CN113061605A

CN113061605A - Construction method and application of fucose transferase 8(FUT8) function-deficient cell strain

Info

Publication number: CN113061605A
Application number: CN202110228894.XA
Authority: CN
Inventors: 袁燕秋; 毛洋; 王圣钧; 王军舰; 何羽骐; 钟雨伦
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2020-07-13
Filing date: 2021-03-02
Publication date: 2021-07-02
Anticipated expiration: 2041-03-02
Also published as: CN113061605B

Abstract

The invention discloses a construction method and application of a fucose transferase 8(FUT8) function-deficient cell strain. The invention achieves the purpose that the constructed mature engineering cells lose the function of fucose transferase (FUT8) by screening and selecting the active site of FUT8 and adopting a base editing technology under the conditions of not introducing exogenous proteins and not destroying the integral structure of endogenous proteins and by changing the minimal gene level, and the invention is changed from a host for normally producing antibody medicines containing fucose into a host for producing antibody medicines containing no fucose, thereby improving the yield and the quality of the produced antibody medicines.

Description

Construction method and application of fucose transferase 8(FUT8) function-deficient cell strain

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to a construction method and application of a fucose transferase 8(FUT8) function-deficient cell strain.

Background

Therapeutic antibody drugs are a rapidly growing field of drug development in recent years. In addition to neutralizing the effect of the antigen, a considerable number of therapeutic antibody drugs, such as anti-tumor antibody drugs, kill target cells after binding them by antibody-dependent cell-mediated cytotoxicity (ADCC). Therefore, improving the ADCC ability of a therapeutic antibody drug is important for improving the efficacy of an anti-tumor antibody drug.

The research shows that: the ADCC of the therapeutic antibody modified by fucose-deficient glycosylation is enhanced by several tens of times compared to a therapeutic antibody containing α 1, 6-linked core fucose. However, therapeutic antibodies produced by the engineered cells, which are not engineered, contain almost all fucose glycosylation modifications. Therefore, the development of engineering cells capable of producing fucose-deficient glycosylated antibodies has important application prospects for the development of therapeutic antibody drugs with enhanced ADCC.

At present, the strategies adopted in the prior art for producing fucose-deficient glycosylated antibodies are mainly as follows: inducing the engineered cell to synthesize a highly bisecting N-acetylglucosamine (bisecting GlcNAc), low fucose antibody molecule by overexpressing N-acetylglucosamine transferase iii (gntiii) in the engineered cell; or reducing or eliminating the expression of fucose transferase 8(FUT8) by the engineering cells through the knockout technology of Zinc Finger Nuclear, TALEN and the like, thereby leading the engineering cells to lose the capability of synthesizing fucose-containing antibody molecules; or reducing the expression of fucose transferase (FUT8) by RNA interference technology (RNAi), thereby making the engineered cell lose the ability to synthesize fucose-containing antibody molecules; or inhibiting the synthesis and transport pathway of fucose by similar molecular biological methods. However, a common problem with these approaches is: the introduction of large amounts of foreign proteins (e.g., overexpression of GnTIII) or the reduction of expression of endogenous proteins (e.g., knock-out of FUT8) may place unnecessary growth stress on the engineered cells that have already been mature, thereby reducing the yield and quality of antibody drug production.

Therefore, there is a need for a method for constructing a fucosyltransferase deficient cell line without introducing a foreign protein and without disrupting the expression of an endogenous protein, so as to improve ADCC of a therapeutic antibody drug and reduce the risk of low yield and quality of the antibody drug.

Disclosure of Invention

The invention aims to provide a targeting sequence for constructing a cell strain with a function of fucosyltransferase 8(FUT8) being deleted;

another objective of the invention is to provide a fucosyltransferase 8 functional deletion cell strain;

another object of the present invention is to provide a method for constructing the above-mentioned fucosyltransferase 8-deficient cell line;

another object of the present invention is to provide a sequencing primer for discriminating the cell line from a wild-type cell line;

the invention also aims to provide the application of the cell strain in preparing a medicament for expressing the antibody.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a targeting sequence for constructing a cell strain with fucose transferase 8 function deficiency is 5'-TGTCAGACGCACTGACAAAG-3' (SEQ ID NO. 1).

The cell strain is obtained by constructing plasmid and transfecting a base editor;

wherein the sequence of the constructed plasmid is shown as SEQ ID NO. 3;

the base editor includes ABEmax adenine base editor, BE3 cytosine base editor.

The ABEmax adenine base editor was synthesized from the whole gene of Shanghai Biotechnology, Inc. according to the sequence reported in the literature.

The BE3 cytosine base editor was synthesized from the whole gene of Shanghai Biotechnology Ltd based on a sequence reported in the literature.

In a second aspect of the present invention, there is provided:

a cell strain with fucose transferase 8 function deletion, the protein sequence expressed by the cell strain is shown in SEQ ID NO. 2.

Based on a CRISPR (clustered regularly interspaced short palindromic repeats) targeting system, the active site of the fucose transferase 8 is selected, and the length of the selected sequence is 20 bp.

In a third aspect of the present invention, there is provided:

the construction method of the fucose transferase 8 function-deficient cell strain comprises the following steps:

(1) culturing the cell strain;

(2) mixing and constructing plasmids and a base editor, and incubating to obtain a transfection reagent;

(3) mixing the transfection reagent and the cultured cell strain, and incubating;

(4) observing cells, judging the transfection condition, and obtaining a fucose transferase function-deficient cell strain after successful transfection;

wherein, the transfection condition is judged as follows:

and (4) observing the fluorescence of the cells, wherein if the fluorescence exists, the cells are successfully transfected.

Further, the construction method further comprises the following steps:

(5) sorting monoclonal cells with fluorescence;

(6) detecting and determining the fucose transferase 8 function-deficient cell strain.

Sorting may be accomplished using sorting methods conventional in the art, including flow cytometry sorting, among others.

Detection determination of fucosyltransferase 8 functionally deficient cell lines can be determined using detection methods routine in the art, including direct sequencing.

The specific operation of sequencing comprises: extracting the genome of the monoclonal cell, and sequencing by using a sequencing primer 5'-TCTGTTGATTC CAGGTTCCCA-3' (SEQ ID No.4) to screen successfully edited engineering cells.

Furthermore, the sequence of the constructed plasmid is shown as SEQ ID NO. 3;

the construction method of the plasmid comprises the following steps:

(1) synthesizing a double-stranded DNA fragment with a restriction enzyme BbsI site;

(2) carrying out enzyme digestion;

(3) carrying out enzyme linkage to obtain a constructed plasmid;

wherein, the carrier plasmid comprises gRNA and fluorescent protein.

The gRNA has the characteristics of targeting the active site of the fucose transferase 8 and high-efficiency gene editing.

