WO2019129215A1 - 谷氨酰胺合成酶基因以及应用 - Google Patents

谷氨酰胺合成酶基因以及应用 Download PDF

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WO2019129215A1
WO2019129215A1 PCT/CN2018/124959 CN2018124959W WO2019129215A1 WO 2019129215 A1 WO2019129215 A1 WO 2019129215A1 CN 2018124959 W CN2018124959 W CN 2018124959W WO 2019129215 A1 WO2019129215 A1 WO 2019129215A1
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gene
seq
mgs
vector
expression
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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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南京金斯瑞生物科技有限公司
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    • C12Y603/01—Acid-ammonia (or amine)ligases (amide synthases)(6.3.1)
    • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

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  • the invention belongs to the field of biotechnology, and particularly relates to a novel glutamine synthetase gene and the application of the glutamine synthetase gene in the construction of a eukaryotic expression vector.
  • the expression system can be divided into a transient expression system and a stable expression system depending on the time difference of the expression of the protein of interest and whether the expression process requires the addition of screening pressure.
  • the transient expression system means that the host cell does not add screening pressure after introducing the expression vector containing the foreign gene, and collects the protein produced by the cell, and the transfected cell gradually loses the target protein as it divides, and the protein production cannot be continued for a long time.
  • the stable expression system refers to the expression vector entering the host cell and selected for culture by screening pressure, selecting the selected cells for protein production, integrating the target gene into the genome of the cell, and with the cell division, the target gene can still exist stably and can last for a long time. Provide the protein of interest. Since stable expression requires pressure selection or even gene amplification, it takes a long time and requires a relatively large amount of manpower.
  • Amplification of foreign genes in mammalian cells is one of the important strategies for increasing the expression level of foreign genes.
  • the entire vector in the expression system consists of two independent and contiguous expression units: a foreign gene expression unit and an amplified gene expression unit. Amplified genes are often also selected markers.
  • the dihydrofolate reductase (DHFR) gene amplification system and the glutamine synthetase (GS) amplification system are the most commonly used gene amplification selection systems.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • glutamine synthetase uses intracellular ammonia and glutamine to add methionine sulfonimide (Methionine sulphoximine, MSX) in the absence of exogenous glutamine. Amplification is carried out to achieve the purpose of increasing the expression level of the target gene. Compared to the DHFR system, the GS system does not require a cumbersome pressurization process to achieve a high level of expression.
  • the existing GS system still has problems in that the expression efficiency cannot meet the production requirements and the high expression cell strain is difficult to screen.
  • the invention provides a novel glutamine synthetase gene (also referred to herein as the GS16034 gene) having the nucleotide sequence set forth in SEQ ID NO:8.
  • the invention provides a eukaryotic expression vector comprising the glutamine synthetase gene.
  • the eukaryotic expression vector is obtained by replacing the initial vector pcDNA3.1 (+) site 2136-2931 bp sequence with the glutamine synthetase gene.
  • the eukaryotic expression vector is prepared by the following steps:
  • the invention provides a method of expressing a protein of interest in a host cell, comprising operably placing the gene encoding the protein of interest and the glutamine synthetase gene in the same eukaryotic expression vector
  • the eukaryotic expression vector is introduced into the host cell.
  • the term "operably” as used herein means that the gene encoding the protein of interest and the glutamine synthetase gene are respectively located in the eukaryotic expression vector, and the eukaryotic expression vector memory can be utilized.
  • the expression elements eg, promoters, enhancers, terminators, etc.
  • the method can further comprise culturing the host cell in the absence of exogenous glutamine and the presence of methionine sulfone imine such that the gene encoding the protein of interest is amplified in the host cell.
  • the host cell is a CHOK1 cell.
  • the protein of interest is an antibody
  • the gene encoding the protein of interest comprises a heavy chain encoding gene and a light chain encoding gene of the antibody.
  • the eukaryotic expression vector containing the glutamine synthetase gene provided by the present invention can significantly improve the expression of the target gene in the host cell, and contributes to the high expression cell line. filter.
  • Figure 1 shows the number of Pools with a level of expression above 20 mg/L in the pools of the vectors pcDNA3.1-mGS-DGV and pcDNA3.1-GS16034-DGV, respectively, when the antibodies mAb01, mAb02, and mAb03 were expressed.
