CN109988777A - Glutamine synthetase gene and application - Google Patents

Glutamine synthetase gene and application Download PDF

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CN109988777A
CN109988777A CN201711481610.8A CN201711481610A CN109988777A CN 109988777 A CN109988777 A CN 109988777A CN 201711481610 A CN201711481610 A CN 201711481610A CN 109988777 A CN109988777 A CN 109988777A
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expression
gene
seq
mgs
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时红星
赵钰
陈明月
祁碧玉
贺伟伟
邹晋晋
张丽华
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Nanjing Jinsirui Science and Technology Biology Corp
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Priority to PCT/CN2018/124959 priority patent/WO2019129215A1/en
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Abstract

The present invention provides a kind of new glutamine synthetase genes, have the nucleotide sequence as shown in SEQ ID NO:8.The present invention also provides the carrier for expression of eukaryon for containing the glutamine synthetase gene.Glutamine synthetase gene provided by the invention can be used in the screening of the expression and overexpression cell line that promote destination protein in GS gene-amplification system.

Description

Glutamine synthetase gene and application
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of new glutamine synthetase gene and the paddy ammonia Application of the amide synthetase gene in construction of eukaryotic expression vector.
Background technique
Whether need to add screening pressure according to the time difference of destination protein expression and expression process, can be by expression System is divided into transient expression system and stablizes expression system.Transient expression system refers to that host cell is being imported containing foreign gene Screening pressure is not added after expression vector, collects the albumen that cell generates, and the cell after transfection gradually loses mesh with division Albumen, permanent carry out protein production cannot be continued.Stablize expression system and refers to that expression vector enters host cell and through sieving Pressure selection culture is selected, the cell after selecting screening carries out protein production, and target gene is integrated into cellular genome, and with thin Born of the same parents' division, target gene remain to be stabilized, can continue muchly to provide destination protein.It need to be through excess pressure due to stablizing expression Selection even gene magnification and etc., it takes a long time and needs to consume relatively more manpower.
In recent years, along with the development of mammaliancellculture technology, monoclonal antibody and other recombination eggs are improved White expression quantity.The building of stable cell lines is the first step of recombinant antibodies industrialization preparation.As antibody industry upstream key How quickly, efficiently technology establishes production cell line, is the critical issue in antibody industry development.
Amplification of the foreign gene in mammalian cell is one of the Critical policies for improving foreign gene expression levels.One As for, entire carrier in expression system is made of two independences and the expression unit that connects together: exogenous gene expression Unit and amplification gene expression unit.Amplification gene is often also selected marker.Dihyrofolate reductase (dihydrofolate Reductase, DHFR) gene-amplification system and glutamine synthelase (glutamine synthetase, GS) amplification system It is most common gene magnification selection system.DHFR system is after target gene and DHFR gene are simultaneously or separately transfected cell Methotrexate (methotrexate, MTX) pressurization amplification is added.GS system is the more effective amplification expression system of newly-developed System.After cell transfecting GS gene and target gene, glutamine synthelase utilizes intracellular ammonia and glutamine, outer lacking Under the condition of culture of source glutamine, methionine sulphoximine (Methionine sulphoximine, MSX) is added and is expanded Increase, achievees the purpose that improve destination gene expression level.Compared with DHFR system, GS system does not need to carry out cumbersome pressurization Process can obtain high-caliber expression quantity.
However, existing GS system still has, expression efficiency is not able to satisfy production requirement and overexpression cell line is difficult to The problem of screening.
Summary of the invention
On the one hand, the present invention provides a kind of new glutamine synthetase gene (also referred to herein as GS16034 bases Cause), there is the nucleotide sequence as shown in SEQ ID NO:8.
On the other hand, the present invention provides a kind of carrier for expression of eukaryon, contain the glutamine synthetase gene.
In one embodiment, the carrier for expression of eukaryon is by by starting vector pcDNA3.1 (+) site 2136- Sequence replaces with the glutamine synthetase gene and obtains between 2931bp.
In another embodiment, the carrier for expression of eukaryon is through the following steps that preparation:
1) sequence between starting vector pcDNA3.1 (+) site 2136-2931bp is replaced with into mGS gene to obtain intermediate load Body pcDNA3.1-mGS, wherein the mGS gene has the nucleotide sequence as shown in SEQ ID NO.7；
2) sequence between starting vector pcDNA3.1 (+) site 232-819bp is cloned into the intermediate vector pcDNA3.1- Between the site 1252-1253bp of mGS, to obtain carrier pcDNA3.1-mGS-DGV；And
It 3) is the glutamine synthetase gene by mGS gene replacement on the carrier pcDNA3.1-mGS-DGV.
On the other hand, the method for express express target protein that the present invention provides a kind of in host cell comprising will be described The encoding gene of destination protein and the glutamine synthetase gene are operably positioned in same carrier for expression of eukaryon and will The carrier for expression of eukaryon imports the host cell.Terminology used here " operationally " refers to the coding of the destination protein Gene and the glutamine synthetase gene are located at the appropriate location in the carrier for expression of eukaryon, can use Existing Expression element (such as promoter, enhancer, terminator etc.) enables own sequence in the carrier for expression of eukaryon It is expressed in host cell, that is, generates corresponding albumen.
The method may additionally include shortage external source glutamine and there are institute is cultivated under conditions of methionine sulphoximine Host cell is stated, to allow the encoding gene of the destination protein to be expanded in the host cell.Preferably, the place Chief cell is CHOK1 cell.
In some embodiments, the destination protein is antibody, and the encoding gene of the destination protein includes described anti- The heavy chain encoding gene and light chain encoding gene of body.
