CN111500629B

CN111500629B - Method for high expression of laminin-511 variant and application thereof

Info

Publication number: CN111500629B
Application number: CN202010307391.7A
Authority: CN
Inventors: 不公告发明人
Original assignee: Nuwacell Ltd
Current assignee: Nuwacell Ltd
Priority date: 2020-04-17
Filing date: 2020-04-17
Publication date: 2021-11-19
Anticipated expiration: 2040-04-17
Also published as: CN111500629A

Abstract

The invention belongs to the field of biology, and particularly relates to a method for highly expressing laminin-511 variant and application thereof. Firstly, an expression vector pPBmL-CCP-TPA is disclosed, which takes PiggyBac transposon plasmid as a framework, and a regulation and control unit comprises: CCP promoter and TPA signal peptide gene. Also discloses a method for constructing a cell strain expressing LM511E8F by using the expression vector pPBmL-CCP-TPA. The invention constructs a plasmid containing a cell expression strong promoter and a secretion signal peptide, obtains a cell strain for efficiently and stably expressing LM511E8F by screening, and optimizes a downstream preparation process, thereby realizing the mass production of the LAM511E8F recombinant protein.

Description

Method for high expression of laminin-511 variant and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a method for highly expressing laminin-511 variant and application thereof.

Background

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hipSCs), are cells with unlimited expansion and multidirectional differentiation potential, and have wide application prospects in the field of regenerative medicine. The evolution of hPSC culture systems has included the development of culture media and extracellular matrix. Wherein the extracellular matrix provides important support for the adhesion, proliferation and differentiation of hPSC. The extracellular matrix is developed in three stages, from feeder layer cells in the first stage, such as Mouse Embryonic Fibroblasts (MEF), to Matrigel in the second stage (Matrigel), and to matriprotein in the third stage, from complex components to definite components, thereby laying the foundation for the production of clinical-grade hPSC and cell preparations.

MEFs act as feeder cells that support the adherence and expansion of hpscs. However, MEFs have significant disadvantages in use: 1) the preparation method needs to prepare 12.5-14.5 days of pregnant mice, and has the disadvantages of limited source, time-consuming manufacture, non-uniform quality and high cost; 2) the life cycle is short, the aging can be realized after the in vitro division is about 20 times, and the capability of the in vitro division for generating the proliferation promoting factors and the differentiation inhibiting factors is gradually weakened or even disappears; 3) the mouse-derived feeder layer cells can bring the pollution of animal-derived pathogens and xenoantigen, and can not be applied to the production of clinical-grade hPSC and derived cell products thereof; 4) MEFs are not resistant and cannot be used as a substrate for screening stem cells transfected with exogenous genes.

Matrigel is a substrate commonly used for culturing hpscs at present. Matrigel is derived from EHS mouse sarcoma and contains, as major components, laminin, type IV collagen, entactin, fibrinogen growth factor, tissue plasminogen activator, and other growth factors. The Matrigel basement membrane matrix is polymerized at room temperature to form a three-dimensional matrix with biological activity, and the structure, the composition and the function of the in vivo cell basement membrane are simulated. The three-dimensional culture substrate formed by the Matrigel basement membrane substrate can promote the adhesion and the amplification of hPSC. Compared with MEF, the Matrigel does not need the fussy maintenance of a mouse feeder layer, and can be compatible with various hPSC cell lines; meanwhile, a feeder layer-free system can reduce the possibility of cell pollution from a feeder layer; can be used for related metabolism or toxicology research. However, Matrigel has certain disadvantages in practical applications: 1) the tumor derived from the EHS mouse rich in the extracellular matrix has the risk of contamination of animal-derived pathogens and xenoantigen, and cannot be applied to the production of clinical-grade hPSC and derived cell products thereof; 2) the components are complex, contain a plurality of undefined components, and have large batch-to-batch difference.

Matrix proteins are a generic term for a class of proteins and their derivatives that can support cell attachment, proliferation and differentiation. At present, a plurality of matrix proteins can support the adherence and the amplification of hPSC in a culture medium with completely clear components, and mainly comprise laminin, glass fibronectin and the like. These matrix proteins can be purified from human tissues (e.g., plasma), or can be recombinantly expressed and purified using host cells by molecular biology techniques. Compared with MEF and Matrigel, matrix proteins have significant advantages: 1) the ingredients are definite, and no animal-derived ingredients are contained; 2) can be produced in large scale by recombinant protein expression technology, and can carry out definite quantification and related quality control, so that the matrix proteins of different batches have stable quality and high repeatability. 3) Simple operation, time saving and greatly simplifies the culture and differentiation of hPSC.

Laminin (LM) is a major component of basement membrane and can support the growth of a variety of cells. LM was originally a non-collagenous glycoprotein isolated from mouse EHS sarcoma by Ruper Timpl equal to the end of the 70 s. Detection by indirect immunofluorescence using purified LM antibodies revealed that LMs, in addition to EHS sarcoma, are widely distributed in the basal membrane matrix of a variety of animal and human cells, primarily in the lamina lucida. LM is a non-collagen glycoprotein peculiar to basement membrane (basal lamina), which is a heterotrimer assembled by three subunits of alpha, beta and gamma chains into a cross shape, has a relative molecular mass of 820kDa and contains 13-15% of sugar, wherein one heavy chain (alpha chain, 400kDa) and two light chains (beta chain, 215 kDa; gamma chain, 205kDa) are in an asymmetric cross shape in structure and are composed of a long arm and three similar broken arms. The beta and gamma cleft arms each have two globular domains and the alpha chain cleft arm has three globular domains, the first of which binds to type iv collagen, the second of which binds to heparin, and a domain which binds to cell surface receptors. LM mediates the binding of cells to the basement membrane through these independent binding sites. LM stimulates cell adhesion, cell expansion during cell development, the growth of the neural axis in the embryo, and promotes regrowth and regeneration after nerve injury in adult animals. As a cell culture matrix, LM can affect cell adhesion, migration, proliferation and differentiation.

The LM in the human genome comprises 5 alpha chains (α 1- α 5), 4 beta chains (β 1- β 4) and 3 gamma chains (γ 1- γ 3), and it has been established that there are a minimum of 15 different LM subtypes assembled from different subunits. The LM subtype has different expression modes in different tissue types and development stages and has different functions. LM and its different isoforms have been shown to have distinct advantages in hPSC culture and differentiation, such as LM511 (. alpha.5. beta.1. gamma.1). LM511 can bind to hPSC cell surface receptors (e.g., integrin α 6 β 1), thereby facilitating the attachment and expansion of hpscs. However, tissue-derived LM511 is very expensive, and the yield of LM511 recombinant protein purified by mammalian cell protein expression is very low, which is not favorable for conventional culture of hPSC. Subsequent studies showed that LM511-E8, which consists of α 5, β 1, γ 1 carboxy-terminal (C-terminal) truncations, supports both adherence and amplification of hPSC as well as wild-type LM 511. In addition, LM511E8F (high expression laminin-511 variant) after the gamma 1 side chain of LM511-E8 chelates Fibronectin (Fibronectin, FN) FN III 7-10(Pro1173-Arg1539) structure can further increase the iPSC adherence efficiency and promote iPSC proliferation. However, the efficiency of preparing recombinant LM511E8F is low, which is mainly characterized by low yield and complicated subsequent treatment.

Disclosure of Invention

The invention aims to construct a plasmid containing a cell expression strong promoter and a secretion signal peptide, obtain a cell strain for efficiently and stably expressing LM511E8F by screening, and optimize a downstream preparation process, thereby realizing the mass production of LAM511E8F recombinant protein.

Specifically, the technical scheme of the invention is as follows:

the invention discloses an expression vector pPBmL-CCP-TPA in a first aspect, which takes PiggyBac transposon plasmid as a skeleton, and a regulation and control unit comprises: CCP promoter and TPA signal peptide gene;

the CCP promoter has the sequence shown in SEQ ID NO: 1 or a variant sequence thereof;

the TPA signal peptide gene has the sequence shown in SEQ ID NO: 2 or a variant sequence thereof.

In the present invention, a "CCP promoter" is used, which is an artificially constructed strong combination promoter consisting of a human cytomegalovirus immediate early enhancer, a human cytomegalovirus immediate early promoter, an HTLV LTR repeat region (to improve protein translation efficiency), a synthetic rabbit beta-globin 3-terminal intron, etc., for driving high-level and continuous expression of genes in mammalian vectors. This promoter was demonstrated in a variety of cells to be able to drive expression of downstream genes with efficiency far beyond the CMV promoter. CCP promoters are capable of long-term expression, driving gene expression without silencing in a variety of cell types.