The fluorescent protein includes green fluorescent protein GFP. Of course, other fluorescent proteins in the cost field can be reasonably replaced according to actual needs.

Further, the reaction system of enzyme digestion in the step (2) is as follows:

vector plasmid	≤1μg
		Restriction enzyme Bbs I	1μL
10×CutSmart Buffer	5μL
		Water (W)	Make up to 50 μ L;

the enzyme digestion in the step (2) is carried out under the conditions of overnight enzyme digestion at 37 ℃ and 20min at 65 ℃ to inactivate the enzyme.

After the enzyme inactivation, the purification treatment was performed by agarose gel (0.8%) electrophoresis.

Further, the reaction system of the enzyme linkage in the step (3) is:

plasmid after enzyme digestion in step (2)	100-500ng
		Targeting sequences	1-2μg
10×T4DNALigaseBuffer	2μL
		T4DNALigase	2 WeissU
Water (W)	Make up to 20 μ L;

the enzyme was inactivated by incubating at 22 ℃ for 1 hour and at 70 ℃ for 5 min.

Further, the above base editor includes ABEmax adenine base editor, BE3 cytosine base editor.

In a fourth aspect of the present invention, there is provided:

a sequencing primer for identifying the cell strain and a wild cell strain, wherein the sequence of the sequencing primer is 5'-TCTGTTGA TTCCAGGTTCCCA-3' (SEQ ID NO. 4).

Further, the cell lines include CHO cells and HEK293 cells. Of course, other engineered cell lines in the art can be substituted as appropriate according to actual needs.

In a fifth aspect of the present invention, there is provided:

the application of the cell strain in preparing a medicine for expressing the antibody.

The invention has the beneficial effects that:

the invention can successfully construct the fucose transferase 8 function-deficient cell strain and simultaneously overcome a plurality of defects in the prior art. The fucose transferase 8 function-deficient cell strain can make the constructed mature engineering cell lose the fucose transferase 8(FUT8) function by the minimum modification of the gene level under the condition of not introducing exogenous protein and not destroying the integral structure of endogenous protein, and can be changed from a host for normally producing antibody medicines containing fucose into a host for producing antibody medicines containing no fucose.

Drawings

FIG. 1 is a map of a vector plasmid (HP180-dCas 9); wherein promoter represents promoter, enhancer represents enhancer, and gRNAscaffold represents gRNA skeleton;

FIG. 2 shows a map (A) and a sequence map (B) of a vector plasmid (HP180-dCas9) at the time of enzyme digestion; wherein promoter represents promoter, enhancer represents enhancer, and gRNAscaffold represents gRNA skeleton;

FIG. 3 is a sequence map of the HP180-dCas9 plasmid of the constructed gRNA 1;

FIG. 4 is a sequence map of the HP180-dCas9 plasmid of the constructed gRNA 2;

FIG. 5 is a sequence map of the HP180-dCas9 plasmid of the constructed gRNA 3;

FIG. 6 is a sequence map of the HP180-dCas9 plasmid of the constructed gRNA 4;

FIG. 7 shows the sequencing results of samples from the gRNA1 group;

FIG. 8 shows the sequencing results of samples from the gRNA2 group;

FIG. 9 shows the sequencing results of samples from the gRNA3 group;

FIG. 10 shows the sequencing results of samples from the gRNA4 group;

FIG. 11 is a graph showing N-sugar chains of rituximab produced by wild-type CHO cells (WT) and CHO mutant cells (CHO-R365G).

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.

EXAMPLE 1 selection of mutation sites

In the invention, the inventors research shows that H363, R365, D368, K369, E373, Y382, D409, D453 and S469 are active sites of FUT8, but not all the mutations of the sites are operable.

The requirement for screening for a suitable active site in the present invention is based on the following conditions:

1. since CRISPR technology relies on the localization of PAM (pro-spacer adjacent motif), this requires that the designed gRNA (guide RNA) must target sequences near PAM;

2. the base editing tool can only effectively realize the conversion of a specific base in an active window which is a fixed distance away from the PAM sequence in the gRNA targeted sequence;

3. due to the degeneracy of the codons, nonsense mutations may occur.

Based on the screening of the above three conditions, the inventors found three operable sites of R365, D368 and D453.

The FUT8 protein sequence is as follows:

MRAWTGSWRWIMLILFAWGTLLFYIGGHLVRDNDHPDHSSRELSKILAKLERLKQQN EDLRRMAESLRIPEGPIDQGTATGRVRVLEEQLVKAKEQIENYKKQARNDLGKDHEILRRRI ENGAKELWFFLQSELKKLKKLEGNELQRHADEILLDLGHHERSIMTDLYYLSQTDGAGEW REKEAKDLTELVQRRITYLQNPKDCSKARKLVCNINKGCGYGCQLHHVVYCFMIAYGTQR TLILESQNWRYATGGWETVFRPVSETCTDRSGLSTGHWSGEVKDKNVQVVELPIVDSLHPR PPYLPLAVPEDLADRLLRVHGDPAVWWVSQFVKYLIRPQPWLEREIEETTKKLGFKHPVIGV HVRRTDKVGTEAAFHPIEEYMVHVEEHFQLLERRMKVDKKRVYLATDDPSLLKEAKTKYS NYEFISDNSISWSAGLHNRYTENSLRGVILDIHFLSQADFLVCTFSSQVCRVAYEIMQTLHPD ASANFHSLDDIYYFGGQNAHNQIAVYPHQPRTKEEIPMEPGDIIGVAGNHWNGYSKGVNRK LGKTGLYPSYKVREKIETVKYPTYPEAEK(SEQ ID NO.5)。

wherein the first underline is markedRThe active site of R365, the second underlinedDThe third underlined, labeled D368 active siteDIs the D453 active site.

Wherein, the gene sequences near the active sites R365 and D368 of FUT8 in the genome are as follows:

5’-AATAACCAACATCTTTAAAAGGCTAGCTTGTCTTAAACTACAGGAAAAGTTCATA TGGATCTTTGTTTTCTTAGATGACTTTAAATTCTATGAACTGAAGTGGTAGTAACTTTACA GGGTAAAATGAAAGAAAAAAATTAATAAACTTTGGCATAAGAATGTTACAAGCATTATCT TTAAGCTTTGAATTCTGTTATGATTTTGGTCTCAAAAACCAAAAAACTTAAATCTGTTGAT TCCAGGTTCCCATATATTCTTGGATATGCCAATTACTTTTTCTGTAAGCAAGTGTTTCATAA AACTTTTACTTAACTTTCATATTGACCTGTACTATTCAACATTCAGCTATGTTAAAGTATTT GTGAAGTGTTTTGAAATGATTTTATATTTTCTAAGGTGAGAATAAATGAGAAAATGTTTTA ATATGTCTCCAGTGCCCCCATGACTAGGGATACTAATTGAGTACCAGTACATTATCAGTGT GCTCTCCACTTCTCCCCAGAGTCCATGTCAGACGCACTGACAAAGTGGGAACAGAAGC AGCCTTCCATCCCATTGAGGAATACATGGTACACGTTGAAGAACATTTTCAGCTTCTCGA ACGCAGAATGAAAGTGGATAAAAAAAGAGTGTATCTGGCCACTGATGACCCTTCTTTGT TAAAGGAGGCAAAGACAAAGTAAGTTAGACCAACAAGTGGTTCTGTATGGGATTATCTC TTAGTTGAAGAAAATCCTTAATTCTGGGAACTTGTGGTTCTTGTTGCTAACTAATAGGTT CCAAAATCAAAGACTACATGTGCAAATATTAATCTAATCAAGTCATACCTTACTAGCTGTA TCTGATGCAAATTAAGAAGTCTAAAATGAATTAGACTGCTGATTTGTGTAGCATCACTAG CAGTCATCATTCAACACAGTACCACACTTCTTAGTACCAAAATCTGTTTAACATACTAGA GTTTCCATAAATCAAATTTTGTAGCCTGGGGCTTAAGTAACAGAAGTTTATGTCTCACAG TTTTGATCTGGGATATT-3’(SEQ ID NO.6)。

the bold AGA and GAC are coding sequences corresponding to the active sites R365 and D368 respectively; wherein the underlined bases areAAndGrespectively, a single base target modified by base editing; targeting sequences designed for it are as follows:

target D368:

gRNA1：5’-CTTTGTCAGTGCGTCTGACA-3’(SEQ ID NO.7)；

target R365:

gRNA2：5’-GTCAGACGCACTGACAAAGT-3’(SEQ ID NO.8)；

gRNA3：5’-TGTCAGACGCACTGACAAAG-3’(SEQ ID NO.1)。

the gene sequence near the active site D453 of FUT8 in the genome is as follows:

5’-GGTCATAATGATGTATGTGTTTAAGATAATATACCCATAATATATTTTTGTAAAACAT GGTTCTGCTACGTAGCCCAGGCCAGCCTTGGACCCGTAGTCCTCTTGTCTCAGCCAGCT GAATATTTGGACTATAGGAATGAATATCTCACCACACCTGGCATCAAGATATTCTGAAGTA GTTTCTTCTCCATTGTTGATTTGAGAGATGCTTCAACTACTCTATTAAATGTTATCAGGAG GGAGCTTTTGATTAAAGAAGTGGTCTCCCTTCTGTAGAATACTTAAGTTTAGTCATGTAC AAATCTCTTTTTTTCAGGTACTCCAATTATGAATTTATTAGTGATAACTCTATTTCTTGGTC AGCTGGACTACACAACCGATACACAGAAAATTCACTTCGGGGCGTGATCCTGGATATAC ACTTTCTCTCCCAGGCTGACTTCCTTGTGTGTACTTTTTCATCCCAGGTAAGTGGTAGCA CTTTTTAGTGGCCAGTACTCAGTGTTGTACTGTGCCAGGAAAATTATTTGTGAGTCTGTG TTTTTTGTGTTTGTTTGTTTGTTTGTTTGTTTGTTTTCATGACAGTGTTTCTCTGTGTAGCT TTGGAGCCTATCCTGGTACTGGCTCTGTAGACCAGGTTAGCCTTGAACTCGAAGAGATC AGCCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCGTGCACCACCAACGCCCAGCAT TAGTCTGTGTTTTTAGTGTTAGTATGATTCCAGTATAATTTCCTTAACTTAGTACTTGAA-3’ (SEQ ID NO.9)。

the thickened GAT is a coding sequence corresponding to the active site D453; wherein,underlined basesAA single base modified by base editing; targeting sequences were designed for this as follows:

target D453: gRNA 4: 5'-GGATATACACTTTCTCTCCC-3' (SEQ ID NO.10)

Example 2 construction of plasmids

1. The synthesis of the targeting sequence DNA fragment with the restriction endonuclease BbsI site is as follows:

for gRNA 1:

forward direction: 5'-CACCGCTTTGTCAGTGCGTCTGACA-3' (SEQ ID NO.11)

And (3) reversing: 5'-AAACTGTCAGACGCACTGACAAAGC-3' (SEQ ID NO.12)

For gRNA 2:

forward direction: 5'-CACCGTCAGACGCACTGACAAAGT-3' (SEQ ID NO.13)

And (3) reversing: 5'-AAACACTTTGTCAGTGCGTCTGAC-3' (SEQ ID NO.14)

For gRNA 3:

forward direction: 5'-CACCTGTCAGACGCACTGACAAAG-3' (SEQ ID NO.15)

And (3) reversing: 5'-AAACCTTTGTCAGTGCGTCTGACA-3' (SEQ ID NO.16)

For gRNA 4:

forward direction: 5'-CACCGGATATACACTTTCTCTCCC-3' (SEQ ID NO.17)

And (3) reversing: 5'-AAACGGGAGAGAAAGTGTATATCC-3' (SEQ ID NO.18)

Oligonucleotide chains of the above sequences were synthesized, and the respective sets of plus and minus chains were mixed to a final concentration of 50. mu.M. Denaturation at 94 ℃ for 5min, and annealing at 50 ℃ to obtain the double-stranded DNA fragment with the BbsI site.

2. The above fragments were loaded into a vector plasmid with small guide RNA (sgRNA) backbone, green fluorescent protein, GFP, as follows:

a) a vector plasmid (designated as HP180-dCas9) having a BbsI cohesive end was prepared by digestion: the map of the HP180-dCas9 plasmid and the BbsI site are shown in figure 1:

the reaction system is as follows:

TABLE 1 restriction reaction System of vector plasmid

Reagent	Volume of
		HP180-dCas9 plasmid	≤1μg
Restriction enzyme Bbs I	1μL(10Units)
		CutSmart Buffer(10×)	5μL
Water (W)	Make up to 50 μ L

The reagents were mixed well and cleaved overnight at 37 ℃ and then the enzyme was inactivated at 65 ℃ for 20min, and the resulting cleaved product was purified by agarose gel (0.8%) electrophoresis.

The map and sequence of the vector plasmid (HP180-dCas9) during enzyme digestion are shown in FIG. 2.

b) Enzyme linking: the reaction system is as follows:

TABLE 2 reaction System for enzyme linkage

Reagent	Volume of
		Digested HP180-dCas9 plasmid	100-500ng
Targeting sequences	1-2μg
		T4DNALigaseBuffer(10×)	2μL
T4DNALigase	2WeissU
		Water (W)	Make up to 20. mu.L

The reagents were mixed, homogenized, incubated at 22 ℃ for 1 hour, and the enzyme was inactivated at 70 ℃ for 5 min.