  • Figure 2 shows that the vectors pcDNA3.1-mGS-DGV-mAb01 and pcDNA3.1-GS16034-DGV-mAb01 were treated by the same transfection method, transfection scale, and the same screening procedure, and the highest expression was evaluated by Batch culture. The cumulative expression level of each pool.
  • Figure 3 shows that the vectors pcDNA3.1-mGS-DGV-mAb02 and pcDNA3.1-GS16034-DGV-mAb02 were treated by the same transfection method, transfection scale, and the same screening procedure, and the highest expression was evaluated by Batch culture. The cumulative expression level of each pool.
  • Figure 4 shows that the vectors pcDNA3.1-mGS-DGV-mAb03 and pcDNA3.1-GS16034-DGV-mAb03 were treated by the same transfection method, transfection scale, and the same screening procedure, and the highest expression was obtained after evaluation by Batch culture. Cumulative expression levels of three pools.
  • the initial vector pcDNA3.1(+) was purchased from Thermo Fisher Scientific, and the nucleotide sequence thereof is shown in SEQ ID NO: 9.
  • the CHOK1 cell line was purchased from ATCC under the product number CCL-61. After acclimatization, the medium was composed of CD CHO + 6 mM L-Glutamine, and the medium was purchased from Thermo Fisher.
  • the mGS gene is a glutamine synthetase gene commonly used in existing GS amplification systems, the sequence of which is shown in SEQ ID NO: 7.
  • the GS gene is one of the key factors affecting the expression ability of the vector.
  • the screening of high expression clones also relies on the screening of high expression pools.
  • the present invention will be further illustrated by the expression of three target antibodies mAb01, mAb02, mAb03 by an eukaryotic expression vector containing the glutamine synthetase gene of the present invention.
  • the light chain nucleotide sequence of the antibody mAb01 is shown in SEQ ID NO: 1, and the heavy chain nucleotide sequence is shown in SEQ ID NO: 2.
  • the light chain nucleotide sequence of the antibody is mAb02 is shown in SEQ ID NO: 3, and the heavy chain nucleotide sequence is shown in SEQ ID NO: 4.
  • the light chain nucleotide sequence of the antibody mAb03 is shown in SEQ ID NO: 5, and the heavy chain nucleotide sequence is shown in SEQ ID NO: 6.
  • the eukaryotic expression vector comprising the GS16034 gene and the antibody sequence of interest can be constructed by the following steps: (1) replacing the sequence of the initial vector pcDNA3.1 (+) site 2136-2931 bp with mGS to obtain the intermediate vector pcDNA3.1-mGS, and then The sequence of 232-819 bp of the original vector pcDNA3.1(+) was cloned into the 1252-1253 bp position of the intermediate vector pcDNA3.1-mGS to obtain a new vector pcDNA3.1-mGS-DGV, and the vector pcDNA3.
  • the mGS gene was replaced with the GS16034 gene on 1-mGS-DGV to obtain the vector pcDNA3.1-GS16034-DGV, and the target antibody light chain sequence mAb-LC was inserted between positions 895-1010, between positions 1916-2031.
  • the target antibody heavy chain sequence mAb-HC was inserted to obtain an expression vector containing the GS16034 gene and the antibody sequence of interest.
  • the assembly of the full length sequences of the respective expression vectors can be accomplished using the Gibson method.
  • the entire vector sequence can be divided into 8 fragments such as fragment A/B/C/D/E/F/G/H.
  • the gene sequences of the 8 fragments are shown in SEQ ID NOS: 10-17, respectively.
  • Fragment A was amplified using primers A1:A2 (see sequences SEQ ID NO: 18 and 19); fragment B was amplified using primers B1:B2 (see sequences SEQ ID NO: 20 and 21); primers C1:C2 were used (see sequence SEQ ID NOS: 22 and 23) amplified fragment C; amplified fragment D using primer D1: D2 (sequences see SEQ ID NO: 24 and 25); using primer E1: E2 (see SEQ ID NO: 26 and 27 for the sequence) Amplification of fragment E; amplification of fragment F using primers F1:F2 (see sequences SEQ ID NO: 28 and 29); amplification of fragment G using primers G1:G2 (see sequences SEQ ID NO: 30 and 31); using primer H1 :H2 (see SEQ ID NO: 32 and 33 for the sequence) amplified fragment H.