In addition, promoters having similar functions and activities as the CCP promoter may also be used in the present invention without departing from the scope of the present invention.

By optimizing the original expression vector PiggyBac transposon plasmid and replacing the original EF1a promoter with the chimeric CCP promoter, the transcription and translation levels of the exogenous gene in 293F cells are improved, and the protein expression amount is increased.

The CCP promoter sequence has the sequence shown in SEQ ID NO: 1 or a series of variants thereof. The variant sequence is similar to SEQ ID NO: 1 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity. The difference is caused by substitution, deletion or addition of a base.

Meanwhile, in the invention, a human Tissue Plasminogen Activator (TPA) signal peptide sequence is introduced to the N end of a target gene to replace a mouse Igk-chain V-J2-C secretion signal peptide, so that the extracellular secretion of a target protein LM511E8F is further promoted, the cell pressure is reduced, and the subsequent collection and purification of the target protein are facilitated.

The TPA signal peptide gene has the sequence shown in SEQ ID NO: 2 or a variant sequence thereof. The variant sequence is similar to SEQ ID NO: 2 (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity. The difference is caused by substitution, deletion or addition of a base.

The second aspect of the invention discloses a method for constructing the expression vector pPBmL-CCP-TPA, wherein the expression vector pPBmL-CCP-TPA is obtained by carrying out restriction enzyme digestion, ligation and recombination on a PiggyBac transposon plasmid skeleton, a CCP promoter and a TPA signal peptide gene.

The third aspect of the invention discloses a cell strain for expressing LM511E8F, and the construction method of the cell strain comprises the following steps:

s1: the genes alpha 5E8, beta 1E8 and FNIII-gamma 1E8 are respectively transferred into the expression vector pPBmL-CCP-TPA to respectively obtain recombinant plasmids pPBmL-CCP-TPA-alpha 5E8 (NeoR)⁺)、pPBmL-CCP-TPA-β1E8(HygR⁺) And pPBmL-CCP-TPA-FNIII-gamma 1E8 (Puror)⁺)；

S2: the recombinant plasmid pPBmL-CCP-TPA-alpha 5E8 (NeoR)⁺)、pPBmL-CCP-TPA-β1E8(HygR⁺) And pPBmL-CCP-TPA-FNIII-gamma 1E8 (Puror)⁺) And (3) transferring the cells into HEK293 cells together, and screening to obtain a positive cell population to obtain a cell strain expressing LM511E 8F.

Wherein the amino acid sequences of alpha 5E8, beta 1E8 and FNIII-gamma 1E8 are respectively shown in SEQ ID NO: 3. SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

The nucleotide sequence of pPBmL-CCP-TPA (NeoR +) is shown in SEQ ID NO: and 6.

The nucleotide sequence of pPBmL-CCP-TPA (HygR +) is shown in SEQ ID NO: shown at 7.

The nucleotide sequence of pPBmL-CCP-TPA (Puror +) is shown in SEQ ID NO: shown in fig. 8.

Preferably, after S2, S3 is further included: and inoculating the obtained positive cell group into a multi-well plate, taking supernate to perform protein quantitative detection when the confluence degree of the cells is 80-90%, and screening to obtain a cell strain with high expression of LM511E 8F.

More preferably, the resulting positive cell population is digested and plated on 96 plates, and high expression monoclonals are selected, with 2, 3, 4, 5 or 6 cells plated per well.

In one embodiment of the invention, the resulting positive cell population is digested and plated on 96 plates, and high expression monoclonals are selected, with 2 or 3 cells plated per well. After the monoclonal amplification is carried out for 8-10 days, the supernatant is taken to carry out expression identification on alpha 5E8 protein, beta 1E8 protein or FNIII-gamma 1E8 protein, the monoclonal cell strain with the highest expression quantity is screened out to be used as a subsequent culture seed cell, and the high expression cell strain is named as HEK293 pPBmL-CCP-TPA-alpha 5E 8/beta 1E 8/FNIII-gamma 1E 8.

The fourth aspect of the invention discloses the LM511E8F recombinant protein produced by the cell strain.

The fifth aspect of the invention discloses an LM511E8F recombinant protein which is assembled by the following three polypeptides:

α 5E8, or a polypeptide comprising at least 80% homology to α 5E 8;

β 1E8, or a polypeptide comprising at least 75% homology to β 1E 8;

FNIII- γ 1E8, or a polypeptide comprising at least 75% homology to FNIII- γ 1E 8;

the FNIII is fibronectin 7-10 structural domain, namely FNIII 7-10, and the sequence is sequentially connected with the amino terminal of gamma 1E8 to form the FNIII-gamma 1E8 sequence.

Preferably, the amino terminal of the alpha 5E8, beta 1E8 and FNIII-gamma 1E8 sequences are respectively connected with a tag protein antibody. The purpose of the tag protein antibody is to facilitate the subsequent expression identification of target genes and the purification of target proteins.

More preferably, His6-taq, Flag-taq and HA-taq are respectively grafted at the amino terminal of the sequences of alpha 5E8, beta 1E8 and FNIII-gamma 1E 8.

In the present invention, the "His 6-taq", also referred to as polyhistidine tag (polyhistidine), His6 tag or hexa histidine tag, is an amino acid sequence consisting of a linkage of at least 6 histidine residues at the C-terminus or N-terminus of a target protein in transfected cells.

In the present invention, "Flag-taq" is a segment consisting of 8 amino acid residues, N-DYKDDDDK-C (1012Da), which functions as a tag. In the research of protein expression and positioning, a target gene to be researched and a FLAG-tag gene sequence can be connected through a genetic engineering technical means and can be connected to the C end or the N end of the target protein, and then the integrated gene is transferred into cells or embryonic stem cells or fertilized eggs. The subsequent detection is mainly realized by the specific combination of an immune determinant formed by the Flag-tag peptide chain and a monoclonal antibody thereof, and the detection means comprises immunofluorescence (immunofluorescence) and immunoblotting (Western Blotting) and the like.

In the invention, the 'HA-taq' is HA tag protein, the tag sequence YPYDVPDYA is derived from hemagglutinin surface antigenic determinant of influenza virus, 9 amino acids have small influence on the spatial structure of an exogenous target protein, the tag protein is easy to form and fuse to the N end or the C end, and the anti-HA antibody detection and ELISA detection are easy to use.

The sixth aspect of the invention discloses a method for producing LM511E8F recombinant protein, which is produced by using the cell strain.

Preferably, the method comprises:

(1) carrying out suspension culture on the cell strain to obtain cell supernatant;

(2) carrying out ultrafiltration concentration on the cell supernatant obtained in the step (1) to obtain a crude protein extracting solution;

(3) and (3) treating the crude protein extract by affinity chromatography to obtain LM511E8F recombinant protein.

In some preferred embodiments of the invention, the culture medium is replaced with PB buffer after ultrafiltration concentration in step (2), the PB buffer being: 20mM PB, 300mM NaCl, 10mM imidazole, pH 7.2.

In a specific embodiment of the invention, the obtained high expression cell strain HEK293 pPBmL-CCP-TPA-alpha 5E 8/beta 1E 8/FNIII-gamma 1E8 is subjected to seed suspension culture, and the culture solution is KOP293 culture solution. Then carrying out amplification culture by using the specific steps of 1 × 10⁶The initial density of cells/ml was expanded to a 1L system. The culture conditions were 37 ℃ and 5% CO ₂100 +/-20 rpm, culturing for 4-5 days until the cell density reaches 8-10 × 10⁶The cells/ml were stopped from culturing. The cell supernatant was collected by centrifugation at 200 g/min for 5 min for subsequent ultrafiltration concentration.

Preparing a buffer solution required by an ultrafiltration concentration replacement system: 20mM PB, 300mM NaCl, 10mM imidazole, pH 7.2.

Concentrating 1L of supernatant to 50ml by using ultrafiltration concentration equipment, adding 450ml of buffer solution for resuspension, and continuing ultrafiltration concentration to 50 ml; 450ml of buffer are then added again and the mixture is concentrated again to 50ml by ultrafiltration. Finally obtaining crude protein extract which contains the target protein and completes the replacement of the buffer solution.

More preferably, one or any combination of Ni-NTA filler, Anti-Flag filler and Anti-HA filler is adopted in the affinity chromatography column; preferably, Ni-NTA packing is adopted in the affinity chromatography column.

