcaacaaga ataatgacga aactacgtgc tcaacaatgt cgattctgga catcattttt 6240
tccaaaagtc ttggaaatga caggaaatat tgatgaagca gaatgggagt ggaaagcagg 6300
attccatcgc tggaacaatt acatgatgga ctggaaaaat caatttaacg attacactag 6360
caagaaagaa agttgtgtgg gtctctaaga attcgatatc aagcttatcg ataatcaacc 6420
tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 6480
gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 6540
cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 6600
tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 6660
cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 6720
ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 6780
tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 6840
tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 6900
ggaccttcct tcccgcggcctgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 6960
ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacccgg 7020
gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag 7080
aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac 7140
cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt 7200
tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtaaaat 7260
cgataaggat cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta 7320
actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 7380
ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 7440
gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 7500
tattgggcgc t 7511
<210>6
<211>6733
<212>DNA
<213>Artificial Sequence
<400>6
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240
ggcgaaaccc gacaggactataaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 540
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1020
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1860
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1920
aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1980
aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2040
atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2100
cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2160
accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2220
taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2280
agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2340
cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2400
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2460
gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2520
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2580
gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2640
gaagagtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt 2700
tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag 2760
tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc 2820
attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag 2880
tcatcgctat taccatggta ttggtgagag acagtcataa ggaaatgctg tgtttattgc 2940
acaatatgta aagcatcttc ctgagaaaat aaaagggaaa tgttgaatgg gaaggatatg 3000
ctttcttttg tattcctttt ctgagaaatc agactttttc accttggcct tggccaccaa 3060
aagctaacaa ataaaggcat atgaagtagc caaggccttt tctagttata tctatgacac 3120
tgagttcatt tcatcattta ttttcctgac ttcctcctgg gtccatatga gcagtcttag 3180
aatgaatatt agctgaataa tccaaataca tagtagatgt tgatttgggt tttctaagca 3240
atccaagact tgtatgacag taagatgtat taccatccaa cacacatctc agcatgatat 3300
aaatgcaagg tatattgtga agaaaaattt ttaattatgt caaagtgctt actttagaag 3360
gtcatctatc tgtcccaaag ctgtgaatat atatattgaa ggtaatgaat agatgaagct 3420
aaccttgtaa aaatgagtag tgtgaaatac aactacaatt atgaacatct gtcactaaag 3480
aggcaaagaa acttgaagat tgcttttgca aatgggctcc tattaataaa aagtactttt 3540
gaggtctggc tcagactcta ttgtagtact tagggtaaga ccctcctcct gtatgggctt 3600
tcattttctt tcttgcttcc ctcatttgcc cttccatgaa tactagctga taaacattga 3660
ctataaaaga tatgaggcca aacttgagct gtcccatttt aataaatctg tataaataat 3720
atttgttcta caaaagtatt atctaaataa atgttacttt ctgtcttaaa atccctcaac 3780
aaatccccac tatctagaga ataagattga cattccctgg aatcacagca tgctttgtct 3840
gccattatct gacccctttc tctttctctc ttctcacctc catctactcc tttttccttg 3900
caattcatga cccagattca ctgtttgatt tggcttgcat gtgtgtgtgc tgagttgcgt 3960
ctgactgtta tcaaccccat gaatgatagt ccaccaggct ctactgtcca tgaaattttc 4020
cagtcaagaa tactggagtg gattgcattt cctactccat ttgattaatt tagtgacttt 4080
taaatttctt tttccatatt cgggagccta ttcttccttt ttagtctata ctctcttcac 4140
tcttcaggtc taaggtatca tcgtgtgctt gttagcttgt tactttctcc attatagctt 4200
aagcactaac aactgttcag gttggcatga aattgtgttc tttgtgtggc ctgtatattt 4260
ctgttgtgta ttagaattta ccccaagatc tcaaagaccc actgaatact aaagagacct 4320
cattgtggtt acaataattt ggggactggg ccaaaacttc cgtgcatccc agccaagatc 4380
tgtagctact ggacaatttc atttccttta tcagattgtg agttattcct gttaaaatgc 4440
tccccagaat ttctggggac agaaaaatag gaaggaattc aaatccactg ttggttttat 4500
ttcaaaccac aaaattagca tgccattaaa tactatatat aaacagccac taaatcagat 4560
cattatccat tcagcttctc cttcacttct tctcccatga aggtcctcat ccttgcctgt 4620
ctggtggctc tggcccttgc acaccatcac caccatcacc tcgagaacct gtacttccaa 4680
ggccaactta cccctacctt ctacgacaat tcatgtccta atgtctctaa catcgtacgg 4740
gatactattg tcaatgagct aagatcagac cctcgtattg ccgcgagcat ccttcgtctt 4800
cacttccacg actgctttgt taatggttgt gacgcatcga tcttgttaga caacacaaca 4860
tcatttcgaa cagagaaaga tgcgtttgga aacgcaaact cggcaagagg atttccagtg 4920
attgatagaa tgaaagccgc ggtggagagt gcatgcccaa gaaccgtttc atgcgcagat 4980
ttgctcacca ttgcagctca acaatctgtc actttggcgg gaggtccttc ttggagagtt 5040
cctttgggca gaagagatag cttacaagca tttctggatc ttgctaatgc aaatcttcca 5100
gctccattct tcacacttcc acaacttaaa gacagcttta gaaatgttgg cctcaaccgt 5160
tcttctgatc tcgttgcact gtccgggggc cacacatttg gtaaatctca gtgtcggttt 5220
attatggaca gattatacaa cttcagcaac accggtttac ccgatcctac tctcaacact 5280
acttatctcc aaactcttcg tggactatgt cccctcgatg gtaatctaag cgctttggtg 5340
gattttgatc tacgtacgcc aacgattttt gacaacaaat actatgtgaa tctcgaagag 5400
caaaaaggac ttatccaaag cgaccaagag ttgttctcta gccccaatgc cactgacaca 5460
atccctttgg tgagatcatt tgctaatagc acacaaacat tcttcaatgc gtttgtggag 5520
gcgatggata ggatgggaaa cattacacct cttacaggaa ctcaaggaca gatcaggttg 5580
aattgtaggg tggtgaactc caactcttga gaattcgata tcaagcttat cgataatcaa 5640
cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt 5700
acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct 5760
ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 5820
gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg 5880
ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc 5940
acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc 6000
actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt 6060
gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca 6120
gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt 6180
cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgaccc 6240
gggcggccgc ttcgagcaga catgataaga tacattgatg agtttggaca aaccacaact 6300
agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 6360
accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag 6420