  • the PCR reaction system was 10 ⁇ L of 10X PBO Buffer, 1 ⁇ L of 10 mM dNTPs, 1 ⁇ L of upstream primer (25 ⁇ M), 1 ⁇ L of downstream primer (25 ⁇ M), 0.5 ⁇ L of template DNA, 1 ⁇ L of PBO Polymerase, and sterile water to 50 ⁇ L.
  • the PCR reaction procedure was: pre-denaturation at 95 ° C for 5 min; 25 cycles (denaturation at 95 ° C for 30 s, annealing at 60 ° C for 90 s, extension at 72 ° C for 1 kb / 30 sec); and finally extension reaction at 72 ° C for 10 min.
  • the PCR product was recovered by gelation and the assembly of the vector was completed by Gibson assembly.
  • the Gibson reaction system was: A 20 ng, B 20 ng, C 20 ng, D 20 ng, E 20 ng, F 20 ng, G 20 ng, H 20 ng, Gibson mix 15 ⁇ l, and water was added to 20 ⁇ l.
  • the reaction procedure was: 50 ° C for 1 hour. 10 ⁇ L of E. coli competent cells DB3.1 was taken out from the Gibson reaction solution, and colonies were picked and identified by PCR.
  • the PCR reaction system was: 10X pfu Buffer 5 ⁇ L, 10 mM dNTPs 0.5 ⁇ L, upstream primer (25 ⁇ M) 0.3 ⁇ L, downstream primer (25 ⁇ M) 0.3 ⁇ L, template DNA 0.15 ⁇ L, PBO Polymerase 0.25 ⁇ L, sterile water to 50 ⁇ L.
  • the PCR reaction procedure was: pre-denaturation at 95 ° C for 3 min; 25 cycles (denaturation at 95 ° C for 20 s, annealing at 60 ° C for 25 s, extension at 72 ° C for 30 s); and finally extension reaction at 72 ° C for 3 min.
  • the obtained expression vector was subjected to full sequence sequencing using the following sequencing primers.
  • CHOK1 cells were cultured in CD CHO medium containing 6 mM L-Glutamine, and cells were seeded at 3-6 x 10 5 cells/ml before transfection. After 24 hours, the following plasmid pcDNA3.1-mGS-DGV-mAb01 was used.
  • the cell suspension was aspirated to remove the cell pellet by centrifugation, and the supernatant was collected for ELISA quantification.
  • the high expression pool obtained by each vector was subjected to batch culture to evaluate the cumulative expression ability of Pool. The evaluation of the cumulative expression level is helpful to make a preliminary judgment on the expression potential and expression stability of Pool.
  • the antibody in the supernatant was captured by a goat anti-human monoclonal antibody in a 96-well plate, and HRP-labeled goat anti-human IgG k was added, and the substrate TMB was added to catalyze the reaction.
  • the absorbance at a wavelength of 450 nm / 650 nm was read.
  • the concentration range of the standard was set to 0-200 ng/ml, and the sample was diluted and subjected to ELISA quantitative detection.
  • the number of highly expressed Pools obtained by the expression vector containing GS16034 gene was significantly increased by 38%, 30%, and compared with the vector containing mGS gene. 110% (as shown in Figure 1).
  • the high expression of the pool obtained by the vector containing the GS16034 gene also showed higher expression levels (as shown in Figures 2-4), in which the average expression level of Top3 was increased, respectively. 25.88%, 42.88% and 87.20%.
  • the expression vector containing the GS16034 gene shows the following advantages: (1) increased the proportion of high expression of Pool; (2) increased the expression level of Pool. With this vector, more high expression levels of Pool can be screened, thereby increasing the efficiency of screening highly expressed cell lines, and screening for highly expressed monoclonal cells. At the same time, the expression level of Pool reflects the expression level of monoclonal cells, so the level of monoclonal expression will be greatly improved. For more high expression of Pool, more pools can be selected for monoclonal screening to enhance the quality of the monoclonal.