In one embodiment of the invention, affinity chromatography is performed using a nickel column. The method comprises the following steps:

(1) preparing a Loading Buffer solution (Loading Buffer) for Ni column chromatography: 20mM PB, 350mM NaCl, 10mM imidazole, pH 7.2.

(2) Preparing an eluent (Elute Buffer) for Ni column chromatography: 20mM PB, 350mM NaCl, 200mM imidazole, pH 7.2.

(3) And (3) treatment of Ni columns: by dH₂The Ni column was washed sequentially with O and Loading Buffer for 10 minutes each at a wash flow rate of 3 ml/min.

(4) Protein purification: and (3) feeding the crude protein extract obtained in the step into a Ni column through a sample loading column at the sample loading speed of 2 ml/min. After the completion of the Loading, the column was washed with Loading Buffer and the impurity-washed protein treatment was carried out at a flow rate of 3 ml/min. And adding protein eluent Elute Buffer after the impurity washing is finished, setting the flow rate to be 2 ml/min, and collecting eluted samples in different tubes. Finally, purified LM511E8F recombinant protein is obtained, and 5mg of LM511E8F recombinant protein can be harvested per liter of culture supernatant after quantification.

The seventh aspect of the invention discloses the application of the LM511E8F recombinant protein or the LM511E8F recombinant protein produced by the method in cell culture; preferably, the cell is a pluripotent stem cell.

The eighth aspect of the invention discloses a culture method of pluripotent stem cells, wherein the pluripotent stem cells are inoculated into a cell culture container coated with the LM511E8F recombinant protein or the LM511E8F recombinant protein produced by the method.

Preferably, the LM511E8F recombinant protein is in the range of 0.1-1 μ g/cm²(e.g., 0.1. mu.g/cm)²、0.2μg/cm²、0.3μg/cm²、0.5μg/cm²、1μg/cm²) Coating the surface of the cell culture vessel;

more preferably, it is in the range of 0.3. mu.g/cm²And (6) coating.

In some embodiments of the invention, the cell culture vessel is a cell culture plate.

More preferably, the method comprises one or more of the following conditions:

(1) inoculating pluripotent stem cells in a cell culture container coated with LM511E8F recombinant protein in a monolayer cell culture manner;

(2) at a level of 150cells/cm or more²(iii) pluripotent stem cells were seeded in a cell culture vessel coated with the LM511E8F recombinant protein.

(3) To be less than or equal to (3-4) multiplied by 10⁴cells/cm²(iii) pluripotent stem cells were seeded in a cell culture vessel coated with the LM511E8F recombinant protein.

In some embodiments of the invention, human pluripotent stem cells from different sources are seeded onto a culture plate coated with the recombinant protein LM511E8F for culture.

The ninth aspect of the invention discloses the pluripotent stem cells obtained by the method.

On the basis of the common general knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily without departing from the concept and the protection scope of the invention.

Patents CN104884468A and US2014/0127806a1 both disclose the preparation of recombinant LM511E8F by constructing an expression plasmid by using a molecular biology technique, transforming a host cell, expressing a protein, and finally purifying by a chromatography column. However, the above method still differs from the present application, specifically as follows:

firstly, US2014/0127806A1 and CN104884468A respectively construct recombinant plasmids containing alpha 5E8, beta 1E8 and gamma 1E8 by using a vector pSecTag2B, introducing mouse Igk-chain V-J2-C signal peptide into the N end of a target gene, and collecting supernatant for protein purification after the three plasmids are cotransformed for 72 hours. The secretory signal peptide introduced by the method is mouse-derived Igk-chain V-J2-C, is not beneficial to extracellular secretion after 293F cells express proteins, and meanwhile, three plasmids are transiently co-transformed for 72 hours to collect supernatant for protein purification, and the short expression time is not beneficial to subsequent protein yield improvement;

the invention optimizes the original expression vector PiggyBac transposon plasmid skeleton, uses the chimeric CP promoter to replace the original EF1a promoter, improves the transcription and translation level of the exogenous gene in 293F cells, increases the protein expression quantity, simultaneously introduces a human Tissue Plasminogen Activator (TPA) signal peptide sequence into the N end of the target gene to replace a mouse Igk-chain V-J2-C secretion signal peptide, further promotes the extracellular secretion of the target protein, reduces the cell pressure and is beneficial to the collection and purification of the subsequent target protein;

secondly, the supernatants are collected after the three plasmids are cotransformed and cultured for 72 hours in US2014/0127806A1 and CN104884468A, and the screening of high-expression cell strains is not carried out, so that the yield is low, and the large-scale application of the recombinant protein cannot be realized. In addition, the transformation modes of the two patents are transient transformation, plasmids are gradually lost or silenced along with cell amplification, so that continuous production cannot be realized, the production time and cost of recombinant proteins are increased, and the expression level of each protein fluctuates along with the transformation efficiency;

three plasmids containing target genes and plasmids expressing transposase are co-transformed into 293F cells, and a monoclonal cell strain which stably and efficiently expresses target proteins is obtained through later-stage antibiotic screening, so that LM511E8F recombinant proteins are continuously and stably produced;

thirdly, the supernatant collected after the protein US2014/0127806A is expressed for 72 hours is directly passed through an affinity chromatography column for protein purification, and when the method is used for protein purification, the binding efficiency of the protein and the filler is influenced by complex components of the culture supernatant, so that the binding efficiency of the protein is reduced, and the protein yield is finally influenced. Moreover, tests of the inventor prove that complex components in the culture supernatant not only influence the binding efficiency of the protein and the filler, but also reduce the service life of the filler. Meanwhile, the method has lower efficiency and increased production cost when being used for processing large-volume culture supernatant.

According to the invention, the operation of concentrating and replacing a buffer system of the cell culture supernatant is realized by optimizing the post-protein purification pretreatment operation and utilizing ultrafiltration concentration equipment to carry out purification pretreatment, so that the production efficiency is obviously improved, and the production cost is reduced.

Its advantages are as follows: firstly, the invention provides a method for constructing recombinant plasmids by selecting high-efficiency secretion signal peptides based on the increase of strong promoters, and proper expression plasmids are designed according to 293F cell strains; secondly, by carrying out resistance screening on different target genes, a stable and efficient cell strain capable of realizing continuous production is obtained; thirdly, the protein purification pretreatment is optimized, the production efficiency of the recombinant LM511E8F is increased, the production cost is reduced, and the large-scale production of the recombinant LM511E8F is promoted; fourthly, the LM511E8F recombinant protein produced by the invention does not contain any animal-derived components, and is suitable for the culture and application of subsequent clinical-grade cells.

The innovation points of the invention are as follows: 1) proper promoters and secretion signal peptides are designed according to the characteristics of 293F cells, so that the expression and extracellular secretion of proteins are obviously improved, and the yield of target proteins is improved; 2) a stable and efficient expression cell strain is obtained by a special resistance screening method, and the continuous production of recombinant protein is realized; 3) the pretreatment method of the purification step of the large-volume culture supernatant is optimized, the production efficiency is improved, and the production cost is reduced.

Compared with the prior art, the invention has the following remarkable advantages and effects:

the invention successfully constructs the recombinant plasmid containing the high-efficiency promoter and the secretory signal peptide combination, and different genes are matched with different screening genes, thereby providing convenience for subsequent screening of positive cell populations. Meanwhile, by inoculating monoclone and screening high-expression cell strains, the expression quantity of LM511E8 recombinant protein is obviously improved. The experiment proves that: through SDS-PAGE analysis, the prepared LM511E8F recombinant protein has better purity and the size accords with the expectation; 5mg of purified LM511E8F per liter of culture supernatant could be obtained, much greater than the previously reported 1mg/L yield. The hPSC culture test shows that the prepared LM511E8F can support the adherence and proliferation of pluripotent stem cells under lower coating concentration, and the cultured human pluripotent stem cells have typical shapes and good states. Therefore, the LM511E8F recombinant protein provided by the invention has a good application prospect in the field of cells.

Drawings

FIG. 1 shows plasmid maps of plasmids PBmL-CCP-TPA-alpha 5E8(NeoR +), pPBmL-CCP-TPA-beta 1E8(HygR +), and pPBmL-CCP-FNIII-gamma 1E8(Puror +).

FIG. 2 shows the photograph under microscope of the positive cell population obtained after the recombinant plasmid is transfected and antibiotic selection (the control cell is not transfected with the resistance gene-containing plasmid).