Compared with existing glutamine synthetase gene, contain the true of glutamine synthetase gene provided by the invention Nuclear expression carrier can be obviously improved expression of the target gene in host cell, facilitate the screening to overexpression cell line.
Detailed description of the invention
Fig. 1 show respectively express antibody mAb01, mAb02 and mAb03 when, carrier pcDNA3.1-mGS- DGV and In the Pool that pcDNA3.1-GS16034-DGV is screened, quantity of the expression in the Pool of 20mg/L or more.
Fig. 2 shows carrier pcDNA3.1-mGS-DGV-mAb01 and pcDNA3.1-GS16034-DGV-mAb01 through identical After transfection method, transfection scale, the processing of identical screening process, is assessed through Batch culture, express highest three Pool Accumulation expression.
Fig. 3 shows carrier pcDNA3.1-mGS-DGV-mAb02 and pcDNA3.1-GS16034-DGV-mAb02 through identical After transfection method, transfection scale, the processing of identical screening process, is assessed through Batch culture, express highest three Pool Accumulation expression.
Fig. 4 shows carrier pcDNA3.1-mGS-DGV-mAb03 and pcDNA3.1-GS16034-DGV-mAb03 through identical After transfection method, transfection scale, the processing of identical screening process, after Batch culture assessment, highest three are expressed The accumulation expression of Pool.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The consumptive material arrived used in following embodiments, reagent etc. are commercially bought unless otherwise specified.
From Thermo Fisher Scientific, nucleotide sequence is joined for starting vector pcDNA3.1 (+) purchase See SEQ ID NO:9.
CHOK1 cell line is purchased from ATCC, production number CCL-61.After domestication, medium component is CD CHO+6mM L- Glutamine, culture medium are purchased from Thermo Fisher company.
MGS gene is common glutamine synthetase gene in existing GS amplification system, and sequence is shown in SEQ ID In NO:7.
For the carrier for expression of eukaryon containing glutamine synthetase gene, GS gene is the key that influence carrier ability to express One of factor, meanwhile, the screening of high-expression clone also depends on the screening to high expression Pool (cell pool).Below by way of containing originally The carrier for expression of eukaryon of invention glutamine synthetase gene carrys out the expression of three target antibodies mAb01, mAb02, mAb03 Further illustrate the present invention.
Embodiment
1. recombinant expression carrier constructs
The light chain nucleotide sequence of antibody mAb01 is as shown in SEQ ID NO:1, heavy chain nucleotide sequence such as SEQ ID NO: Shown in 2.Antibody is the light chain nucleotide sequence of mAb02 as shown in SEQ ID NO:3, heavy chain nucleotide sequence such as SEQ ID NO: Shown in 4.The light chain nucleotide sequence of antibody mAb03 is as shown in SEQ ID NO:5, heavy chain nucleotide sequence such as SEQ ID NO:6 It is shown.
The carrier for expression of eukaryon comprising GS16034 gene and object antibody sequence can be constructed by following steps: (1) will Sequence replaces with mGS and obtains intermediate vector pcDNA3.1- mGS between starting vector pcDNA3.1 (+) site 2136-2931bp, then Sequence between starting vector pcDNA3.1 (+) site 232-819bp is cloned into the 1252- into intermediate vector pcDNA3.1- mGS Between the site 1253bp, new carrier pcDNA3.1-mGS-DGV is obtained, by mGS gene on carrier pcDNA3.1-mGS-DGV GS16034 gene is replaced with, to obtain carrier pcDNA3.1- GS16034-DGV, is inserted into mesh between the site 895-1010 Labeling antibody sequence of light chain mAb-LC is inserted into target antibody sequence of heavy chain mAb-HC, to obtain between the site 1916-2031 The expression vector of gene containing GS16034 and object antibody sequence.
Specifically, it can use the assembling that Gibson method completes each expression vector full length sequence.For example, for Complete vector sequence can be divided into segment A/B/C/D/E/F/G/H etc. by the assembling of pcDNA3.1-GS16034-DGV-mAb01 8 segments.The gene order of 8 segments is respectively referring to SEQ ID NO:10-17.
Utilize primer A1:A2 (sequence is shown in SEQ ID NO:18 and 19) amplified fragments A；Using primer B1:B2, (sequence is shown in SEQ ID NO:20 and 21) amplified fragments B；Utilize primer C1:C2 (sequence is shown in SEQ ID NO:22 and 23) amplified fragments C；Benefit With primer D1:D2 (sequence is shown in SEQ ID NO:24 and 25) amplified fragments D；Using primer E1:E2 (sequence is shown in SEQ ID NO: 26 and 27) amplified fragments E；Utilize primers F 1:F2 (sequence is shown in SEQ ID NO:28 and 29) amplified fragments F；Utilize primer G1:G2 (sequence is shown in SEQ ID NO:30 and 31) amplified fragments G；It is expanded using primer H1:H2 (sequence is shown in SEQ ID NO:32 and 33) Segment H.
PCR reaction system be 5 μ L, 10mM dNTPs of 10X PBO Buffer, 1 μ L, upstream primer (25 μM) 1 μ L, under Swim primer (25 μM) 1 μ L, 0.5 μ L of template DNA, 1 μ L of PBO Polymerase, sterile water to 50 μ L.PCR response procedures are as follows: 95 DEG C of initial denaturation 5min；25 circulations (95 DEG C of denaturation 30s, 60 DEG C of annealing 90s, 72 DEG C of extension 1kb/30sec)；Last 72 DEG C are prolonged Stretch reaction 10min.PCR product is recycled by glue, the method assembled using Gibson completes the assembling of carrier.