  • Primer C1 GTTGACATTGATTATTGACTAGTTA
  • Primer C2 CGGCCGCCACTGTGCTGGATATCTG
  • Primer F2 TCAGTTCTTGTACTGGAAGGGCTCG
  • Primer H2 CTCGAGGAGCTCGGTACCAAGCTTA
  • Primer JJ-CD-F GTACCACATCCGGGCCTACGAT

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Abstract

本发明提供了一种新的谷氨酰胺合成酶基因，其具有如SEQ ID NO：8所示的核苷酸序列。本发明还提供了含有所述谷氨酰胺合成酶基因的真核表达载体。本发明提供的谷氨酰胺合成酶基因可用在GS基因扩增系统中促进目的蛋白的表达以及高表达细胞株的筛选。

Description

谷氨酰胺合成酶基因以及应用 技术领域
本发明属于生物技术领域，具体涉及一种新的谷氨酰胺合成酶基因，以及该谷氨酰胺合成酶基因在真核表达载体构建中的应用。
背景技术
根据目的蛋白表达的时间差异以及表达过程是否需要添加筛选压力，可将表达系统分为瞬时表达系统和稳定表达系统。瞬时表达系统是指宿主细胞在导入含有外源基因的表达载体后不添加筛选压力，收集细胞产生的蛋白，转染后的细胞随着分裂而逐渐丢失目的蛋白，不能持续长久的进行蛋白生产。稳定表达系统是指表达载体进入宿主细胞并经筛选压力选择培养，挑选筛选后的细胞进行蛋白生产，目的基因整合进细胞基因组，并随着细胞分裂，目的基因仍能稳定存在，可以持续长久地提供目的蛋白。由于稳定表达需经过压力选择甚至基因扩增等步骤，需要较长的时间且需要消耗相对多的人力。
近年，伴随着哺乳动物细胞培养技术的发展，提高了单克隆抗体以及其他重组蛋白的表达量。稳定细胞系的构建是重组抗体产业化制备的第一步。作为抗体产业上游关键技术，如何快速、高效建立生产细胞系，是抗体产业发展中的关键问题。
外源基因在哺乳动物细胞内的扩增是提高外源基因表达水平的重要策略之一。一般来讲，表达系统中的整个载体是由两个独立且连在一起的表达单元组成：外源基因表达单元和扩增基因表达单元。扩增基因往往亦为选择标记。二氢叶酸还原酶(dihydrofolate reductase，DHFR)基因扩增系统和谷氨酰胺合成酶(glutamine synthetase，GS)扩增系统是最常用的基因扩增选择系统。DHFR系统是将目的基因和DHFR基因同时或分别转染细胞后加入氨甲喋呤(methotrexate，MTX)加压扩增。GS系统是新近发展的更有效的扩增表达系统。细胞转染GS基因及目的基因后，谷氨酰胺合成酶利用细胞内的氨和谷氨酰胺，在缺乏外源谷氨酰胺的培养条件下，加入甲硫氨酸砜亚胺(Methionine sulphoximine,MSX)进行扩增，达到提高目的基因表达水平的目的。与DHFR系统相比，GS系统不需要进行繁琐的加压过程便可以得到高水平的表达量。
然而，现有的GS系统仍然存在表达效率不能满足生产要求以及高表达细胞株难以筛选的问题。
发明内容
在一方面，本发明提供了一种新的谷氨酰胺合成酶基因(本文中也称作GS16034基因)，其具有如SEQ ID NO：8所示的核苷酸序列。
另一方面，本发明提供了一种真核表达载体，其含有所述谷氨酰胺合成酶基因。
在一个实施方案中，所述真核表达载体是通过将初始载体pcDNA3.1(+)位点2136-2931bp间序列替换为所述谷氨酰胺合成酶基因而得到。
在另一实施方案中，所述真核表达载体是通过以下步骤制备：
1)将初始载体pcDNA3.1(+)位点2136-2931bp间序列替换为mGS基因以获得中间载体pcDNA3.1-mGS，其中所述mGS基因具有如SEQ ID NO.7所示的核苷酸序列；
2)将初始载体pcDNA3.1(+)位点232-819bp间序列克隆进所述中间载体pcDNA3.1-mGS的1252-1253bp位点之间，以获得载体pcDNA3.1-mGS-DGV；以及
3)将所述载体pcDNA3.1-mGS-DGV上mGS基因替换为所述谷氨酰胺合成酶基因。
另一方面，本发明提供了一种在宿主细胞中表达目的蛋白的方法，其包括将所述目的蛋白的编码基因和所述谷氨酰胺合成酶基因可操作地置于同一真核表达载体中并将所述真核表达载体导入所述宿主细胞。这里所用的术语“可操作地”指所述目的蛋白的编码基因和所述所述谷氨酰胺合成酶基因分别位于所述真核表达载体中的适当位置，可以利用所述真核表达载体内存在的表达元件(例如启动子、增强子、终止子等)使得自身序列能够在宿主细胞中得到表达，即产生相应的蛋白。
所述方法还可包括在缺乏外源谷氨酰胺并存在甲硫氨酸砜亚胺的条件下培养所述宿主细胞，以便让所述目的蛋白的编码基因在所述宿主细胞中得到扩增。优选地，所述宿主细胞为CHOK1细胞。