FIG. 3 shows the results of SDS-PAGE analysis of LM511E8F obtained after elution by affinity chromatography (pre-column sample after pre-treatment of the supernatant, flow-through after passing through the affinity chromatography column, and elution samples for #1- # 4).

FIG. 4 shows hPSC culture at 100 cells/cm after plating well plates with LM511E8F prepared according to the present invention²Images under a cell microscope (randomly picked three fields of view) were taken after 5 days of incubation.

FIG. 5 shows the cells/cm according to 150²The number of clones was counted in a histogram after 5 days of culture by inoculating a Matrigel (Corning, 354230) coated well plate, a Vitronectin (Nuwacell, RP01002) coated well plate, and an LM511E8F coated well plate prepared according to the present invention.

FIG. 6 shows that the LM511E8F prepared according to the present invention was used to coat the well plates and then cultured pluripotent stem cells according to 3X 10⁴Cells/cm²Pictures under a cell microscope from day 1 to day 4 of inoculation and culture.

FIG. 7 shows the results of 3X 10⁴Cells/cm²ESCs (H9) and hipSCs (EP-iPSC, MSC-iPSC, NCB19001) from different sources were inoculated into LM511E 8F-coated well plates prepared according to the present invention, and cultured until the photograph under a cell microscope at day 4.

Detailed Description

The technical solutions of the present invention are described in detail below with reference to the drawings and the embodiments, but the present invention is not limited to the scope of the embodiments.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

The strains, cell lines, materials and sources used in the present invention are described:

HEK293 cells were purchased from zhahai kary biotechnology limited.

G418, Hygromycin and Puromycin were purchased from Sigma. The Ni-NTA Agarose used was purchased from QIAGEN. The serum-free, high-density medium KOP293 used for the scale-up culture was purchased from Kyoto Biotech, Inc. The ultrafiltration concentration unit was purchased from Satorius.

EXAMPLE 1 construction of high expression vector pPBmL-CCP-TPA

The embodiment discloses a method for constructing a high expression vector pPBmL-CCP-TPA, which comprises the following steps:

taking PiggyBac transposon plasmid pPBmL-PNEE and artificially synthesized CCP-TPA sequence, and carrying out enzyme digestion for 2 hours at 37 ℃, wherein the enzyme digestion system is shown in the following tables 1 and 2:

TABLE 1

Components	Volume of
		pPBmL-PNEE	10μl
NdeI	1.5μl
		BamHI	1.5μl
Cutsmart	3μl
		dH₂O	14μl

TABLE 2

Components	Volume of
		CP-TPA	30μl
NdeI	1.5μl
		XbaI	1.5μl
Cutsmart	5μl
		dH₂O	12μl

The ligation was performed after recovery with a DNA purification recovery kit, and the ligation system is shown in Table 3:

TABLE 3

The reaction was carried out at 22 ℃ for 60 minutes, the ligation product was added to Trans10 competent cells on ice, ice-washed for 40 minutes, heat shock for 45 seconds at 42 ℃ for 2 minutes, 500. mu.l of TB liquid medium was added, shaking culture was carried out at 37 ℃ for 1 hour, culture was carried out on LB solid agar plate containing ampicillin resistance for 17 to 20 hours, single colonies were picked, and NdeI and XbaI double-restriction-verified and sequenced positive clones. Finally, the pPBmL-CP-TPA carrier which is successfully constructed is obtained. The nucleotide sequence of the pPBmL-CP-TPA carrier is shown as SEQ ID NO: shown at 9.

Example 2

The embodiment discloses a cell strain for expressing LM511E8F, and the construction method of the cell strain comprises the following steps:

The detailed steps in S1 are as follows:

the pPBmL-CCP-TPA vector constructed in example 1 and the artificially synthesized NeoR gene, HygR gene and Puror gene were digested with NheI and BglII at 37 ℃ for 2 hours, respectively, and the digestion systems are shown in the following tables 4 and 5:

TABLE 4

Components	Volume of
		pPBmL-CCP-TPA	10μl
NheI	1.5μl
		BglII	1.5μl
Cutsmart	3μl
		dH₂O	14μl

TABLE 5

The ligation was performed after recovery with a DNA purification recovery kit, and the ligation system is shown in Table 6:

TABLE 6

Components	Volume of
		pPBmL-CCP-TPA glue recovery product	7.5μl
Recovery product of NeoR (HygR or Puror) glue	1μl
		T4DNA ligase	0.5μl
T4DNA ligase buffer	1μl

Reacting at 22 ℃ for 60 minutes, adding the obtained ligation product into Trans10 competent cells on ice, carrying out ice bath for 40 minutes, carrying out heat shock for 45 seconds at 42 ℃, carrying out ice bath for 2 minutes, adding 500 mu l of TB liquid culture medium, carrying out shake culture for 1 hour at 37 ℃, carrying out culture for 17-20 hours on an LB solid agar plate containing ampicillin resistance, picking out a single colony, carrying out NheI and BglII double enzyme digestion verification and sequencing positive clones. Finally, successfully constructed pPBmL-CCP-TPA (NeoR) and pPBmL-CCP-TPA (HygR) are obtained⁺) And pPBmL-CCP-TPA (Puror)⁺) Three vectors.

The plasmid maps of plasmids PBmL-CCP-TPA-alpha 5E8(NeoR +), pPBmL-CCP-TPA-beta 1E8(HygR +) and pPBmL-CCP-TPA-FNIII-gamma 1E8(Puror +) are shown in FIG. 1.

In S2, the electrical rotator is shown in table 7 below:

TABLE 7

Components
		PBmL-CCP-TPA-α5E8(NeoR+)	2μg
pPBmL-CCP-TPA-β1E8(HygR+)	2μg
		pPBmL-CCP-TPA-FNIII-γ1E8(PuroR+)	2μg
HEK293 cell	2×10⁶An
		Electrotransfer buffer	150μl

Electrotransfer program HEK293(ATCC) was selected for electrotransfer according to the Lonza electrotransfer built-in program. After the electrotransformation is finished, 200 mul of DMEM + 10% FBS culture medium is added to resuspend the electrotransformation system and then inoculated into one hole of a 6-hole plate, the temperature is 37 ℃, and 5% CO is added₂And culturing for 24 hours in saturated humidity. Digesting cells after 24 hours, wherein the number of the digested cells can be selected from 3 × 10³Cells/cm²、5×10³Cells/cm²、10×10³Cells/cm²、15×10³Cells/cm²Or 20X 10³Cells/cm²The inoculation density is preferably 10X 10³Cells/cm²。

The antibiotics used include: g418, Hygromycin and Puromycin, wherein G418 is used for screening alpha 5E8, Hygromycin is used for screening beta 1E8, and Puromycin is used for screening FNIII-gamma 1E 8.

Antibiotic screening concentration: g418 can be 200. mu.g/ml, 400. mu.g/ml, 600. mu.g/ml, 800. mu.g/ml, 1000. mu.g/ml and 1200. mu.g/ml, preferably 800. mu.g/ml, and the screening time is 8 days; hygromycin can be screened for 50. mu.g/ml, 100. mu.g/ml, 150. mu.g/ml, 200. mu.g/ml and 250. mu.g/ml, preferably 200. mu.g/ml, with a screening time of 5 days; puromycin can be selected from 0.1. mu.g/ml, 0.3. mu.g/ml, 0.5. mu.g/ml, 0.7. mu.g/ml and 1. mu.g/ml, preferably 0.5. mu.g/ml, with a screening period of 4 days.

Antibiotic addition sequence: the three antibiotics can be added sequentially or in any two combinations at a time or simultaneously, preferably sequentially, and are screened, namely G418, Hygromycin and Puromycin are added sequentially. Finally, a positive cell population containing the recombinant plasmid is obtained. The photograph under a microscope of the positive cell population is shown in FIG. 2.

Example 3

The embodiment discloses a screening method of a high expression cell strain HEK293 pPBmL-CCP-TPA-alpha 5E 8/beta 1E 8/FNIII-gamma 1E 8.

And (3) obtaining a positive cell population after screening antibiotics, inoculating a 96-well plate after digestion, culturing by using DMEM + 10% FBS, and taking the cultured supernatant to perform ELISA to detect the expression quantity of each clone so as to preferably select a high-expression cell strain.

The method comprises the following specific steps:

(1) digested cells were seeded in 96-well plates: the number of cells to be seeded per well upon seeding may be selected from 1 cell/well, 2 cells/well, 3 cells/well, 4 cells/well and 5 cells/well, preferably 3 cells/well.