Gibson reaction system are as follows: A 20ng, B 20ng, C 20ng, D 20ng, E 20ng, F 20ng, G 20ng, H 20ng, 15 μ l of Gibson mix, add water to 20 μ l.Response procedures are as follows: 50 DEG C 1 hour.10 μ will be taken out in Gibson reaction solution L converts competent escherichia coli cell DB3.1, and picking colony identified using PCR.Utilize primer JJ-EA-F and JJ- EA-R (see SEQ ID NO:34 and 35) carries out the PCR identification of EA connector；Using primer JJ-BC-F and JJ-BC-R (see SEQ ID NO:36 and the PCR identification for 37) carrying out BC connector；Using primer JJ-CD-F and JJ-CD-R (see SEQ ID NO:38 and 39) into The PCR of row CD connector is identified.PCR reaction system are as follows: 5 μ L, 10mM dNTPs of 10X pfu Buffer, 0.5 μ L, upstream primer (25 μM) 0.3 μ L, downstream primer (25 μM) 0.3 μ L, 0.15 μ L of template DNA, 0.25 μ L of PBO Polymerase, sterile water To 50 μ L.PCR response procedures are as follows: 95 DEG C of initial denaturation 3min；(95 DEG C of denaturation 20s, 60 DEG C of annealing 25s, 72 DEG C are prolonged 25 circulations Stretch 30s)；Last 72 DEG C of extension 3min.
Complete sequence sequencing identification is carried out using the expression vector that following sequencing primer pair obtains.
Primer Sequence
Seq-1 GTGTACGGTGGGAGGTCTATATAAGCA
Seq-2 GCCAAGGTGCAGTGGAAGGTGGACAACG
Seq-3 GAGTTCCGCGTTACATAACTTACG
Seq-4 GCTGGTCCTGCATCATCCTGT
Seq-5 GACGGCTCATTCTTCCTGTAC
Seq-6 GTGGTGGTTACGCGCAGCGTGA
Seq-7 GCCGATTTCGGCCTATTGGTTA
Seq-8 GAGCCTAAGTGCGTGGAAGAAC
Seq-9 GCCACACCAACTTCTCCACCAA
Seq-10 GCATTTTTTTCACTGCATTCTA
Seq-11 GCGCTCTCCTGTTCCGACCCT
Seq-12 GCTTAATCAGTGAGGCACCTAT
Seq-13 GTCAATACGGGATAATACCGCG
Seq-14 GCTTGACCGACAATTGCATGAAGAA
Seq-15 GTGTACGGTGGGAGGTCTATATAAGCA
Similarly, for expression vector pcDNA3.1-mGS-DGV-mAb01, pcDNA3.1-mGS-DGV- mAb02, PcDNA3.1-GS16034-DGV-mAb02, pcDNA3.1-mGS-DGV-mAb03 and pcDNA3.1-GS16034-DGV- MAb03 all obtains complete vector using the method for above-mentioned Gibson assembling.
2. Transfected Recombinant Plasmid CHOK1 cell
CHOK1 cell is cultivated with the CD CHO culture medium containing 6mM L-Glutamine, and cell is pressed 3- before transfection 6x105Cell/ml is inoculated with, after 24 hours, for following plasmid pcDNA3.1-mGS-DGV- mAb01, pcDNA3.1- mGS-DGV-mAb02、pcDNA3.1-mGS-DGV-mAb03、pcDNA3.1- GS16034-DGV-mAb01、pcDNA3.1- GS16034-DGV-mAb02 and pcDNA3.1-GS16034- DGV-mAb03 draws 40 μ g plasmids and preparation 10 respectively7Carefully Born of the same parents are for transfecting.Electricity, which is completed, using Bio-Rad electroporation rotates into capable transfection.
3. quantitative ELISA detects the antibody expression in cells and supernatant
By Transfected cells after MSX pressure is screened 2 weeks, draws cell suspension and collected after centrifugation removes cell precipitation The clear ELISA that carries out is quantified.Meanwhile batch culture is carried out to assess the accumulation of Pool to each carrier high expression Pool obtained Ability to express.Expression potentiality to Pool are conducive to the assessment of accumulation expression and expression stability does and preliminary sentences It is disconnected.
Specifically, the antibody in supernatant is captured with 96 orifice plate endoperidium goat-anti people monoclonal antibodies, adds the goat-anti of HRP label Substrate TMB catalysis reaction is added in human IgG κ.Read the light absorption value of 450nm/650nm wavelength.In this experiment, the standard of setting Product concentration range is 0-200ng/ml, and ELISA quantitative detection is carried out after sample is diluted.
According to quantitative result, analyzes pcDNA3.1-mGS-DGV and pcDNA3.1-GS16034-DGV expression vector and expressing The quantity of the high expression Pool of the expression >=20mg/L obtained respectively when mAb01, mAb02, mAb03, as shown in Figure 1.Together When compare pcDNA3.1-mGS-DGV and pcDNA3.1-GS16034-DGV expression vector when expressing mAb01, mAb02, mAb03 Highest 3 Pool of the accumulation level obtained respectively, as shown in Fig. 2-4.
Expression for mAb01, mAb02, mAb03, under identical screening system, relative to the carrier of the gene containing mGS, The expression vector of the gene containing GS16034 high expression Pool quantity obtained is obviously improved, increased separately 38%, 30% with And 110% (as shown in Figure 1).After carrying out accumulation expression proficiency assessment to these height expression Pool, the load of the gene containing GS16034 Body high expression Pool obtained also shows that higher expression (as in Figure 2-4), wherein the average expression water of Top3 It is flat to be respectively increased 25.88%, 42.88% and 87.20%.