在一些实施方案中，所述目的蛋白为抗体，所述目的蛋白的编码基因包括所述抗体的重链编码基因和轻链编码基因。
与现有的谷氨酰胺合成酶基因相比，含有本发明提供的谷氨酰胺合成酶基因的真核表达载体可以明显改善目的基因在宿主细胞中的表达，有助于对高表达细胞株的筛选。
附图说明
图1显示分别表达抗体mAb01、mAb02、和mAb03时，载体pcDNA3.1-mGS-DGV和pcDNA3.1-GS16034-DGV所筛选到的Pool中，表达水平在20mg/L以上的Pool的数量。
图2显示载体pcDNA3.1-mGS-DGV-mAb01和pcDNA3.1-GS16034-DGV-mAb01经相同的转染方法、转染规模、相同的筛选流程处理后，经Batch culture评估，表达最高的三个Pool的累积表达水平。
图3显示载体pcDNA3.1-mGS-DGV-mAb02和pcDNA3.1-GS16034-DGV-mAb02经相同的转染方法、转染规模、相同的筛选流程处理后，经Batch culture评估，表达最高的三个Pool的累积表达水平。
图4显示载体pcDNA3.1-mGS-DGV-mAb03和pcDNA3.1-GS16034-DGV-mAb03经相同的转染方法、转染规模、相同的筛选流程处理后，经Batch culture评估后，表达最高的三个Pool的累积表达水平。
具体实施方式
下述实施实例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施实例中所使用到的耗材，试剂等，如无特殊说明，均可从商业途径购买。
所述初始载体pcDNA3.1(+)购买自Thermo Fisher Scientific，其核苷酸序列参见SEQ ID NO：9。
CHOK1细胞系购自ATCC，产品号CCL-61。经过驯化后，培养基成分为CD CHO+6mM L-Glutamine，培养基皆购自于Thermo Fisher公司。
mGS基因为现有GS扩增系统中常用的谷氨酰胺合成酶基因，其序列显示在SEQ ID NO：7中。
对于含谷氨酰胺合成酶基因的真核表达载体，GS基因是影响载体表达能力的关键因素之一，同时，高表达克隆的筛选也依靠对高表达Pool(细胞池)的筛选。以下通过 含有本发明谷氨酰胺合成酶基因的真核表达载体对三个目标抗体mAb01、mAb02、mAb03的表达来进一步说明本发明。
实施例
1.重组表达载体构建
抗体mAb01的轻链核苷酸序列如SEQ ID NO：1所示，重链核苷酸序列如SEQ ID NO：2所示。抗体为mAb02的轻链核苷酸序列如SEQ ID NO：3所示，重链核苷酸序列如SEQ ID NO：4所示。抗体mAb03的轻链核苷酸序列如SEQ ID NO：5所示，重链核苷酸序列如SEQ ID NO：6所示。
可以通过以下步骤构建包含GS16034基因和目标抗体序列的真核表达载体：(1)将初始载体pcDNA3.1(+)位点2136-2931bp间序列替换为mGS获得中间载体pcDNA3.1-mGS，再将初始载体pcDNA3.1(+)位点232-819bp间序列克隆进中间载体pcDNA3.1-mGS的1252-1253bp位点之间，获得新的载体pcDNA3.1-mGS-DGV，将载体pcDNA3.1-mGS-DGV上mGS基因替换为GS16034基因，从而获得载体pcDNA3.1-GS16034-DGV，在895-1010位点之间插入目标抗体轻链序列mAb-LC，在1916-2031位点之间插入目标抗体重链序列mAb-HC，从而获得含GS16034基因和目标抗体序列的表达载体。
具体地，可以利用Gibson方法完成各个表达载体全长序列的组装。例如，对于pcDNA3.1-GS16034-DGV-mAb01的组装，可以将完整载体序列分为片段A/B/C/D/E/F/G/H等8个片段。8个片段的基因序列分别参见SEQ ID NO:10-17。
利用引物A1:A2(序列见SEQ ID NO：18和19)扩增片段A；利用引物B1:B2(序列见SEQ ID NO：20和21)扩增片段B；利用引物C1:C2(序列见SEQ ID NO：22和23)扩增片段C；利用引物D1:D2(序列见SEQ ID NO：24和25)扩增片段D；利用引物E1:E2(序列见SEQ ID NO：26和27)扩增片段E；利用引物F1:F2(序列见SEQ ID NO：28和29)扩增片段F；利用引物G1:G2(序列见SEQ ID NO：30和31)扩增片段G；利用引物H1:H2(序列见SEQ ID NO：32和33)扩增片段H。