(2) And (3) ELISA detection: and (3) performing monoclonal amplification until the confluence is 80-90%, and identifying the supernatant according to laminin-related side chain protein antibodies or tag protein antibodies, such as Anti-His6Taq, Anti-Flag Taq or Anti-HA Taq, preferably His6 Taq.

The monoclonal expression amount is detected by ELISA, high-expression cell strains are screened, and partial experimental results are shown in Table 8, wherein a positive control is protein coating of His6Taq, and a negative control is cell supernatant transfected with empty plasmids.

TABLE 8

Finally, the cell strain with the highest yield of the LM511E8F recombinant protein is obtained and named as HEK293 pPBmL-CAG-TPA-alpha 5E 8/beta 1E 8/FNIII-gamma 1E 8.

Example 4

The embodiment discloses a method for separating and purifying LM511E8F recombinant protein, which comprises the following steps:

the high expression cell line HEK293 pPBmL-CCP-TPA-alpha 5E 8/beta 1E 8/FNIII-gamma 1E8 obtained in example 3 was suspension cultured in KOP293, and the suspension culture was amplified to 1X 10⁶The initial density of cells/ml was expanded to a 1L system. The culture conditions were 37 ℃ and 5% CO ₂100 +/-20 rpm, culturing for 4-5 days until the cell density reaches 8-10 × 10⁶The cells/ml were stopped from culturing. 200 g/min, centrifuge for 5 min and collect the supernatant for subsequent ultrafiltration concentration.

Preparing a Loading Buffer solution for Ni column chromatography: 20mM PB, 350mM NaCl, 10mM imidazole, pH 7.2.

Preparing an eluent Elute Buffer for Ni column chromatography: 20mM PB, 350mM NaCl, 200mM imidazole, pH 7.2.

And (3) treatment of Ni columns: by dH₂The Ni column was washed sequentially with O and Loading Buffer for 10 minutes each at a wash flow rate of 3 ml/min.

Protein purification: and (3) feeding the crude protein extract obtained in the step into a Ni column through a sample loading column at the sample loading speed of 2 ml/min. After the completion of the Loading, the column was washed with Loading Buffer and the impurity-washed protein treatment was carried out at a flow rate of 3 ml/min. And adding protein eluent Elute Buffer after the impurity washing is finished, setting the flow rate to be 2 ml/min, collecting the eluted samples in tubes, wherein 1ml of each tube is marked as #1, #2, #3 and the like. Finally, purified LM511E8F recombinant protein is obtained, and 5mg of LM511E8F recombinant protein can be harvested per liter of culture supernatant after quantification.

The SDS-PAGE analysis results of LM511E8F obtained after elution by affinity chromatography are shown in FIG. 3 (pre-column sample after pre-treatment of supernatant, flow-through solution after passing through affinity chromatography column, and elution samples for #1- # 4).

Example 5

This example investigates the culture test of hPSC on LM511E8F recombinant protein. The method comprises the following steps:

the LM511E8F recombinant protein obtained in example 3 was coated on 6-well plates, where the LM511E8F recombinant protein was in the range of 0.1. mu.g/cm²、0.2μg/cm²、0.3μg/cm²、0.5μg/cm²Or 1. mu.g/cm²Coating, preferably 0.3. mu.g/cm²And (6) coating.

Pluripotent stem cells were seeded in monolayer cell culture onto 6-well coated plates.

4 kinds of hPSCs (H9-ESC, EP-iPSC, MSC-iPSC, NCB19001) were used at a high density of 3X 10⁴Cells/cm²) Seeding and Low Density (150 cells/cm)²) Inoculating on the coating. Changing the liquid in the high-density inoculated holes every day, culturing for 4 days, and observing the cell confluency and the cell state; the culture medium is changed every two days after the culture medium is not changed 4 days before the low-density inoculated wells, and the clone state is observed and the clone is counted after the culture is cultured for 6 days.

According to 150cells/cm²A histogram of the number of clones after 5 days of culture was generated by inoculating a Matrigel (Corning, 354230) coated well plate, a Vitronectin (Nuwacell, RP01002) coated well plate, and an LM511E8F coated well plate prepared according to the present invention, as shown in FIG. 5.

Under the condition of high-density inoculation, the four kinds of hPSCs are cultured for 4 days, the cell confluence reaches 85-90%, the cell mass is compact, the shape is typical, no differentiation is caused, and the cell shape is shown in figure 6; the low-density inoculation culture is carried out for 6 days, and the clone morphology is compact, typical and undifferentiated. The counting results showed that the number of clones in wells coated with LM511E8F recombinant protein was significantly higher than in Matrigel and viral coated wells, showing a clear advantage.

FIG. 7 shows the data in terms of 3X 10, respectively⁴Cells/cm²ESCs (H9) and hipSCs (EP-iPSC, MSC-iPSC, NCB19001) from different sources were inoculated into LM511E 8F-coated well plates prepared according to the present invention, and cultured until the photograph under a cell microscope at day 4.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Shang Sheng Yuan Biotechnology Co., Ltd in Anhui

<120> a method for highly expressing laminin-511 variant and application thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1048

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<213> Artificial Sequence (Artificial Sequence)

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ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 60

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 120

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 180

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 240

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 300

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 360

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 420

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 480

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 540

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 600

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 660

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 720

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 780

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 840

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 900

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 960

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 1020

aagacgaaga agacaaaccg tcgtcgac 1048

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atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60

tcgcccagc 69

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Asp Ala Ala Glu Asp Ala Ala Gly Gln Ala Leu Gln Gln Ala Asp His