To sum up, the expression vector of the gene containing GS16034 embodies advantage below, and (1) improves high expression Pool's Ratio；(2) expression of Pool is improved.Using the carrier, the Pool of more more high expression levels can be screened, from And the efficiency of screening high-expression cell line is improved, it is easier to screen highly expressed monoclonal cell.Meanwhile the expression water of Pool It is flat to embody the expression of wherein monoclonal cell, therefore the monoclonal expression screened also has biggish promotion.More The acquisitions of more high expression Pool, can choose more Pool and carry out monoclonal screenings, to promote monoclonal in terms of quality Diversity.
Fields technician of the present invention should be understood that process as described above and material, be merely exemplary, without that should regard To limit the scope of the invention.
Correlated series
SEQ ID NO:1
The light chain of mAb01
ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACCGGCGTGCACTCCGAGATT GTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGTCCTGGCGAGAGAGCCACCCTGTCCTGC AGAGCCTCTCAGTCCGTGGGCTCCTCTTACCTGGCCTGTCAGCAGAAGCCCGGCCAGGCTCCC CGGCTGCTGATCTACGGCGCCTTTTCTAGAGCCACCGGCATCCCCGACCGGTTCTCCGGATCT GGCTCTGGCACCGACTTCACCCTGACCATCTCTCGGCTGGAACCCGAGGACTTCGCCGTGTAC TACTGCCAGCAGTACGGCTCCTCCCCCTGGACCTTTGGCCAGGGCACCAAGGTGGAAATCAAG CGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTCCGGC ACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAG GTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCAAGGA CAGCACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGT GTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGG CGAGTGCTGA
SEQ ID NO:2
The heavy chain of mAb01
ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACCGGCGTGCACTCTCAGGTGC AGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCAGATCCCTGAGACTGTCTTGTGCCG CCTCCGGCTTCACCTTCTCCAGCTACACCATGCACTGGCGACAGGCCCCTGGCAAGGGACTGG AATGGGTCACCTTCATCTCTTACGACGGCAACAACAAGTACTACGCCGACTCCGTGAAGGGCC GGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGCGGG CCGAGGACACCGCCATCTACTACTGTGCTAGAACCGGCTGGCTGGGCCCCTTCGATTATTGGG GCCAGGGCACCCTCGTGACCGTGTCCTCTGCTTCTACCAAGGGCCCCTCCGTGTTCCCTCTGGC CCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCGTGAAGGACTACTT CCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGAGTGCACACCTTTCC AGCAGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGTGCCCTCCAGCTCT CTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACACCAAGGTGGACAA GAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAACT GCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCTAAGGACACCCTGATGATCTCCCG GACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCA ATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTAC AACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAA GAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTCCAA GGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCAGGGACGAGCTGA CCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCGTGG AATGGGAGTCCAACGGCCAGCCTGAGAACAACTATAAGACCACCCCCCCTGTGCTGGACTCC GACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGGCAGCAGGGCAA CGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTC CCTGAGCCCCGGCAAATGA
SEQ ID NO:3
The light chain of mAb02
ATGGATATCGTGATGACCCAGACCCCCCTGTCCCTGCCTGTGACACCTGGCGAGCCTGCCATC TCCTGCCGGTCCTCTAAGTCCCTGCTGCACTCCAACGGCATCACCTACCTGTACTGGTATCTGC AGAAGCCCGGCCAGTCCCCCCAGCTGCTGATCTACCAGATGTCCAACCTGGTGTCCGGCGTGC CCGACAGATTCTCCGGCTCTGGCTCTGGCACCGACTTCACCCTGAAGATCTCCCGGGTGGAAG CCGAGGACGTGGGCGTGTACTACTGCGCCCAGAACCTGGAACTGCCCTACACCTTTGGCGGAG GCACCAAGGTGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCG ACGAGCAGCTGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCG AGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTG ACCGAGCAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCC GACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGT GACCAAGTCTTTCAACCGGGGCGAGTGCTGA
SEQ ID NO:4
The heavy chain of mAb02
ATGCAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCCGGCTCCTCCGTGAAGTC CTGCAAGGCTTCCGGCTACGCCTTCTCCTACTCCTGGATCAACTGGGTGCGACAGGCCCCTGG ACAGGGCCTGGAATGGATGGGCAGAATCTTCCCTGGCGACGGCGACACCGACTACAACGGCA AGTTCAAGGGCAGAGTGACCATCACCGCCGACAAGTCCACCTCCACCGCCTACATGGAACTGT CCTCCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGAACGTGTTCGACGGCTACT GGCTGGTGTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTTCTACCAAGGGCCCCT CCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCT CGTGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCAGCGG AGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACT GTGCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAAC ACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTG TCCTGCCCCTGAACTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACAC CCTGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCC TGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTA GAGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATT GGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAA AAGACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAG CAGGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTC CGATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCC CTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGT GGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCC AGAAGTCCCTGTCCCTGAGCCCCGGCAAGTGA
SEQ ID NO:5
The light chain of mAb03