The sequence map of the HP180-dCas9 plasmid of the constructed gRNA1 is shown in FIG. 3;

the sequence map of the HP180-dCas9 plasmid of the constructed gRNA2 is shown in FIG. 4;

the sequence map of the HP180-dCas9 plasmid of the constructed gRNA3 is shown in FIG. 5;

the sequence map of the HP180-dCas9 plasmid of the constructed gRNA4 is shown in FIG. 6;

the obtained HP180-dCas9 plasmid (HP180-dCas9-gRNA1) for coding the gRNA1 has a sequence shown as SEQ ID NO. 19.

The obtained HP180-dCas9 plasmid (HP180-dCas9-gRNA2) for coding the gRNA2 has a sequence shown as SEQ ID NO. 20.

The obtained HP180-dCas9 plasmid (HP180-dCas9-gRNA3) of the gRNA3 has a sequence shown in SEQ ID NO. 3.

The obtained HP180-dCas9 plasmid (HP180-dCas9-gRNA4) for coding the gRNA4 has a sequence shown as SEQ ID NO. 21.

EXAMPLE 3 transfection of CHO engineered cells

The HP180-dCas9 plasmid encoding gRNA (HP180-dCas9-gRNA1, 2, 3, 4) was co-transfected with the base editor plasmid (ABEmax or BE3) into CHO engineered cells as follows:

1. preparation of cells

At 37 ℃ 5% CO₂Culturing CHO cells in CHO Fusion culture medium (containing 2% Glutamax and 1% penicillin and streptomycin (P/S) double antibody) in cell culture box with concentration, counting cell density after growth reaches logarithmic growth phase, and collecting cell number of 2 × 10⁶The cell fluid was centrifuged for 5min at 100g in a 15ml centrifuge tube, the supernatant was discarded, and the cells were resuspended in 1.8ml Opti MEM medium (containing 2% GlutaMax and 1% P/S double antibody).

2. Incubation of transfection reagents

After vortexing FectoPRO reagent (available from Polyplus Transfection Co.) for 5S, 2. mu.L was added to a 1.5ml EP tube, Opti MEM medium (containing 2% GlutaMax and 1% P/S double antibody) was added to another 1.5ml EP tube, and 0.5. mu.g of HP180-dCas9-gRNA plasmid and 0.5. mu.g of plasmid with base editor (gRNA1 co-transfected with BE 3; gRNA2, 3, 4 co-transfected with ABEmax) were added to the tube containing Opti MEM medium and mixed. The liquids in the two tubes were then mixed and incubated for 10 min.

3. Mixing transfection reagent and cell sap

And pouring the cell sap into a culture dish, adding the incubated transfection reagent into the culture dish, and uniformly mixing.

4. Changing culture medium

After 24 hours, the transfection was successful if there was green fluorescence, as observed under a fluorescence microscope. The culture was carefully aspirated using a pipette gun, discarded, and CHO Fusion medium (containing 2% Glutamax and 1% P/S double antibody) was added.

EXAMPLE 4 cloning of cell lines

1. Floor board

Mu.l PBS buffer was added to 36 wells at the peripheral edge of the 96-well plate and 150. mu.l medium (50% CHO Fusion + 37% CHO cloning + 10% FBS + 2% Glutamax + 1% double antibody) was added to the middle 60 wells.

2. Preparation of cells

Counting and counting 3-5 days after the transfection success is confirmedCalculating the cell density of the transfected CHO cell sap and the wild-type CHO cell sap, and respectively taking the cell containing number of 2 multiplied by 10⁶The cell fluid was centrifuged at 100g for 5min in a flow centrifuge tube, the supernatant was discarded and resuspended in 4ml PBS.

3. Flow cytometer sorting

4. Culture of monoclonal

The 96-well plate was placed at 37 ℃ in 5% CO₂Culturing in cell culture box at concentration for 1-2 weeks to obtain wild type CHO cell and CHO mutant cell (CHO-R365G).

Example 5 sequencing validation

After the cells in example 4 were grown to an appropriate density, the genome of the selected CHO monoclonal (CHO-R365G) and wild-type CHO cells were extracted with FlexiGene DNA kit (purchased from QIAGEN, cat # 51206), respectively. Since the positions of the grnas 1, 2, and 3 are substantially coincident, a sequencing primer can be shared, and the sequence of the primer is as follows: 5'-TCTGTTGATTCCAGGTTCCCA-3' (SEQ ID NO. 4).

Sequencing results of samples from the gRNA1 group are shown in fig. 7;

and (4) conclusion: all sequencing samples of the gRNA1 group did not show base mutations as in fig. 7, indicating that gRNA1 is not an ideal targeting sequence.

Sequencing results of samples from the gRNA2 group are shown in fig. 8;

and (4) conclusion: although the group of gRNA2 has base mutation, the base mutation is not on the target site, and although the base mutation is in the sequence coding for R365, the base mutation is nonsense mutation and cannot change the amino acid sequence of the site, which indicates that the gRNA2 is not an ideal target sequence.

Sequencing results of samples from the gRNA3 group are shown in fig. 9;

and (4) conclusion: as can be seen from fig. 9, the gRNA3 has only one nucleotide shift compared to gRNA2, but can be accurately edited to the target site, and is a unimodal mutation, i.e., both alleles are mutated, which finally results in the mutation of the R365 site of the FUT8 protein to glycine (SEQ ID No.2), and the loss of the function of FUT8, indicating that gRNA3 is an ideal targeting sequence.

The amino acid sequence of FUT8 after mutation is as follows:

MRAWTGSWRWIMLILFAWGTLLFYIGGHLVRDNDHPDHSSRELSKILAKLERLKQQN EDLRRMAESLRIPEGPIDQGTATGRVRVLEEQLVKAKEQIENYKKQARNDLGKDHEILRRRI ENGAKELWFFLQSELKKLKKLEGNELQRHADEILLDLGHHERSIMTDLYYLSQTDGAGEW REKEAKDLTELVQRRITYLQNPKDCSKARKLVCNINKGCGYGCQLHHVVYCFMIAYGTQR TLILESQNWRYATGGWETVFRPVSETCTDRSGLSTGHWSGEVKDKNVQVVELPIVDSLHPR PPYLPLAVPEDLADRLLRVHGDPAVWWVSQFVKYLIRPQPWLEREIEETTKKLGFKHPVIGV HVGRTDKVGTEAAFHPIEEYMVHVEEHFQLLERRMKVDKKRVYLATDDPSLLKEAKTKYS NYEFISDNSISWSAGLHNRYTENSLRGVILDIHFLSQADFLVCTFSSQVCRVAYEIMQTLHPD ASANFHSLDDIYYFGGQNAHNQIAVYPHQPRTKEEIPMEPGDIIGVAGNHWNGYSKGVNRK LGKTGLYPSYKVREKIETVKYPTYPEAEK(SEQ ID NO.2)。

wherein, the bold and underlined G is used as the active site mutation generation of R365 in the original sequence. Finally, the full-length sequence of the optimal HP180-dCas9-gRNA plasmid is shown in SEQ ID NO. 3.