1 5 10 15

Thr Trp Ala Thr Val Val Arg Gln Gly Leu Val Asp Arg Ala Gln Gln

20 25 30

Leu Leu Ala Asn Ser Thr Ala Leu Glu Glu Ala Met Leu Gln Glu Gln

35 40 45

Gln Arg Leu Gly Leu Val Trp Ala Ala Leu Gln Gly Ala Arg Thr Gln

50 55 60

Leu Arg Asp Val Arg Ala Lys Lys Asp Gln Leu Glu Ala His Ile Gln

65 70 75 80

Ala Ala Gln Ala Met Leu Ala Met Asp Thr Asp Glu Thr Ser Lys Lys

85 90 95

Ile Ala His Ala Lys Ala Val Ala Ala Glu Ala Gln Asp Thr Ala Thr

100 105 110

Arg Val Gln Ser Gln Leu Gln Ala Met Gln Glu Asn Val Glu Arg Trp

115 120 125

Gln Gly Gln Tyr Glu Gly Leu Arg Gly Gln Asp Leu Gly Gln Ala Val

130 135 140

Leu Asp Ala Gly His Ser Val Ser Thr Leu Glu Lys Thr Leu Pro Gln

145 150 155 160

Leu Leu Ala Lys Leu Ser Ile Leu Glu Asn Arg Gly Val His Asn Ala

165 170 175

Ser Leu Ala Leu Ser Ala Ser Ile Gly Arg Val Arg Glu Leu Ile Ala

180 185 190

Gln Ala Arg Gly Ala Ala Ser Lys Val Lys Val Pro Met Lys Phe Asn

195 200 205

Gly Arg Ser Gly Val Gln Leu Arg Thr Pro Arg Asp Leu Ala Asp Leu

210 215 220

Ala Ala Tyr Thr Ala Leu Lys Phe Tyr Leu Gln Gly Pro Glu Pro Glu

225 230 235 240

Pro Gly Gln Gly Thr Glu Asp Arg Phe Val Met Tyr Met Gly Ser Arg

245 250 255

Gln Ala Thr Gly Asp Tyr Met Gly Val Ser Leu Arg Asp Lys Lys Val

260 265 270

His Trp Val Tyr Gln Leu Gly Glu Ala Gly Pro Ala Val Leu Ser Ile

275 280 285

Asp Glu Asp Ile Gly Glu Gln Phe Ala Ala Val Ser Leu Asp Arg Thr

290 295 300

Leu Gln Phe Gly His Met Ser Val Thr Val Glu Arg Gln Met Ile Gln

305 310 315 320

Glu Thr Lys Gly Asp Thr Val Ala Pro Gly Ala Glu Gly Leu Leu Asn

325 330 335

Leu Arg Pro Asp Asp Phe Val Phe Tyr Val Gly Gly Tyr Pro Ser Thr

340 345 350

Phe Thr Pro Pro Pro Leu Leu Arg Phe Pro Gly Tyr Arg Gly Cys Ile

355 360 365

Glu Met Asp Thr Leu Asn Glu Glu Val Val Ser Leu Tyr Asn Phe Glu

370 375 380

Arg Thr Phe Gln Leu Asp Thr Ala Val Asp Arg Pro Cys Ala Arg Ser

385 390 395 400

Lys Ser Thr Gly Asp Pro Trp Leu Thr Asp Gly Ser Tyr Leu Asp Gly

405 410 415

Thr Gly Phe Ala Arg Ile Ser Phe Asp Ser Gln Ile Ser Thr Thr Lys

420 425 430

Arg Phe Glu Gln Glu Leu Arg Leu Val Ser Tyr Ser Gly Val Leu Phe

435 440 445

Phe Leu Lys Gln Gln Ser Gln Phe Leu Cys Leu Ala Val Gln Glu Gly

450 455 460

Ser Leu Val Leu Leu Tyr Asp Phe Gly Ala Gly Leu Lys Lys Ala Val

465 470 475 480

Pro Leu Gln Pro Pro Pro Pro Leu Thr Ser Ala Ser Lys Ala Ile Gln

485 490 495

Val Phe Leu Leu Gly Gly Ser Arg Lys Arg Val Leu Val Arg Val Glu

500 505 510

Arg Ala Thr Val Tyr Ser Val Glu Gln Asp Asn Asp Leu Glu Leu Ala

515 520 525

Asp Ala Tyr Tyr Leu Gly Gly Val Pro Pro Asp Gln Leu Pro Pro Ser

530 535 540

Leu Arg Arg Leu Phe Pro Thr Gly Gly Ser Val Arg Gly Cys Val Lys

545 550 555 560

Gly Ile Lys Ala Leu Gly Lys Tyr Val Asp Leu Lys Arg Leu Asn Thr

565 570 575

Thr Gly Val Ser Ala Gly Cys Thr Ala Asp Leu Leu Val Gly Arg Ala

580 585 590

Met Thr Phe His Gly His Gly Phe Leu Arg Leu Ala Leu Ser Asn Val

595 600 605

Ala Pro Leu Thr Gly Asn Val Tyr Ser Gly Phe Gly Phe His Ser Ala

610 615 620

Gln Asp Ser Ala Leu Leu Tyr Tyr Arg Ala Ser Pro Asp Gly Leu Cys

625 630 635 640

Gln Val Ser Leu Gln Gln Gly Arg Val Ser Leu Gln Leu Leu Arg Thr

645 650 655

Glu Val Lys Thr Gln Ala Gly Phe Ala Asp Gly Ala Pro His Tyr Val

660 665 670

Ala Phe Tyr Ser Asn Ala Thr Gly Val Trp Leu Tyr Val Asp Asp Gln

675 680 685

Leu Gln Gln Met Lys Pro His Arg Gly Pro Pro Pro Glu Leu Gln Pro

690 695 700

Gln Pro Glu Gly Pro Pro Arg Leu Leu Leu Gly Gly Leu Pro Glu Ser

705 710 715 720

Gly Thr Ile Tyr Asn Phe Ser Gly Cys Ile Ser Asn Val Phe Val Gln

725 730 735

Arg Leu Leu Gly Pro Gln Arg Val Phe Asp Leu Gln Gln Asn Leu Gly

740 745 750

Ser Val Asn Val Ser Thr Gly Cys Ala Pro Ala Leu Gln Ala Gln Thr

755 760 765

Pro Gly Leu Gly Pro Arg Gly Leu Gln Ala Thr Ala Arg Lys Ala Ser

770 775 780

Arg Arg Ser Arg Gln Pro Ala Arg His Pro Ala

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<210> 4

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<213> Artificial Sequence (Artificial Sequence)

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Asp Leu Gln His Ser Ala Ala Asp Ile Ala Arg Ala Glu Met Leu Leu

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Glu Glu Ala Lys Arg Ala Ser Lys Ser Ala Thr Asp Val Lys Val Thr

20 25 30

Ala Asp Met Val Lys Glu Ala Leu Glu Glu Ala Glu Lys Ala Gln Val

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Ala Ala Glu Lys Ala Ile Lys Gln Ala Asp Glu Asp Ile Gln Gly Thr

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Gln Asn Leu Leu Thr Ser Ile Glu Ser Glu Thr Ala Ala Ser Glu Glu

65 70 75 80

Thr Leu Phe Asn Ala Ser Gln Arg Ile Ser Glu Leu Glu Arg Asn Val

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Glu Glu Leu Lys Arg Lys Ala Ala Gln Asn Ser Gly Glu Ala Glu Tyr

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Ile Glu Lys Val Val Tyr Thr Val Lys Gln Ser Ala Glu Asp Val Lys

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Lys Thr Leu Asp Gly Glu Leu Asp Glu Lys Tyr Lys Lys Val Glu Asn

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Leu Ile Ala Lys Lys Thr Glu Glu Ser Ala Asp Ala Arg Arg Lys Ala

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Glu Met Leu Gln Asn Glu Ala Lys Thr Leu Leu Ala Gln Ala Asn Ser

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Lys Leu Gln Leu Leu Lys Asp Leu Glu Arg Lys Tyr Glu Asp Asn Gln

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Arg Tyr Leu Glu Asp Lys Ala Gln Glu Leu Ala Arg Leu Glu Gly Glu

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Val Arg Ser Leu Leu Lys Asp Ile Ser Gln Lys Val Ala Val Tyr Ser

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Thr Cys Leu

225

<210> 5

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Asp Pro Leu Ser Pro Pro Thr Asn Leu His Leu Glu Ala Asn Pro Asp

1 5 10 15

Thr Gly Val Leu Thr Val Ser Trp Glu Arg Ser Thr Thr Pro Asp Ile

20 25 30

Thr Gly Tyr Arg Ile Thr Thr Thr Pro Thr Asn Gly Gln Gln Gly Asn

35 40 45

Ser Leu Glu Glu Val Val His Ala Asp Gln Ser Ser Cys Thr Phe Asp

50 55 60

Asn Leu Ser Pro Gly Leu Glu Tyr Asn Val Ser Val Tyr Thr Val Lys

65 70 75 80

Asp Asp Lys Glu Ser Val Pro Ile Ser Asp Thr Ile Ile Pro Ala Val

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Pro Pro Pro Thr Asp Leu Arg Phe Thr Asn Ile Gly Pro Asp Thr Met

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Arg Val Thr Trp Ala Pro Pro Pro Ser Ile Asp Leu Thr Asn Phe Leu

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Val Arg Tyr Ser Pro Val Lys Asn Glu Glu Asp Val Ala Glu Leu Ser

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Ile Ser Pro Ser Asp Asn Ala Val Val Leu Thr Asn Leu Leu Pro Gly

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Thr Glu Tyr Val Val Ser Val Ser Ser Val Tyr Glu Gln His Glu Ser

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Thr Pro Leu Arg Gly Arg Gln Lys Thr Gly Leu Asp Ser Pro Thr Gly

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Ile Asp Phe Ser Asp Ile Thr Ala Asn Ser Phe Thr Val His Trp Ile