gttcaggggg agatgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa 6480
atcgataagg atcttcctag agcatggcta cgtagataag tagcatggcg ggttaatcat 6540
taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 6600
cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 6660
gagcgagcga gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg 6720
cgtattgggc gct 6733
<210>7
<211>5400
<212>DNA
<213>Artificial Sequence
<400>7
cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 60
cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 120
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 180
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 240
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 300
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 360
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 420
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 480
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 540
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 600
ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 660
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 720
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 780
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 840
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 900
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 960
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 1020
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 1080
gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1140
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1200
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1260
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1320
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1380
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1440
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1500
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1560
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1620
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1680
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1740
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1800
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1860
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1920
aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta 1980
aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga 2040
atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa 2100
cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga 2160
accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc 2220
taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga 2280
agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg 2340
cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt 2400
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct 2460
gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg 2520
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 2580
gggttccttg tagttaatga ttaacccgcc atgctactta tctacgtagc catgctctag 2640
gaagagttga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 2700
tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 2760
accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 2820
aatagggact ttccattgac gtcaatgggt ggactattta cggtaaactg cccacttggc 2880
agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 2940
gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 3000
ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 3060
tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 3120
tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 3180
gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgctagca 3240
taacgcgtac tggtaccgcc accatgaagg tcctcatcct tgcctgtctg gtggctctgg 3300
cccttgcaca ccatcaccac catcacctcg agaacctgta cttccaaggc caacttaccc 3360
ctaccttcta cgacaattca tgtcctaatg tctctaacat cgtacgggat actattgtca 3420
atgagctaag atcagaccct cgtattgccg cgagcatcct tcgtcttcac ttccacgact 3480
gctttgttaa tggttgtgac gcatcgatct tgttagacaa cacaacatca tttcgaacag 3540
agaaagatgc gtttggaaac gcaaactcgg caagaggatt tccagtgatt gatagaatga 3600
aagccgcggt ggagagtgca tgcccaagaa ccgtttcatg cgcagatttg ctcaccattg 3660
cagctcaaca atctgtcact ttggcgggag gtccttcttg gagagttcct ttgggcagaa 3720
gagatagctt acaagcattt ctggatcttg ctaatgcaaa tcttccagct ccattcttca 3780
cacttccaca acttaaagac agctttagaa atgttggcct caaccgttct tctgatctcg 3840
ttgcactgtc cgggggccac acatttggta aatctcagtg tcggtttatt atggacagat 3900
tatacaactt cagcaacacc ggtttacccg atcctactct caacactact tatctccaaa 3960
ctcttcgtgg actatgtccc ctcgatggta atctaagcgc tttggtggat tttgatctac 4020
gtacgccaac gatttttgac aacaaatact atgtgaatct cgaagagcaa aaaggactta 4080
tccaaagcga ccaagagttg ttctctagcc ccaatgccac tgacacaatc cctttggtga 4140
gatcatttgc taatagcaca caaacattct tcaatgcgtt tgtggaggcg atggatagga 4200
tgggaaacat tacacctctt acaggaactc aaggacagat caggttgaat tgtagggtgg 4260
tgaactccaa ctcttgagaa ttcgatatca agcttatcga taatcaacct ctggattaca 4320
aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat 4380
acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct 4440
ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac 4500
gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca 4560
cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca 4620
tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg 4680
tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga 4740
ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt 4800
cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga 4860
gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacccggg cggccgcttc 4920
gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 4980
aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 5040
gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga 5100
tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc gataaggatc 5160
ttcctagagc atggctacgt agataagtag catggcgggt taatcattaa ctacaaggaa 5220
cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg 5280
cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg 5340
cgcagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 5400

Claims (15)

1. A method of producing a recombinant protein of interest, comprising: providing an overexpression vector comprising a gene for a target protein and an adeno-associated viral vector; viral packaging said overexpression vector to form a viral suspension; introducing the virus suspension into mammary tissue or mammary duct of a temporary non-human mammal by means of adeno-associated virus-mediated transduction to complete virus introduction, and allowing the non-human mammal to express and secrete the recombinant target protein through mammary cells in a lactation process; obtaining milk secreted by said non-human mammal during the lactation period, and obtaining said recombinant protein of interest from said milk.