The gRNA4 group sequencing primers were: 5'-GGGAGCTTTTGATTAAAGAAGTGGT-3' (SEQ ID NO.22)

The sequencing results for the gRNA4 panel are shown in fig. 10:

and (4) conclusion: only one target site mutation occurred in the gRNA4 group, but a bimodal mutation, i.e., only one allele mutation, and mutations in non-target regions, indicating that gRNA4 is not an ideal targeting sequence.

Example 6 Fucosyltransferase 8 function-deficient cell line production feasibility verification experiment

This example uses the antibody drug Rituximab (Rituximab) as an example, and shows the feasibility of producing the fucosyltransferase 8-deficient cell strain in this example. Of course, those skilled in the art can prepare fucosyltransferase 8 deficient cell lines expressing other antibodies according to the protocol in this example.

1. Production expression of antibody drug Rituximab (Rituximab):

the medium of wild type CHO cells (CHO-WT) and the CHO mutant cells selected in the above example (CHO-R365G) was replaced with OptiMEM medium (containing 2% GlutaMax and 1% P/S double antibody) and diluted to 1X 10⁶Individual cells/mL, 5% CO at 37 ℃₂After culturing for 24h in the incubator, extracting the coded antibody drugAn expression plasmid of Rituximab (Rituximab). An expression plasmid encoding an antibody drug Rituximab (Rituximab) is transfected by using a FectoPro transfection reagent, and the specific steps are as follows: adding 10 μ g of expression plasmid containing Rituximab and 20 μ l FectoPro reagent into 2mLOptiMEM culture medium, mixing, incubating for 10min, adding into 18mL of cell suspension culture solution, and returning the cells to 37 deg.C and 5% CO₂The incubator of (2) for cultivation. After 72 hours of cell culture, the cells were centrifuged at 2000g for 10min, and the cell supernatant was collected, and the expressed antibody was present in the cell supernatant.

2. Purification of production expressed antibody drug Rituximab (Rituximab):

the expressed recombinant antibody was purified using Protein a affinity chromatography column as follows: the Protein A affinity chromatographic column is equilibrated by PBS Buffer solution, the cell supernatant collected in the step (1) is injected into the chromatographic column, the chromatographic column is washed by 10mlL of 1M NaCl solution, the Protein bound by the chromatographic column is eluted by IgG Elution Buffer (purchased from Thermo Fisher Scientific company), the expression and purification conditions of the antibody are verified by SDS-PAGE and Coomassie blue staining of partial eluted products, and the antibody after the successful purification is preserved and used for subsequent experiments.

3. Glycosylation modification analysis for production of expressed antibody drug Rituximab (Rituximab):

(1) isolation and purification of N-sugar chain modified by antibody:

the IgG Elution buffer in the antibody eluate was replaced with 50mM TEAB buffer using an ultrafiltration tube with a molecular weight cut-off (MWCO) of 10kDa and the solution was concentrated to 100. mu.L. 5 μ L of LPNGaseF glycosidase was added and the digestion was carried out overnight at 37 ℃. The reaction solution was then passed through a C18 Solid Phase Extraction (SPE) cartridge, and the filtrate was collected to obtain deproteinized N-sugar chains.

(2) Fluorescence labeling of N-sugar chains with procainamide:

the separated N-sugar chains were evaporated to dryness using a centrifugal concentration apparatus, and 20. mu.L of a mixed solution of 0.4M procainamide and 1M sodium borocyanide was added, followed by incubation at 65 ℃ for 1 hour. After the reaction, the reaction solution was poured into NH equilibrated with 85% acetonitrile₂After a cartridge of Solid Phase Extraction (SPE) was washed with 85% acetonitrile, the labeled N-sugar chain was eluted with 5% acetonitrile in 100mM ammonium acetate, and then the purified N-sugar chain was evaporated to dryness by a centrifugal concentration apparatus to obtain procainamide-labeled N-sugar chain.

(3) Analysis of fluorescently labeled N-sugar chains using Ultra Performance Liquid Chromatography (UPLC):

the purified fluorescently labeled N-sugar chains were redissolved with 20. mu.L of deionized water. mu.L of the reconstituted N-sugar chain sample was mixed with 30. mu.L of a mixed solution of dimethylformamide and acetonitrile (the volume ratio of dimethylformamide to acetonitrile was 1:2), and the sample was analyzed by Waters ACQUITY UPLC system under the analysis conditions shown in Table 3:

TABLE 3 Ultra Performance Liquid Chromatography (UPLC) analysis conditions

The results are shown in FIG. 11.

By comparing the spectra of the N-sugar chain of Rituximab prepared by wild-type CHO cells (WT) and CHO mutant cells (CHO-R365G), it was found that, when the wild-type CHO cells were subjected to base editing using gRNA3 to obtain CHO mutant cells (CHO-R365G) in which the R365 active site encoded by the FUT8 gene was mutated, the glycosylation modification of the antibody drug Rituximab (Rituximab) produced was deficient in fucose (red triangle in the sugar chain in the figure) relative to the wild-type CHO cells, thereby confirming that it lost the function of fucose transferase (FUT8) and had the ability to produce antibody drugs containing no fucose.

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> Zhongshan university

<120> construction method and application of fucosyltransferase 8(FUT8) function-deficient cell strain