195 200 205

Ala Pro Arg Ala Thr Ile Thr Gly Tyr Arg Ile Arg His His Pro Glu

210 215 220

His Phe Ser Gly Arg Pro Arg Glu Asp Arg Val Pro His Ser Arg Asn

225 230 235 240

Ser Ile Thr Leu Thr Asn Leu Thr Pro Gly Thr Glu Tyr Val Val Ser

245 250 255

Ile Val Ala Leu Asn Gly Arg Glu Glu Ser Pro Leu Leu Ile Gly Gln

260 265 270

Gln Ser Thr Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala

275 280 285

Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val

290 295 300

Arg Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val

305 310 315 320

Gln Glu Phe Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly

325 330 335

Leu Lys Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly

340 345 350

Arg Gly Asp Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg

355 360 365

Thr Asp Asn Asp Ile Leu Asn Asn Leu Lys Asp Phe Asp Arg Arg Val

370 375 380

Asn Asp Asn Lys Thr Ala Ala Glu Glu Ala Leu Arg Lys Ile Pro Ala

385 390 395 400

Ile Asn Gln Thr Ile Thr Glu Ala Asn Glu Lys Thr Arg Glu Ala Gln

405 410 415

Gln Ala Leu Gly Ser Ala Ala Ala Asp Ala Thr Glu Ala Lys Asn Lys

420 425 430

Ala His Glu Ala Glu Arg Ile Ala Ser Ala Val Gln Lys Asn Ala Thr

435 440 445

Ser Thr Lys Ala Glu Ala Glu Arg Thr Phe Ala Glu Val Thr Asp Leu

450 455 460

Asp Asn Glu Val Asn Asn Met Leu Lys Gln Leu Gln Glu Ala Glu Lys

465 470 475 480

Glu Leu Lys Arg Lys Gln Asp Asp Ala Asp Gln Asp Met Met Met Ala

485 490 495

Gly Met Ala Ser Gln Ala Ala Gln Glu Ala Glu Ile Asn Ala Arg Lys

500 505 510

Ala Lys Asn Ser Val Thr Ser Leu Leu Ser Ile Ile Asn Asp Leu Leu

515 520 525

Glu Gln Leu Gly Gln Leu Asp Thr Val Asp Leu Asn Lys Leu Asn Glu

530 535 540

Ile Glu Gly Thr Leu Asn Lys Ala Lys Asp Glu Met Lys Val Ser Asp

545 550 555 560

Leu Asp Arg Lys Val Ser Asp Leu Glu Asn Glu Ala Lys Lys Gln Glu

565 570 575

Ala Ala Ile Met Asp Tyr Asn Arg Asp Ile Glu Glu Ile Met Lys Asp

580 585 590

Ile Arg Asn Leu Glu Asp Ile Arg Lys Thr Leu Pro Ser Gly Cys Phe

595 600 605

Asn Thr Pro Ser Ile Glu Lys Pro

610 615

<210> 6

<211> 5741

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctagtcgagt aaagcgcaaa tcttttttaa ccctagaaag atagtctgcg taaaattgac 60

gcatgcattc ttgaaatatc gctctctctt tctaaatagc gcgaatccgt cgctgtgcat 120

ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgtttgtcaa tgcggtaagt 180

gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac 240

gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt 300

gataacttat tatatatata ttttcttgtt atagatatcg tgactcatca cactggcggc 360

cgcataactt cgtatagcat acattatacg aagttatcag ccatcgatgg taccccggct 420

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 480

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 540

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 600

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 660

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 720

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 780

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 840

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 900

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 960

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 1020

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 1080

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 1140

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 1200

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 1260

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 1320

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 1380

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 1440

aagacgaaga agacaaaccg tcgtcgactc tagagccacc atggatgcaa tgaagagagg 1500

gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt tcgcccagcg cggcccagcc 1560

ggccaggcgc gcgcgccgta cgaagcttgg atccgctgat cagcctcgac tgtgccttct 1620

agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 1680

actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 1740

cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat 1800

agcaggcatg ctggggatgc ggtgggctct atgaagctta aatcccggcg cgccctaccg 1860

ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg 1920

cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg 1980

ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag 2040

gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg 2100

tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg 2160

gggcagcggc caatagcagc tttggctcct tcgctttctg ggctcagagg ctgggaaggg 2220

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 2280

ccggaagccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 2340

cctcatctcc gggcctttcg acctgcagcc gctagcatgg gatcggccat tgaacaagat 2400

ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 2460

caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 2520

gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg 2580

cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 2640

gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 2700

caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 2760

cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 2820

actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 2880

gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga tgatctcgtc 2940

gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 3000

ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 3060

cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 3120

atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 3180

agatcttaag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat 3240

gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata 3300

aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg 3360

aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat gatccggctg 3420

cctcgcgcca tatgtgataa cttcgtatag catacattat acgaagttat gaaaagtttt 3480

gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa taaacataaa 3540

taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa acttaatatc 3600

tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc gtcaatttta 3660

cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt aaataatagt 3720

ttctaatttt tttattattc agaattcttg aagacgaaag ggcctcgtga tacgcctatt 3780

tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg 3840

aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 3900

catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 3960

tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 4020

tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 4080

ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 4140

ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga 4200

cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 4260

ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 4320

tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 4380

gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 4440

ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc 4500

aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 4560

acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 4620

tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 4680

cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 4740

gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat 4800

taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 4860

tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 4920

cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 4980

ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 5040

accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 5100

cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca 5160

cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 5220

tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 5280

taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 5340

gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 5400

agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 5460

ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 5520

acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 5580

caacgcggcc tttttacggt tcctggcctt ttgctggcct tgaagctgtc cctgatggtc 5640

gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc 5700

gagaagaatc ataatgggga aggccatcca gcctcgcgtc g 5741

<210> 7

<211> 5972

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctagtcgagt aaagcgcaaa tcttttttaa ccctagaaag atagtctgcg taaaattgac 60

gcatgcattc ttgaaatatc gctctctctt tctaaatagc gcgaatccgt cgctgtgcat 120

ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgtttgtcaa tgcggtaagt 180

gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac 240

gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt 300

gataacttat tatatatata ttttcttgtt atagatatcg tgactcatca cactggcggc 360

cgcataactt cgtatagcat acattatacg aagttatcag ccatcgatgg taccccggct 420

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 480

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 540

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 600

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 660

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 720

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 780

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 840

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 900

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 960

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 1020

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 1080

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 1140

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 1200

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 1260

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 1320

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 1380

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 1440

aagacgaaga agacaaaccg tcgtcgactc tagagccacc atggatgcaa tgaagagagg 1500

gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt tcgcccagcg cggcccagcc 1560

ggccaggcgc gcgcgccgta cgaagcttgg atccgctgat cagcctcgac tgtgccttct 1620

agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 1680

actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 1740

cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat 1800

agcaggcatg ctggggatgc ggtgggctct atgaagctta aatcccggcg cgccctaccg 1860

ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg 1920

cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg 1980

ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag 2040

gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg 2100

tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg 2160

gggcagcggc caatagcagc tttggctcct tcgctttctg ggctcagagg ctgggaaggg 2220

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 2280

ccggaagccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 2340

cctcatctcc gggcctttcg acctgcagcc gctagcatgg atagatccgg aaagcctgaa 2400

ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg 2460

atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga 2520

tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg 2580

cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag 2640

agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa 2700

accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc 2760

gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact 2820

acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg 2880

atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc 2940

gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg 3000

acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc 3060

caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag 3120

acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat 3180

atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat 3240

gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg 3300

cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc 3360

gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gagatcttaa 3420

gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt 3480

gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta 3540

acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt 3600

aaagcaagta aaacctctac aaatgtggta tggctgatta tgatccggct gcctcgcgcc 3660

atatgtgata acttcgtata gcatacatta tacgaagtta tgaaaagttt tgttacttta 3720

tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa ataaattgtt 3780

tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat 3840

taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt acgcatgatt 3900

atctttaacg tacgtcacaa tatgattatc tttctagggt taaataatag tttctaattt 3960

ttttattatt cagaattctt gaagacgaaa gggcctcgtg atacgcctat ttttataggt 4020

taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 4080

cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 4140

ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 4200

ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 4260

aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 4320

actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 4380

gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca 4440

agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 4500

cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 4560

catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 4620

aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 4680

gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac 4740

aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 4800

agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 4860

ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 4920

actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 4980

aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 5040

gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 5100

atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 5160

tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 5220

tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 5280

ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 5340

agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa 5400

ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 5460

tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 5520

gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 5580

cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 5640

ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 5700

agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 5760

tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 5820

ctttttacgg ttcctggcct tttgctggcc ttgaagctgt ccctgatggt cgtcatctac 5880

ctgcctggac agcatggcct gcaacgcggg catcccgatg ccgccggaag cgagaagaat 5940

cataatgggg aaggccatcc agcctcgcgt cg 5972

<210> 8

<211> 5534

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ctagtcgagt aaagcgcaaa tcttttttaa ccctagaaag atagtctgcg taaaattgac 60

gcatgcattc ttgaaatatc gctctctctt tctaaatagc gcgaatccgt cgctgtgcat 120

ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgtttgtcaa tgcggtaagt 180

gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac 240

gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt 300

gataacttat tatatatata ttttcttgtt atagatatcg tgactcatca cactggcggc 360

cgcataactt cgtatagcat acattatacg aagttatcag ccatcgatgg taccccggct 420

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 480

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 540

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 600

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 660

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 720

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 780

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 840

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 900

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 960

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 1020

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 1080

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 1140

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 1200

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 1260

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 1320

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 1380

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 1440

aagacgaaga agacaaaccg tcgtcgactc tagagccacc atggatgcaa tgaagagagg 1500

gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt tcgcccagcg cggcccagcc 1560