ATGGATATCCAGATGACCCAGTCCCCCTCCTCCGTGTCTGCCTCTGTGGGCGACAGAGTGACC ACCTGTCGGGCCTCCCAGGGCATCTCTTCTTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAG GCCCCCAAGCTGCTGATCTACGGCGCCTCCTCTCTGGAATCCGGCGTGCCCTCTAGATTCTCCG GCTCTGGCTCTGGCACCGACTTTACCCTGACCATCTCCAGCCTGCAGCCCGAGGACTTCGCCTC CTACTACTGCCAGCAGGCCAACTCCTTCCCCTACACCTTTGGCCAGGGCACCAAGCTGGAAAT CAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGCTGAAGTC CGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTG GAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAGGACTCCA AGGACAGCACCTACTCCCTGTCCTCCACCCTGACCCTGTCCAAGGCCGACTACGAGAAGCACA AGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACC GGGGCGAGTGCTGA
SEQ ID NO:6
The heavy chain of mAb03
ATGGAAGTGCAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTGGCAGATCCCTGCTGTCT TGCGCCGCCTCCCGGTTCACCTTCGACGACTACGCTATGCACTGGGTGCGACAGGCCCCTGGC AAGGGACTGGAATGGGTGTCCGGCATCTCCTGGAACTCTGGCAGAATCGGCTACGCCGACTCC GTGAAGGGCAGATTCACCATCTCCCGGGACAACGCCGAGAACTCCCTGTTCCTGCAGATGAAC GGCCTGCGGGCCGAGGATACCGCCCTGTACTATTGCGCCAAGGGCCGGGACTCCTTCGACATC TGGGGCCAGGGCACAATGGTCACCGTGTCCTCCGCTTCCACCAAGGGCCCCTCTGTGTTTCCT CTGGCCCCCTCCAGCAAGTCCACCTCTGGTGGAACAGCCGCCCTGGGCTGCCTCGTGAAGGAC TACTTTCCCGAGCCCGTGACCGTGTCTTGGAACTCCGGCGCTCTGACCTCTGGCGTGCACACCT TTCCAGCCGTGCTGCAGTCTAGCGGCCTGTACTCCCTGTCCTCCGTCGTGACAGTGCCCTCCAG CTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAATACCAAGGTGGA CAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGA ACTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCTC CCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGT TCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAG TACAACTCCACCTACCGGGTGGTGTCCGTGCTGACAGTGCTGCATCAGGACTGGCTGAACGGC AAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAAGACCATCTC CAAGGCTAAGGGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCTCCATCTCGGGACGAGC TGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCGATATCGCCG TGGAATGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGAC TCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGC AACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTG TCCCTGAGCCCCGGCAAATGA
SEQ ID NO:7
MGS sequence
ATGGCCACCTCAGCAAGTTCCCACTTGAACAAAAACATCAAGCAAATGTACTTGTGCCTGCCC CAGGGTGAGAAAGTCCAAGCCATGTATATCTGGGTTGATGGTACTGGAGAAGGACTGCGCTG CAAAACCCGCACCCTGGACTGTGAGCCCAAGTGTGTAGAAGAGTTACCTGAGTGGAATTTTGA TGGCTCTAGTACCTTTCAGTCTGAGGGCTCCAACAGTGACATGTATCTCAGCCCTGTTGCCATG TTTCGGGACCCCTTCCGCAGAGATCCCAACAAGCTGGTGTTCTGTGAAGTTTTCAAGTACAAC CGGAAGCCTGCAGAGACCAATTTAAGGCACTCGTGTAAACGGATAATGGACATGGTGAGCAA CCAGCACCCCTGGTTTGGAATGGAACAGGAGTATACTCTGATGGGAACAGATGGGCACCCTTT TGGTTGGCCTTCCAATGGCTTTCCTGGGCCCCAAGGTCCGTATTACTGTGGTGTGGGCGCAGA CAAAGCCTATGGCAGGGATATCGTGGAGGCTCACTACCGCGCCTGCTTGTATGCTGGGGTCAA GATTACAGGAACAAATGCTGAGGTCATGCCTGCCCAGTGGGAGTTCCAAATAGGACCCTGTG AAGGAATCCGCATGGGAGATCATCTCTGGGTGGCCCGTTTCATCTTGCATCGAGTATGTGAAG ACTTTGGGGTAATAGCAACCTTTGACCCCAAGCCCATTCCTGGGAACTGGAATGGTGCAGGCT GCCATACCAACTTTAGCACCAAGGCCATGCGGGAGGAGAATGGTCTGAAGTAAGTAGCTTCC TCTGGAGCCATCTTTATTCTCATGGGGTGGAAGGGCTTTGTGTTAGGGTTGGGAAAGTTGGAC TTCTCACAAACTACATGCCATGCTCTTCGTGTTTGTCATAAGCCTATCGTTTTGTACCCGTTGG AGAAGTGACAGTACTCTAGGAATAGAATTACAGCTGTGATATGGGAAAGTTGTCACGTAGGT TCAAGCATTTAAAGGTCTTTAGTAAGAACTAAATACACATACAAGCAAGTGGGTGACTTAATT CTTACTGATGGGAAGAGGCCAGTGATGGGGGTCTTCCCATCCAAAAGATAATTGGTATTACAT GTTGAGGACTGGTCTGAAGCACTTGAGACATAGGTCACAAGGCAGACACAGCCTGCATCAAG TATTTATTGGTTTCTTATGGAACTCATGCCTGCTCCTGCCCTTGAAGGACAGGTTTCTAGTGAC AAGGTCAGACCCTCACCTTTACTGCTTCCACCAGGCACATCGAGGAGGCCATCGAGAAACTAA GCAAGCGGCACCGGTACCACATTCGAGCCTACGATCCCAAGGGGGGCCTGGACAATGCCCGT CGTCTGACTGGGTTCCACGAAACGTCCAACATCAACGACTTTTCTGCTGGTGTCGCCAATCGC AGTGCCAGCATCCGCATTCCCCGGACTGTCGGCCAGGAGAAGAAAGGTTACTTTGAAGACCG CCGCCCCTCTGCCAATTGTGACCCCTTTGCAGTGACAGAAGCCATCGTCCGCACATGCCTTCTC AATGAGACTGGCGACGAGCCCTTCCAATACAAAAACTAA
SEQ ID NO:8
GS16034 sequence
ATGACCACCTCCGCCTCCAGCCACCTGAACAAGGGCATCAAACAGGTGTACATGAGCCTGCCC CAGGGCGAGAAGGTGCAGGCCATGTATATCTGGATCGACGGCACCGGCGAGGGCCTGAGATG CAAGACCAGAACCCTGGACTCCGAGCCTAAGTGCGTGGAAGAACTGCCCGAGTGGAACTTCG ACGGCTCCTCCACCCTGCAGTCCGAGGGCTCCAACTCCGACATGTACCTGGTGCCTGCCGCCA TGTTCCGGGACCCTTTCCGGAAGGACCCCAACAAGCTGGTGCTGTGCGAGGTGTTCAAGTACA ACAGACGGCCTGCCGAGACAAACCTGCGGCATACCTGCAAGCGGATCATGGACATGGTGTCC AACCAGCACCCTTGGTTTGGCATGGAACAGGAGTACACCCTGATGGGCACCGACGGCCACCC TTTCGGCTGGCCTTCTAACGGCTTTCCTGGCCCCCAGGGCCCTTACTATTGTGGCGTGGGCGCC GATAGAGCCTACGGCAGAGATATCGTGGAAGCCCACTACCGGGCCTGCCTGTACGCTGGCGT GAAGATCGCTGGCACCAACGCCGAAGTGATGCCCGCCCAGTGGGAGTTCCAGATCGGCCCTT GCGAGGGCATCTCCATGGGCGATCATCTGTGGGTGGCCCGGTTCATCCTGCACAGAGTGTGCG AGGATTTCGGCGTGATCGCCACCTTCGACCCCAAGCCCATCCCCGGCAATTGGAACGGCGCTG GCTGCCACACCAACTTCTCCACCAAGGCCATGCGGGAAGAGAACGGCCTGAAGTACATCGAG GAAGCCATCGAGAAGCTGTCCAAGCGGCACCAGTACCACATCCGGGCCTACGATCCTAAGGG CGGCCTGGACAATGCCAGACGGCTGACCGGCTTTCACGAGACATCCAACATCAACGACTTCTC TGCCGGCGTGGCCAACAGATCCGCCAGCATCAGAATCCCTCGGACCGTGGGACAGGAAAAGA AGGGCTACTTCGAGGACAGGCGGCCCTCCGCCAACTGCGATCCATTCTCTGTGACCGAGGCCC TGATCCGGACCTGCCTGCTGAATGAGACAGGCGACGAGCCCTTCCAGTACAAGAACTGA
SEQ ID NO:9
pcDNA3.1(+)