<130>

<160> 22

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<400> 1

tgtcagacgc actgacaaag 20

<210> 2

<211> 575

<212> PRT

<213> Artificial sequence

<400> 2

Met Arg Ala Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile Leu Phe

1 5 10 15

Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp

20 25 30

Asn Asp His Pro Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala

35 40 45

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

50 55 60

Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Thr Ala Thr

65 70 75 80

Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln

85 90 95

Ile Glu Asn Tyr Lys Lys Gln Ala Arg Asn Asp Leu Gly Lys Asp His

100 105 110

Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe

115 120 125

Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Lys Leu Glu Gly Asn Glu

130 135 140

Leu Gln Arg His Ala Asp Glu Ile Leu Leu Asp Leu Gly His His Glu

145 150 155 160

Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala

165 170 175

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

180 185 190

Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Arg

195 200 205

Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu

210 215 220

His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr

225 230 235 240

Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu

245 250 255

Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Leu

260 265 270

Ser Thr Gly His Trp Ser Gly Glu Val Lys Asp Lys Asn Val Gln Val

275 280 285

Val Glu Leu Pro Ile Val Asp Ser Leu His Pro Arg Pro Pro Tyr Leu

290 295 300

Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Leu Arg Val His

305 310 315 320

Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile

325 330 335

Arg Pro Gln Pro Trp Leu Glu Arg Glu Ile Glu Glu Thr Thr Lys Lys

340 345 350

Leu Gly Phe Lys His Pro Val Ile Gly Val His Val Gly Arg Thr Asp

355 360 365

Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val

370 375 380

His Val Glu Glu His Phe Gln Leu Leu Glu Arg Arg Met Lys Val Asp

385 390 395 400

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

405 410 415

Ala Lys Thr Lys Tyr Ser Asn Tyr Glu Phe Ile Ser Asp Asn Ser Ile

420 425 430

Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg

435 440 445

Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val

450 455 460

Cys Thr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln

465 470 475 480

Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp Asp Ile

485 490 495

Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Val Tyr Pro

500 505 510

His Gln Pro Arg Thr Lys Glu Glu Ile Pro Met Glu Pro Gly Asp Ile

515 520 525

Ile Gly Val Ala Gly Asn His Trp Asn Gly Tyr Ser Lys Gly Val Asn

530 535 540

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

545 550 555 560

Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Glu Lys

565 570 575

<210> 3

<211> 4845

<212> DNA

<213> Artificial sequence

<400> 3

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacacct gtcagacgca ctgacaaagg ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagagccac catggcggcc gctagttatt aatagtaatc aattacgggg 480

tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 540

cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 600

gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 660

cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 720

ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 780

cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 840

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 900

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 960

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 1020

ggtttagtga accgtcagat ccgctagcat ggtgagcaag ggcgaggagc tgttcaccgg 1080

ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 1140

cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 1200

cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg 1260

cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 1320

aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 1380

cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 1440

caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 1500

ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 1560

catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 1620

cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 1680

ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 1740

tctcggcatg gacgagctgt acaagtaagg atccgggatg cagaaattga tgatctatta 1800

aacaataaag atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg 1860

agaacagagt acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg 1920

ggattagata aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt 1980

tcatagttgg atatcataat ttaaacaagc aaaaccaaat taagggccag ctcattcctc 2040

ccactcatga tctatagatc tatagatctc tcgtgggatc attgtttttc tcttgattcc 2100

cactttgtgg ttctaagtac tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg 2160