PCR反应体系为10X PBO Buffer 5μL,10mM dNTPs 1μL、上游引物(25μM)1μL、下游引物(25μM)1μL、模板DNA 0.5μL、PBO Polymerase 1μL、无菌水至50μL。PCR反应程序为：95℃预变性5min；25个循环(95℃变性30s,60℃退火90s,72℃ 延伸1kb/30sec)；最后72℃延伸反应10min。通过胶回收PCR产物，利用Gibson组装的方法，完成载体的组装。
Gibson反应体系为：A 20ng、B 20ng、C 20ng、D 20ng、E 20ng、F 20ng、G20ng、H 20ng、Gibson mix 15μl，加水至20μl。反应程序为：50℃1小时。将Gibson反应液中取出10μL转化大肠杆菌感受态细胞DB3.1，挑取菌落进行利用PCR进行鉴定。利用引物JJ-EA-F和JJ-EA-R(见SEQ ID NO：34和35)进行EA接头的PCR鉴定；利用引物JJ-BC-F和JJ-BC-R(见SEQ ID NO：36和37)进行BC接头的PCR鉴定；利用引物JJ-CD-F和JJ-CD-R(见SEQ ID NO：38和39)进行CD接头的PCR鉴定。PCR反应体系为：10X pfu Buffer 5μL,10mM dNTPs 0.5μL、上游引物(25μM)0.3μL、下游引物(25μM)0.3μL、模板DNA 0.15μL、PBO Polymerase 0.25μL、无菌水至50μL。PCR反应程序为：95℃预变性3min；25个循环(95℃变性20s,60℃退火25s,72℃延伸30s)；最后72℃延伸反应3min。
利用下面的测序引物对获得的表达载体进行全序列测序鉴定。
引物 序列
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
类似地，对于表达载体pcDNA3.1-mGS-DGV-mAb01、pcDNA3.1-mGS-DGV-mAb02、pcDNA3.1-GS16034-DGV-mAb02、pcDNA3.1-mGS-DGV-mAb03和pcDNA3.1-GS16034-DGV-mAb03，皆采用上述Gibson组装的方法获得完整载体。
2.重组质粒转染CHOK1细胞
CHOK1细胞用含有6mM L-Glutamine的CD CHO培养基进行培养，转染前将细胞按3-6x10 5细胞/ml进行接种，24小时后，对于下述质粒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以及pcDNA3.1-GS16034-DGV-mAb03，分别吸取40μg质粒和准备10 7细胞用于转染。利用Bio-Rad电转仪完成电转进行转染。
3.定量ELISA检测细胞培养上清中的的抗体表达
将转染后细胞经MSX压力筛选2周后，吸取细胞悬液经离心去除细胞沉淀后收集上清进行ELISA定量。同时，对各个载体所获得的高表达Pool进行批培养以评估Pool的累积表达能力。对累积表达水平的评估有利于对Pool的表达潜力以及表达稳定性做初步的判断。
具体地，以96孔板内包被羊抗人单抗来捕获上清中的抗体，再加入HRP标记的羊抗人IgGκ，加入底物TMB催化反应。读取450nm/650nm波长的吸光值。在该实验中，设置的标准品浓度范围为0-200ng/ml,将样品进行稀释后进行ELISA定量检测。
根据定量结果，分析pcDNA3.1-mGS-DGV和pcDNA3.1-GS16034-DGV表达载体在表达mAb01、mAb02、mAb03时分别获得的表达水平≥20mg/L的高表达Pool的数量，如图1所示。同时比较pcDNA3.1-mGS-DGV和pcDNA3.1-GS16034-DGV表达载体在表达mAb01、mAb02、mAb03时分别获得的累积水平最高的3个Pool，如图2-4所示。
对于mAb01、mAb02、mAb03的表达，在相同的筛选体系下，相对于含mGS基因的载体，含GS16034基因的表达载体所获得的高表达Pool数量有明显提升，分别增加了38％、30％以及110％(如图1所示)。在对这些高表达Pool进行累积表达水平评估后，含GS16034基因的载体所获得的高表达Pool也显示出更高的表达水平(如图2-4所示)，其中Top3的平均表达水平分别提高了25.88％、42.88％以及87.20％。
综上，含GS16034基因的表达载体体现出来以下的优势，(1)提高了高表达Pool的比例；(2)提高了Pool的表达水平。利用该载体，可以筛选到更多更高表达水平的Pool，从而提高筛选高表达细胞系的效率，更易筛选到高表达的单克隆细胞。同时，Pool的表达水平体现了其中单克隆细胞的表达水平，故筛选到的单克隆表达水平也会 有较大的提升。更多高表达Pool的获得，可以选择更多Pool进行单克隆筛选，从而提升单克隆在质量方面的多样性。