ggccaggcgc gcgcgccgta cgaagcttgg atccgctgat cagcctcgac tgtgccttct 1620

agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 1680

actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 1740

cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat 1800

agcaggcatg ctggggatgc ggtgggctct atgaagctta aatcccggcg cgccctaccg 1860

ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag ccccgctggg 1920

cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca ccggtaggcg 1980

ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag 2040

gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg 2100

tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg 2160

gggcagcggc caatagcagc tttggctcct tcgctttctg ggctcagagg ctgggaaggg 2220

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 2280

ccggaagccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 2340

cctcatctcc gggcctttcg acctgcagcc gctagcatga ccgagtacaa gcccacggtg 2400

cgcctcgcca cccgcgacga cgtcccccgg gccgtacgca ccctcgccgc cgcgttcgcc 2460

gactaccccg ccacgcgcca caccgtcgac ccggaccgcc acatcgagcg ggtcaccgag 2520

ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac 2580

gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc 2640

gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag 2700

atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc 2760

ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg 2820

gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc 2880

cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgagtgccc gaaggaccgc 2940

gcgacctggt gcatgacccg caagcccggt gccagatctt aagatacatt gatgagtttg 3000

gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta 3060

ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc 3120

attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag taaaacctct 3180

acaaatgtgg tatggctgat tatgatccgg ctgcctcgcg ccatatgtga taacttcgta 3240

tagcatacat tatacgaagt tatgaaaagt tttgttactt tatagaagaa attttgagtt 3300

tttgtttttt tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag 3360

tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat 3420

atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac 3480

aatatgatta tctttctagg gttaaataat agtttctaat ttttttatta ttcagaattc 3540

ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 3600

ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 3660

atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 3720

tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 3780

cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 3840

agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 3900

taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 3960

tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg 4020

catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 4080

ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 4140

ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 4200

catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 4260

aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt 4320

aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 4380

taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 4440

atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 4500

gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 4560

tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 4620

ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 4680

gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 4740

agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 4800

aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 4860

agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 4920

tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 4980

atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 5040

taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 5100

gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 5160

gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 5220

aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 5280

tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 5340

gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 5400

cttttgctgg ccttgaagct gtccctgatg gtcgtcatct acctgcctgg acagcatggc 5460

ctgcaacgcg ggcatcccga tgccgccgga agcgagaaga atcataatgg ggaaggccat 5520

ccagcctcgc gtcg 5534

<210> 9

<211> 6347

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctagtcgagt aaagcgcaaa tcttttttaa ccctagaaag atagtctgcg taaaattgac 60

gcatgcattc ttgaaatatc gctctctctt tctaaatagc gcgaatccgt cgctgtgcat 120

ttaggacatc tcagtcgccg cttggagctc ccgtgaggcg tgtttgtcaa tgcggtaagt 180

gtcactgatt ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttttac 240

gtgactttta agatttaact catacgataa ttatattgtt atttcatgtt ctacttacgt 300

gataacttat tatatatata ttttcttgtt atagatatcg tgactcatca cactggcggc 360

cgcataactt cgtatagcat acattatacg aagttatcag ccatcgatgg taccccggct 420

ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 480

gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 540

tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 600

aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 660

caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 720

acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 780

ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 840

gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 900

gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 960

tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc gcctggagac 1020

gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc ctccgcggct 1080

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 1140

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 1200

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 1260

agccggctct ccacgctttg cctgaccctg cttgctcaac tctagttctc tcgttaactt 1320

aatgagacag atagaaactg gtcttgtaga aacagagtag tcgcctgctt ttctgccagg 1380

tgctgacttc tctcccctgg gcttttttct ttttctcagg ttgaaaagaa gaagacgaag 1440

aagacgaaga agacaaaccg tcgtcgactc tagaatggtg agcaagggcg aggagctgtt 1500

caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag 1560

cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg 1620

caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt 1680

gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat 1740

gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac 1800

ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat 1860

cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 1920

caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg 1980

ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat 2040

cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag 2100

caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg 2160

gatcactctc ggcatggacg agctgtacaa gtaaggatcc gctgatcagc ctcgactgtg 2220

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 2280

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 2340

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 2400

gacaatagca ggcatgctgg ggatgcggtg ggctctatga agcttaaatc ccggcgcgcc 2460

ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 2520

gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 2580

taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 2640

agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 2700

agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 2760

cctttggggc agcggccaat agcagctttg gctccttcgc tttctgggct cagaggctgg 2820

gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa 2880

ggtcctccgg aagcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct 2940

cctcttcctc atctccgggc ctttcgacct gcagccgcta gcatgggatc ggccattgaa 3000

caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac 3060

tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg 3120

cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag 3180

gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt 3240

gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg 3300

tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg 3360

catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga 3420

gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag 3480

gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgatgat 3540

ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt 3600

tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg 3660

gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt 3720

tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc 3780

ttctgaagat cttaagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa 3840

aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct 3900

gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg 3960

tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct gattatgatc 4020

cggctgcctc gcgccatatg tgataacttc gtatagcata cattatacga agttatgaaa 4080

agttttgtta ctttatagaa gaaattttga gtttttgttt ttttttaata aataaataaa 4140

cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat aataaaactt 4200

aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac acatgcgtca 4260

attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct agggttaaat 4320

aatagtttct aattttttta ttattcagaa ttcttgaaga cgaaagggcc tcgtgatacg 4380

cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt 4440

tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 4500

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 4560

gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 4620

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 4680

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 4740

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 4800

tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 4860

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 4920

cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 4980

aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 5040

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 5100

tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 5160

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 5220

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 5280

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 5340

gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 5400

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 5460

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 5520

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 5580

aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 5640

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 5700

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 5760

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 5820

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 5880

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 5940

gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 6000

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 6060

cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 6120

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 6180

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttgaa gctgtccctg 6240

atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc 6300

ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcg 6347

Claims

1. A cell strain for expressing LM511E8F, wherein the construction method of the cell strain comprises:

s1: the genes alpha 5E8, beta 1E8 and FNIII-gamma 1E8 are respectively transferred into an expression vector pPBmL-CCP-TPA to respectively obtain a recombinant plasmid pPBmL-CCP-TPA-alpha 5E8 (NeoR)⁺）、pPBmL-CCP-TPA-β1E8（HygR⁺) And pPBmL-CCPPA-FNIII-gamma 1E8 (Puror)⁺）；

S2: the recombinant plasmid pPBmL-CCP-TPA-alpha 5E8 (NeoR)⁺）、pPBmL-CCP-TPA-β1E8（HygR⁺) And pPBmL-CCP-TPA-FNIII-gamma 1E8 (Puror)⁺) Transferring the cells into HEK293 cells together, and screening to obtain a positive cell population to obtain a cell strain expressing LM511E 8F;

the expression vector pPBmL-CCP-TPA takes a vector PiggyBac transposon plasmid as a framework, and the regulation and control unit comprises: CCP promoter and TPA signal peptide gene; the CCP promoter has a nucleotide sequence shown as SEQ ID NO: 1 is shown in the specification; the nucleotide sequence of the TPA signal peptide gene is shown as SEQ ID NO: 2 is shown in the specification;

the expression vector pPBmL-CCP-TPA is obtained by carrying out restriction enzyme digestion, connection and recombination on a PiggyBac transposon plasmid skeleton, a CCP promoter and a TPA signal peptide gene.

2. The cell strain expressing LM511E8F of claim 1, further comprising, after S2, S3: and inoculating the obtained positive cell group into a multi-well plate, taking supernate to perform protein quantitative detection when the confluence degree of the cells is 80-90%, and screening to obtain a cell strain with high expression of LM511E 8F.

3. A method for producing a LM511E8F recombinant protein, which is produced using the cell line according to any one of claims 1 to 2.

4. The method of claim 3, comprising:

(1) subjecting the cell strain of any one of claims 1-2 to suspension culture to obtain a cell supernatant;

5. The method according to claim 4, wherein the culture medium is replaced with PB buffer after ultrafiltration concentration in step (2), wherein the PB buffer is: 20mM PB, 300mM NaCl, 10mM imidazole, pH = 7.2.

6. The method of claim 4, wherein the affinity chromatography column is filled with one or any combination of Ni-NTA filler, Anti-Flag filler and Anti-HA filler.

7. The method of claim 6, wherein Ni-NTA packing is used in the affinity chromatography column.