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCG CATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCA AAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTT AGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACT AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAG TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT CTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGG GAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCACTAGTCCAG TGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTA AACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCC GTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATT GCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG GGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGA GGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAA GCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCG CTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAAT CGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGAT TAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTG GAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCG GTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGA TTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTC CCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGT GTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCA GCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATT CTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGA GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGG AGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGA ACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTG GGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCC GGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCG GCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGC GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGC TCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGC TACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAG CCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTG TTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCC TGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTG GGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGC GGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATC GCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACC AAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGG GCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGG AGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCAT CACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATC AATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCAT AGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCA TAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCAC TGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGG GGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATC AGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAA AAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGA CGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTC GTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCC GGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC TAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGT TTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTAC GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATA TGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTG TCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTA TCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGC CTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTT GCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCA TTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCG GTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGG TTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGG TGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAAC GTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCA CTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATA CTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATAT TTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC CTGACGTC
SEQ ID NO:10
Segment A:
CCCGCCACCATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACCGGCGTGCACT CCGAGATTGTGCTGACCCAGTCTCCCGGCACCCTGTCTCTGAGTCCTGGCGAGAGAGCCACCC TGTCCTGCAGAGCCTCTCAGTCCGTGGGCTCCTCTTACCTGGCCTGGTATCAGCAGAAGCCCG GCCAGGCTCCCCGGCTGCTGATCTACGGCGCCTTTTCTAGAGCCACCGGCATCCCCGACCGGT TCTCCGGATCTGGCTCTGGCACCGACTTCACCCTGACCATCTCTCGGCTGGAACCCGAGGACT TCGCCGTGTACTACTGCCAGCAGTACGGCTCCTCCCCCTGGACCTTTGGCCAGGGCACCAAGG TGGAAATCAAGCGGACCGTGGCCGCTCCCTCCGTGTTCATCTTCCCACCTTCCGACGAGCAGC TGAAGTCCGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGG TGCAGTGGAAGGTGGACAACGCCCTGCAGTCCGGCAACTCCCAGGAATCCGTGACCGAGCAG GACTCCAAGGACAGCACCTACTCCCTGTCCTCTACCCTGACCCTGTCCAAGGCCGACTACGAG AAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGTC TTTCAACCGGGGCGAGTGCTGA
SEQ ID NO:11
Segment B:
CTCGAGCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGC CATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCT TTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGG TGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT GCGGTGGGCTCTATGG
SEQ ID NO:12
Segment C:
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCC ATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGA CCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCA TATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCA GTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTT CCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTT CCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA GGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAAT TAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAG CTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCG
SEQ ID NO:13
Segment D:
GCCGCCACCATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACCGGCGTGCAC TCTCAGGTGCAGCTGGTGGAATCTGGCGGCGGAGTGGTGCAGCCTGGCAGATCCCTGAGACT GTCTTGTGCCGCCTCCGGCTTCACCTTCTCCAGCTACACCATGCACTGGGTGCGACAGGCCCCT GGCAAGGGACTGGAATGGGTCACCTTCATCTCTTACGACGGCAACAACAAGTACTACGCCGA CTCCGTGAAGGGCCGGTTCACCATCTCCCGGGACAACTCCAAGAACACCCTGTACCTGCAGAT GAACTCCCTGCGGGCCGAGGACACCGCCATCTACTACTGTGCTAGAACCGGCTGGCTGGGCCC CTTCGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCCTCTGCTTCTACCAAGGGCCCCTCC GTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCTGGCGGCACAGCCGCTCTGGGCTGCCTCG TGAAGGACTACTTCCCCGAGCCCGTGACAGTGTCTTGGAACTCTGGCGCCCTGACCAGCGGAG TGCACACCTTTCCAGCAGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTCGTGACTGT GCCCTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCTCCAACAC CAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCCCCTTGTC CTGCCCCTGAACTGCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCAAAGCCTAAGGACACCC TGATGATCTCCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTG AAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGA GAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGG CTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCCCCCATCGAAAA GACCATCTCCAAGGCCAAGGGCCAGCCCCGGGAACCCCAGGTGTACACACTGCCCCCTAGCA GGGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTCGTGAAAGGCTTCTACCCCTCCG ATATCGCCGTGGAATGGGAGTCCAACGGCCAGCCTGAGAACAACTATAAGACCACCCCCCCT GTGCTGGACTCCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGTCCCGGTGG CAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAG AAGTCCCTGTCCCTGAGCCCCGGCAAATGA
SEQ ID NO:14
Segment E:
GAATTCAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCC CTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAG GAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGAC AGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGC TTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCG CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAG CGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCT CTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAA CTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA CGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTAT CTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG CTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAA AGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACC AGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTA GTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGC CCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCC TCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTC CCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGC
SEQ ID NO:15
Segment F:
ATGACCACCTCCGCCTCCAGCCACCTGAACAAGGGCATCAAACAGGTGTACATGAGCCTGCCC CAGGGCGAGAAGGTGCAGGCCATGTATATCTGGATCGACGGCACCGGCGAGGGCCTGAGATG CAAGACCAGAACCCTGGACTCCGAGCCTAAGTGCGTGGAAGAACTGCCCGAGTGGAACTTCG ACGGCTCCTCCACCCTGCAGTCCGAGGGCTCCAACTCCGACATGTACCTGGTGCCTGCCGCCA TGTTCCGGGACCCTTTCCGGAAGGACCCCAACAAGCTGGTGCTGTGCGAGGTGTTCAAGTACA ACAGACGGCCTGCCGAGACAAACCTGCGGCATACCTGCAAGCGGATCATGGACATGGTGTCC AACCAGCACCCTTGGTTTGGCATGGAACAGGAGTACACCCTGATGGGCACCGACGGCCACCC TTTCGGCTGGCCTTCTAACGGCTTTCCTGGCCCCCAGGGCCCTTACTATTGTGGCGTGGGCGCC GATAGAGCCTACGGCAGAGATATCGTGGAAGCCCACTACCGGGCCTGCCTGTACGCTGGCGT GAAGATCGCTGGCACCAACGCCGAAGTGATGCCCGCCCAGTGGGAGTTCCAGATCGGCCCTT GCGAGGGCATCTCCATGGGCGATCATCTGTGGGTGGCCCGGTTCATCCTGCACAGAGTGTGCG AGGATTTCGGCGTGATCGCCACCTTCGACCCCAAGCCCATCCCCGGCAATTGGAACGGCGCTG GCTGCCACACCAACTTCTCCACCAAGGCCATGCGGGAAGAGAACGGCCTGAAGTACATCGAG GAAGCCATCGAGAAGCTGTCCAAGCGGCACCAGTACCACATCCGGGCCTACGATCCTAAGGG CGGCCTGGACAATGCCAGACGGCTGACCGGCTTTCACGAGACATCCAACATCAACGACTTCTC TGCCGGCGTGGCCAACAGATCCGCCAGCATCAGAATCCCTCGGACCGTGGGACAGGAAAAGA AGGGCTACTTCGAGGACAGGCGGCCCTCCGCCAACTGCGATCCATTCTCTGTGACCGAGGCCC TGATCCGGACCTGCCTGCTGAATGAGACAGGCGACGAGCCCTTCCAGTACAAGAACTGA
SEQ ID NO:16
Segment G:
GCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTC GATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGG ATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAG CTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCAC TGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGAC CTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTC ACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGT GAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTG CCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTC CGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCA CTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAG CAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGG CTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGC TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATT TGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGG CAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAA CTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAAT TAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAA TGC
SEQ ID NO:17
Segment H:
GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCG CATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCA AAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTT AGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACT AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCA ATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAG TATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTG ACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACT CCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT CTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGG GAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCCTCGAGG
SEQ ID NO:18
Primer A1:
GCTAGCGTTTAAACTTAAGCTTGGTACCGAGCTCCTCGAGGCCGCCACCATGGGCTGGTCCTG CA
SEQ ID NO:19
Primer A2:
TCAGCACTCGCCCCGGTTGAAAGAC
SEQ ID NO:20
Primer B1:
AGGGCCTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGCTGACTCGAGTCTAGAG GGCCCGTTT
SEQ ID NO:21
Primer B2:
TGACCCCGTAATTGATTACTATTAATAACTAGTCAATAATCAATGTCAACCCATAGAGCCCAC CGCATCCCCAGC
SEQ ID NO:22
Primer C1:GTTGACATTGATTATTGACTAGTTA
SEQ ID NO:23
Primer C2:CGGCCGCCACTGTGCTGGATATCTG
SEQ ID NO:24
Primer D1:
ACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCCGGCCGCCACCATGG GCTGGTCCTGCA
SEQ ID NO:25
Primer D2:GGGTTTGAATTCTCATTTGCCGGGGCTCAGGGACA
SEQ ID NO:26
Primer E1:
CTACACCCAGAAGTCCCTGTCCCTGAGCCCCGGCAAATGAGAATTCAAACCCGCTGATCAGCC TCGACTGTGCCT
SEQ ID NO:27
Primer E2:GCGAAACGATCCTCATCCTGTCTCT
SEQ ID NO:28
Primers F 1:
GTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGACCACCTCCG CCTCCAGCCACC
SEQ ID NO:29
Primers F 2:TCAGTTCTTGTACTGGAAGGGCTCG
SEQ ID NO:30
Primer G1:
CCTGCCTGCTGAATGAGACAGGCGACGAGCCCTTCCAGTACAAGAACTGAGCGGGACTCTGG GGTTCGAAATGAC
SEQ ID NO:31
Primer G2:
GCATTGGTAACTGTCAGACCAAGTT
SEQ ID NO:32
Primer H1:
AATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAG
SEQ ID NO:33
Primer H2:CTCGAGGAGCTCGGTACCAAGCTTA
SEQ ID NO:34
Primer JJ-EA-F:GAGCAAAAACAGGAAGGCAAAA
SEQ ID NO:35
Primer JJ-EA-R:CATCGCGAAGCAGCGCAAAACG
SEQ ID NO:36
Primer JJ-BC-F:GACGTTGGAGTCCACGTTCTTT
SEQ ID NO:37
Primer JJ-BC-R:CTTGCATCTCAGGCCCTCGCCG
SEQ ID NO:38
Primer JJ-CD-F:GTACCACATCCGGGCCTACGAT
SEQ ID NO:39
Primer JJ-CD-R:GCTCTAGCTAGAGGTCGACGGT.

Claims (8)

1. a kind of glutamine synthetase gene has the nucleotide sequence as shown in SEQ ID NO:8.
2. a kind of carrier for expression of eukaryon contains glutamine synthetase gene described in claim 1.
It is by by starting vector pcDNA3.1 (+) site 2136- 3. carrier for expression of eukaryon as claimed in claim 2 Sequence replaces with the glutamine synthetase gene and obtains between 2931bp.
4. carrier for expression of eukaryon as claimed in claim 2 or claim 3, through the following steps that preparation:
1) sequence between starting vector pcDNA3.1 (+) site 2136-2931bp is replaced with into mGS gene to obtain intermediate vector PcDNA3.1-mGS, wherein the mGS gene has the nucleotide sequence as shown in SEQ ID NO.7；
2) sequence between starting vector pcDNA3.1 (+) site 232-819bp is cloned into the intermediate vector pcDNA3.1-mGS The site 1252-1253bp between, to obtain carrier pcDNA3.1-mGS-DGV；And
It 3) is the glutamine synthetase gene by mGS gene replacement on the carrier pcDNA3.1-mGS-DGV.
5. a kind of method of the express express target protein in host cell, including wanting the encoding gene of the destination protein and right Glutamine synthetase gene described in asking 1 is operably positioned in same carrier for expression of eukaryon and by the carrier for expression of eukaryon Import the host cell.
6. method as claimed in claim 5, wherein further including lacking external source glutamine and there are methionine sulphoximines Under conditions of cultivate the host cell, to allow the encoding gene of the destination protein to be expanded in the host cell Increase.
7. such as method described in claim 5 or 6, wherein the host cell is CHOK1 cell.
8. method as claimed in claim 5, wherein the destination protein is antibody, the encoding gene of the destination protein includes The heavy chain encoding gene and light chain encoding gene of the antibody.
CN201711481610.8A 2017-12-29 2017-12-29 Glutamine synthetase gene and application Pending CN109988777A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112037853A (en) * 2019-10-30 2020-12-04 东莞太力生物工程有限公司 Method for screening cell strain capable of expressing expected product
WO2023125389A1 (en) * 2021-12-27 2023-07-06 Shanghai Zhenge Biotechnology Co., Ltd. Novel markers for recombinant production system

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3064352A1 (en) 2017-05-24 2018-11-29 Thoeris Gmbh Use of glutamine synthetase for treating hyperammonemia

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468742A (en) * 2012-11-22 2013-12-25 苏州康宁杰瑞生物科技有限公司 GS-DHFRmut double-gene screening expression vector, and preparation method and application thereof
CN104540950A (en) * 2012-06-14 2015-04-22 赛诺菲 Cho expression system

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7244616B2 (en) * 2003-06-27 2007-07-17 Bayer Pharmaceuticals Corporation Use of molecular chaperones for the enhanced production of secreted, recombinant proteins in mammalian cells
EP2791160B1 (en) * 2011-12-16 2022-03-02 ModernaTX, Inc. Modified mrna compositions
CN109219660B (en) * 2016-05-11 2023-01-06 美国安进公司 Direct selection of cells expressing high levels of heteromultimers using complementary vectors in the glutamine synthetase gene

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104540950A (en) * 2012-06-14 2015-04-22 赛诺菲 Cho expression system
CN103468742A (en) * 2012-11-22 2013-12-25 苏州康宁杰瑞生物科技有限公司 GS-DHFRmut double-gene screening expression vector, and preparation method and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112037853A (en) * 2019-10-30 2020-12-04 东莞太力生物工程有限公司 Method for screening cell strain capable of expressing expected product
CN112037853B (en) * 2019-10-30 2021-12-10 深圳太力生物技术有限责任公司 Method for screening cell strain capable of expressing expected product
WO2023125389A1 (en) * 2021-12-27 2023-07-06 Shanghai Zhenge Biotechnology Co., Ltd. Novel markers for recombinant production system

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Application publication date: 20190709