2. The method according to claim 1, wherein the promoter sequence in the overexpression vector is a sequence of any one of CMV, SV40, RSV, CAG, EF, whey acidic protein, α S1-casein, α S2-casein, β -casein, κ -casein, β -lactoglobulin, and α -lactalbumin.
3. The method of claim 1, wherein the enhancer sequence in the overexpression vector is a sequence of any one of CMV, SV40 and RSV.
4. The method according to claim 1, wherein the signal peptide coding sequence secreted by the expressed recombinant target protein provided in the overexpression vector is selected from the group consisting of a sequence of any one of whey acidic protein, α S1-casein, α S2-casein, β -casein, κ -casein, β -lactoglobulin, α -lactalbumin, human alkaline phosphatase, melittin, and immunoglobulin light chain protein, wherein the immunoglobulin light chain protein is Igk.
5. The method of claim 1, wherein the sequence of the overexpression vector further comprises a woodchuck hepatitis virus transcriptional regulatory element sequence and an SV40 polyadenylation signal sequence.
6. The method according to claim 1, wherein the sequence of the overexpression vector is as shown in any one of SEQ ID Nos. 1 to 7.
7. The method of claim 1, wherein the recombinant target protein is any one of cholinesterase, peroxidase, growth hormone, antiviral protein, immunoglobulin, viral structural protein, viroid, and membrane protein, or a fragment or derivative of any one of the foregoing.
8. The method of claim 1, wherein the adeno-associated viral vector serotype comprises any one of AAV1-AAV4 and AAV7-AAV 9.
9. The method of claim 1, wherein 293T cells are co-transfected with the overexpression vector, a first helper plasmid, the pAAV-RC plasmid, and a second helper plasmid, the phelprer plasmid, and the resulting transfection solution is then purified to complete the viral package.
10. The method of claim 1, wherein the titer of the viral suspension is not less than 1 x 1012Gene copy number/ml.
11. The method of claim 1, wherein the temporary non-human mammal is a healthy non-human mammal 2-10 days from the expected date of labor, and wherein the temporary non-human mammal is anesthetized prior to the viral introduction.
12. The method of claim 11, further comprising electrical stimulation of the mammary region of the parturient non-human mammal for 1-2 minutes using a stimulation device after introduction of the virus is complete to facilitate rapid transfection.
13. The method of claim 1, wherein the non-human mammal is any one of a mouse, rat, rabbit, goat, sheep, pig, and cow.
14. An overexpression vector, characterized in that the overexpression vector comprises a gene for a target protein and an adeno-associated viral vector, and the overexpression vector is used for forming a viral suspension by viral packaging;
the virus suspension is used for being introduced into mammary tissue or lactiferous ducts of a non-human mammal in labor through an adeno-associated virus-mediated transduction mode to complete virus introduction, the non-human mammal expresses and secretes the recombinant target protein through mammary cells in a lactation process, and the recombinant target protein is obtained from milk of the non-human mammal in a lactation period by obtaining the milk.
15. A viral suspension formed from the over-expression vector of claim 14 packaged with a virus.
CN202010977350.9A 2020-09-17 2020-09-17 Method for producing recombinant target protein, overexpression vector and virus suspension Pending CN111826397A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114317408A (en) * 2021-12-30 2022-04-12 上海桀蒙生物技术有限公司 Milk fat globule membrane organelle and preparation method thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114317408A (en) * 2021-12-30 2022-04-12 上海桀蒙生物技术有限公司 Milk fat globule membrane organelle and preparation method thereof

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