agatcagcag cctctgttcc acatacactt cattctcagt attgttttgc caagttctaa 2220

ttccatcaga agctggtcga cctgcagggg cgcctgatgc ggtattttct ccttacgcat 2280

ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg 2340

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2400

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 2460

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 2520

accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 2580

tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 2640

gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt 2700

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 2760

tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 2820

taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 2880

cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 2940

caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 3000

ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 3060

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3120

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 3180

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 3240

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 3300

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 3360

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 3420

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 3480

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 3540

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 3600

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 3660

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 3720

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 3780

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 3840

gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag 3900

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 3960

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 4020

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 4080

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 4140

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 4200

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 4260

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 4320

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 4380

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4440

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 4500

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 4560

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 4620

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 4680

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 4740

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 4800

cctttttacg gttcctggcc ttttgctggc cttttgctca catgt 4845

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

tctgttgatt ccaggttccc a 21

<210> 5

<211> 575

<212> PRT

<213> Artificial sequence

<400> 5

Met Arg Ala Trp Thr Gly Ser Trp Arg Trp Ile Met Leu Ile Leu Phe

1 5 10 15

Ala Trp Gly Thr Leu Leu Phe Tyr Ile Gly Gly His Leu Val Arg Asp

20 25 30

Asn Asp His Pro Asp His Ser Ser Arg Glu Leu Ser Lys Ile Leu Ala

35 40 45

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

50 55 60

Glu Ser Leu Arg Ile Pro Glu Gly Pro Ile Asp Gln Gly Thr Ala Thr

65 70 75 80

Gly Arg Val Arg Val Leu Glu Glu Gln Leu Val Lys Ala Lys Glu Gln

85 90 95

Ile Glu Asn Tyr Lys Lys Gln Ala Arg Asn Asp Leu Gly Lys Asp His

100 105 110

Glu Ile Leu Arg Arg Arg Ile Glu Asn Gly Ala Lys Glu Leu Trp Phe

115 120 125

Phe Leu Gln Ser Glu Leu Lys Lys Leu Lys Lys Leu Glu Gly Asn Glu

130 135 140

Leu Gln Arg His Ala Asp Glu Ile Leu Leu Asp Leu Gly His His Glu

145 150 155 160

Arg Ser Ile Met Thr Asp Leu Tyr Tyr Leu Ser Gln Thr Asp Gly Ala

165 170 175

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

180 185 190

Arg Arg Ile Thr Tyr Leu Gln Asn Pro Lys Asp Cys Ser Lys Ala Arg

195 200 205

Lys Leu Val Cys Asn Ile Asn Lys Gly Cys Gly Tyr Gly Cys Gln Leu

210 215 220

His His Val Val Tyr Cys Phe Met Ile Ala Tyr Gly Thr Gln Arg Thr

225 230 235 240

Leu Ile Leu Glu Ser Gln Asn Trp Arg Tyr Ala Thr Gly Gly Trp Glu

245 250 255

Thr Val Phe Arg Pro Val Ser Glu Thr Cys Thr Asp Arg Ser Gly Leu

260 265 270

Ser Thr Gly His Trp Ser Gly Glu Val Lys Asp Lys Asn Val Gln Val

275 280 285

Val Glu Leu Pro Ile Val Asp Ser Leu His Pro Arg Pro Pro Tyr Leu

290 295 300

Pro Leu Ala Val Pro Glu Asp Leu Ala Asp Arg Leu Leu Arg Val His

305 310 315 320

Gly Asp Pro Ala Val Trp Trp Val Ser Gln Phe Val Lys Tyr Leu Ile

325 330 335

Arg Pro Gln Pro Trp Leu Glu Arg Glu Ile Glu Glu Thr Thr Lys Lys

340 345 350

Leu Gly Phe Lys His Pro Val Ile Gly Val His Val Arg Arg Thr Asp

355 360 365

Lys Val Gly Thr Glu Ala Ala Phe His Pro Ile Glu Glu Tyr Met Val

370 375 380

His Val Glu Glu His Phe Gln Leu Leu Glu Arg Arg Met Lys Val Asp

385 390 395 400

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

405 410 415

Ala Lys Thr Lys Tyr Ser Asn Tyr Glu Phe Ile Ser Asp Asn Ser Ile

420 425 430

Ser Trp Ser Ala Gly Leu His Asn Arg Tyr Thr Glu Asn Ser Leu Arg

435 440 445

Gly Val Ile Leu Asp Ile His Phe Leu Ser Gln Ala Asp Phe Leu Val

450 455 460

Cys Thr Phe Ser Ser Gln Val Cys Arg Val Ala Tyr Glu Ile Met Gln

465 470 475 480

Thr Leu His Pro Asp Ala Ser Ala Asn Phe His Ser Leu Asp Asp Ile

485 490 495

Tyr Tyr Phe Gly Gly Gln Asn Ala His Asn Gln Ile Ala Val Tyr Pro

500 505 510

His Gln Pro Arg Thr Lys Glu Glu Ile Pro Met Glu Pro Gly Asp Ile

515 520 525

Ile Gly Val Ala Gly Asn His Trp Asn Gly Tyr Ser Lys Gly Val Asn

530 535 540

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

545 550 555 560

Lys Ile Glu Thr Val Lys Tyr Pro Thr Tyr Pro Glu Ala Glu Lys

565 570 575

<210> 6

<211> 1037

<212> DNA

<213> Artificial sequence

<400> 6

aataaccaac atctttaaaa ggctagcttg tcttaaacta caggaaaagt tcatatggat 60

ctttgttttc ttagatgact ttaaattcta tgaactgaag tggtagtaac tttacagggt 120

aaaatgaaag aaaaaaatta ataaactttg gcataagaat gttacaagca ttatctttaa 180

gctttgaatt ctgttatgat tttggtctca aaaaccaaaa aacttaaatc tgttgattcc 240

aggttcccat atattcttgg atatgccaat tactttttct gtaagcaagt gtttcataaa 300

acttttactt aactttcata ttgacctgta ctattcaaca ttcagctatg ttaaagtatt 360

tgtgaagtgt tttgaaatga ttttatattt tctaaggtga gaataaatga gaaaatgttt 420

taatatgtct ccagtgcccc catgactagg gatactaatt gagtaccagt acattatcag 480

tgtgctctcc acttctcccc agagtccatg tcagacgcac tgacaaagtg ggaacagaag 540

cagccttcca tcccattgag gaatacatgg tacacgttga agaacatttt cagcttctcg 600

aacgcagaat gaaagtggat aaaaaaagag tgtatctggc cactgatgac ccttctttgt 660

taaaggaggc aaagacaaag taagttagac caacaagtgg ttctgtatgg gattatctct 720

tagttgaaga aaatccttaa ttctgggaac ttgtggttct tgttgctaac taataggttc 780

caaaatcaaa gactacatgt gcaaatatta atctaatcaa gtcatacctt actagctgta 840

tctgatgcaa attaagaagt ctaaaatgaa ttagactgct gatttgtgta gcatcactag 900

cagtcatcat tcaacacagt accacacttc ttagtaccaa aatctgttta acatactaga 960

gtttccataa atcaaatttt gtagcctggg gcttaagtaa cagaagttta tgtctcacag 1020

ttttgatctg ggatatt 1037

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

ctttgtcagt gcgtctgaca 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

gtcagacgca ctgacaaagt 20

<210> 9

<211> 780

<212> DNA

<213> Artificial sequence

<400> 9

ggtcataatg atgtatgtgt ttaagataat atacccataa tatatttttg taaaacatgg 60

ttctgctacg tagcccaggc cagccttgga cccgtagtcc tcttgtctca gccagctgaa 120

tatttggact ataggaatga atatctcacc acacctggca tcaagatatt ctgaagtagt 180

ttcttctcca ttgttgattt gagagatgct tcaactactc tattaaatgt tatcaggagg 240

gagcttttga ttaaagaagt ggtctccctt ctgtagaata cttaagttta gtcatgtaca 300

aatctctttt tttcaggtac tccaattatg aatttattag tgataactct atttcttggt 360

cagctggact acacaaccga tacacagaaa attcacttcg gggcgtgatc ctggatatac 420

actttctctc ccaggctgac ttccttgtgt gtactttttc atcccaggta agtggtagca 480

ctttttagtg gccagtactc agtgttgtac tgtgccagga aaattatttg tgagtctgtg 540

ttttttgtgt ttgtttgttt gtttgtttgt ttgttttcat gacagtgttt ctctgtgtag 600

ctttggagcc tatcctggta ctggctctgt agaccaggtt agccttgaac tcgaagagat 660

cagcctgcct ctgcctcccg agtgctggga ttaaaggcgt gcaccaccaa cgcccagcat 720

tagtctgtgt ttttagtgtt agtatgattc cagtataatt tccttaactt agtacttgaa 780

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence

<400> 10

ggatatacac tttctctccc 20

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence

<400> 11

caccgctttg tcagtgcgtc tgaca 25

<210> 12

<211> 25

<212> DNA

<213> Artificial sequence

<400> 12

aaactgtcag acgcactgac aaagc 25

<210> 13

<211> 24

<212> DNA

<213> Artificial sequence

<400> 13

caccgtcaga cgcactgaca aagt 24

<210> 14

<211> 24

<212> DNA

<213> Artificial sequence

<400> 14

aaacactttg tcagtgcgtc tgac 24

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<400> 15

cacctgtcag acgcactgac aaag 24

<210> 16

<211> 24

<212> DNA

<213> Artificial sequence

<400> 16

aaacctttgt cagtgcgtct gaca 24

<210> 17

<211> 24

<212> DNA

<213> Artificial sequence

<400> 17

caccggatat acactttctc tccc 24

<210> 18

<211> 24

<212> DNA

<213> Artificial sequence

<400> 18

aaacgggaga gaaagtgtat atcc 24

<210> 19

<211> 4845

<212> DNA

<213> Artificial sequence

<400> 19

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ctttgtcagt gcgtctgaca gttttagagc tagaaatagc aagttaaaat 300

aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga 360

gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 420

gacaaatggc tctagagcca ccatggcggc cgctagttat taatagtaat caattacggg 480

gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 540

gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat 600

agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 660

ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 720

cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 780

gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 840

caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 900

caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 960

cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 1020

tggtttagtg aaccgtcaga tccgctagca tggtgagcaa gggcgaggag ctgttcaccg 1080

gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 1140

ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 1200

ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt 1260

gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 1320

aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 1380

ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 1440

tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 1500

tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 1560

acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 1620

acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag 1680

accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 1740

ctctcggcat ggacgagctg tacaagtaag gatccgggat gcagaaattg atgatctatt 1800

aaacaataaa gatgtccact aaaatggaag tttttcctgt catactttgt taagaagggt 1860

gagaacagag tacctacatt ttgaatggaa ggattggagc tacgggggtg ggggtggggt 1920

gggattagat aaatgcctgc tctttactga aggctcttta ctattgcttt atgataatgt 1980

ttcatagttg gatatcataa tttaaacaag caaaaccaaa ttaagggcca gctcattcct 2040

cccactcatg atctatagat ctatagatct ctcgtgggat cattgttttt ctcttgattc 2100

ccactttgtg gttctaagta ctgtggtttc caaatgtgtc agtttcatag cctgaagaac 2160

gagatcagca gcctctgttc cacatacact tcattctcag tattgttttg ccaagttcta 2220

attccatcag aagctggtcg acctgcaggg gcgcctgatg cggtattttc tccttacgca 2280

tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 2340

gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 2400

ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 2460

cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 2520

gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 2580

gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 2640

ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 2700

tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 2760

atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 2820

ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 2880

ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 2940

tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 3000

gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 3060

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 3120

caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 3180

ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 3240

gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 3300

gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 3360

atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 3420

caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 3480

gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 3540

accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 3600

ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 3660

gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 3720

acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 3780

atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 3840

ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 3900

gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 3960

gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 4020

tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 4080

taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 4140

cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 4200

gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 4260

gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 4320

agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 4380

aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 4440

agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 4500

cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 4560

accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 4620

aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 4680

ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 4740

cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 4800

gcctttttac ggttcctggc cttttgctgg ccttttgctc acatg 4845

<210> 20

<211> 4845

<212> DNA

<213> Artificial sequence

<400> 20

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg tcagacgcac tgacaaagtg ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagagccac catggcggcc gctagttatt aatagtaatc aattacgggg 480

tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 540

cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 600

gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 660

cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 720

ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 780

cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 840

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 900

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 960

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 1020

ggtttagtga accgtcagat ccgctagcat ggtgagcaag ggcgaggagc tgttcaccgg 1080

ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 1140

cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 1200

cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg 1260

cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 1320

aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 1380

cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 1440

caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 1500

ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 1560

catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 1620

cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 1680

ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 1740

tctcggcatg gacgagctgt acaagtaagg atccgggatg cagaaattga tgatctatta 1800

aacaataaag atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg 1860

agaacagagt acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg 1920

ggattagata aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt 1980

tcatagttgg atatcataat ttaaacaagc aaaaccaaat taagggccag ctcattcctc 2040

ccactcatga tctatagatc tatagatctc tcgtgggatc attgtttttc tcttgattcc 2100

cactttgtgg ttctaagtac tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg 2160

agatcagcag cctctgttcc acatacactt cattctcagt attgttttgc caagttctaa 2220

ttccatcaga agctggtcga cctgcagggg cgcctgatgc ggtattttct ccttacgcat 2280

ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg 2340

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2400

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 2460

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 2520

accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 2580

tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 2640

gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt 2700

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 2760

tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 2820

taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 2880

cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 2940

caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 3000

ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 3060

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3120

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 3180

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 3240

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 3300

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 3360

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 3420

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 3480

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 3540

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 3600

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 3660

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 3720

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 3780

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 3840

gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag 3900

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 3960

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 4020

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 4080

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 4140

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 4200

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 4260

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 4320

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 4380

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4440

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 4500

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 4560

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 4620

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 4680

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 4740

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 4800

cctttttacg gttcctggcc ttttgctggc cttttgctca catgt 4845

<210> 21

<211> 4845

<212> DNA

<213> Artificial sequence

<400> 21

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg gatatacact ttctctcccg ttttagagct agaaatagca agttaaaata 300

aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt tgttttagag 360

ctagaaatag caagttaaaa taaggctagt ccgtttttag cgcgtgcgcc aattctgcag 420

acaaatggct ctagagccac catggcggcc gctagttatt aatagtaatc aattacgggg 480

tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 540

cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 600

gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 660

cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 720

ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 780

cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 840

aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 900

aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 960

gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 1020

ggtttagtga accgtcagat ccgctagcat ggtgagcaag ggcgaggagc tgttcaccgg 1080

ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 1140

cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 1200

cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg 1260

cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 1320

aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 1380

cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 1440

caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 1500

ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 1560

catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 1620

cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 1680

ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 1740

tctcggcatg gacgagctgt acaagtaagg atccgggatg cagaaattga tgatctatta 1800

aacaataaag atgtccacta aaatggaagt ttttcctgtc atactttgtt aagaagggtg 1860

agaacagagt acctacattt tgaatggaag gattggagct acgggggtgg gggtggggtg 1920

ggattagata aatgcctgct ctttactgaa ggctctttac tattgcttta tgataatgtt 1980

tcatagttgg atatcataat ttaaacaagc aaaaccaaat taagggccag ctcattcctc 2040

ccactcatga tctatagatc tatagatctc tcgtgggatc attgtttttc tcttgattcc 2100

cactttgtgg ttctaagtac tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg 2160

agatcagcag cctctgttcc acatacactt cattctcagt attgttttgc caagttctaa 2220

ttccatcaga agctggtcga cctgcagggg cgcctgatgc ggtattttct ccttacgcat 2280

ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg 2340

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2400

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 2460

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 2520

accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 2580

tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 2640

gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt 2700

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 2760

tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt 2820

taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 2880

cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 2940

caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 3000

ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 3060

gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3120

aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 3180

tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 3240

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 3300

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 3360

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 3420

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 3480

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 3540

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 3600

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 3660

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 3720

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 3780

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 3840

gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt atcattgcag 3900

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 3960

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 4020

ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 4080

aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 4140

gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 4200

atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 4260

tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 4320

gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga 4380

actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 4440

gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 4500

agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 4560

ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 4620

aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 4680

cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 4740

gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 4800

cctttttacg gttcctggcc ttttgctggc cttttgctca catgt 4845

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence

<400> 22

gggagctttt gattaaagaa gtggt 25

Claims

1. A targeting sequence for constructing a cell strain with fucose transferase 8 function loss is characterized in that the sequence of the targeting sequence is 5'-TGTCAGACGCACTGACAAAG-3' (SEQ ID NO. 1).

2. A cell strain with a function deletion of fucose transferase 8 is characterized in that a protein sequence of the fucose transferase 8 expressed by the cell strain is shown as SEQ ID NO. 2.

3. The method for constructing the fucosyltransferase 8-deficient cell line of claim 2, comprising the steps of:

(1) culturing the cell strain;

wherein, the transfection condition is judged as follows:

4. The building method according to claim 3, characterized in that the building method further comprises the steps of:

(5) sorting monoclonal cells with fluorescence;

5. The method according to any one of claims 3 to 4, wherein the sequence of the plasmid is as shown in SEQ ID NO. 3.

6. The method of constructing a recombinant plasmid according to any one of claims 3 to 4, wherein the base editor comprises the ABEmax adenine base editor, BE3 cytosine base editor.

7. A sequencing primer for identifying the cell line of claim 2 from a wild-type cell line, wherein the sequencing primer has the sequence of 5'-TCTGTTGATTCCAGGTTCCCA-3' (SEQ ID No. 4).

8. The sequencing primer of claim 7, wherein the cell line comprises CHO cells and HEK293 cells.

9. Use of the cell line of claim 2 for the preparation of an antibody medicament.