本发明所属领域技术员应理解，以上描述的方法和材料，仅是示例性的，而不应视为限定本发明的范围。
相关序列
SEQ ID NO：1
mAb01的轻链
Figure PCTCN2018124959-appb-000001
SEQ ID NO：2
mAb01的重链
Figure PCTCN2018124959-appb-000002
Figure PCTCN2018124959-appb-000003
SEQ ID NO：3
mAb02的轻链
Figure PCTCN2018124959-appb-000004
SEQ ID NO：4
mAb02的重链
Figure PCTCN2018124959-appb-000005
Figure PCTCN2018124959-appb-000006
SEQ ID NO：5
mAb03的轻链
Figure PCTCN2018124959-appb-000007
SEQ ID NO：6
mAb03的重链
Figure PCTCN2018124959-appb-000008
Figure PCTCN2018124959-appb-000009
SEQ ID NO：7
mGS序列
Figure PCTCN2018124959-appb-000010
SEQ ID NO：8
GS16034序列
Figure PCTCN2018124959-appb-000011
SEQ ID NO：9
pcDNA3.1(+)
Figure PCTCN2018124959-appb-000012
Figure PCTCN2018124959-appb-000013
Figure PCTCN2018124959-appb-000014
SEQ ID NO：10
片段A:
Figure PCTCN2018124959-appb-000015
SEQ ID NO：11
片段B:
Figure PCTCN2018124959-appb-000016
SEQ ID NO：12
片段C:
Figure PCTCN2018124959-appb-000017
SEQ ID NO：13
片段D:
Figure PCTCN2018124959-appb-000018
SEQ ID NO：14
片段E:
Figure PCTCN2018124959-appb-000019
Figure PCTCN2018124959-appb-000020
SEQ ID NO：15
片段F:
Figure PCTCN2018124959-appb-000021
SEQ ID NO：16
片段G:
Figure PCTCN2018124959-appb-000022
Figure PCTCN2018124959-appb-000023
SEQ ID NO：17
片段H:
Figure PCTCN2018124959-appb-000024
SEQ ID NO：18
引物A1：
Figure PCTCN2018124959-appb-000025
SEQ ID NO：19
引物A2：
Figure PCTCN2018124959-appb-000026
SEQ ID NO：20
引物B1:
Figure PCTCN2018124959-appb-000027
SEQ ID NO：21
引物B2:
Figure PCTCN2018124959-appb-000028
SEQ ID NO：22
引物C1:GTTGACATTGATTATTGACTAGTTA
SEQ ID NO：23
引物C2:CGGCCGCCACTGTGCTGGATATCTG
SEQ ID NO：24
引物D1:
Figure PCTCN2018124959-appb-000029
SEQ ID NO：25
引物D2:GGGTTTGAATTCTCATTTGCCGGGGCTCAGGGACA
SEQ ID NO：26
引物E1:
Figure PCTCN2018124959-appb-000030
SEQ ID NO：27
引物E2:GCGAAACGATCCTCATCCTGTCTCT
SEQ ID NO：28
引物F1:
Figure PCTCN2018124959-appb-000031
SEQ ID NO：29
引物F2:TCAGTTCTTGTACTGGAAGGGCTCG
SEQ ID NO：30
引物G1:
Figure PCTCN2018124959-appb-000032
SEQ ID NO：31
引物G2:
Figure PCTCN2018124959-appb-000033
SEQ ID NO：32
引物H1:
Figure PCTCN2018124959-appb-000034
SEQ ID NO：33
引物H2:CTCGAGGAGCTCGGTACCAAGCTTA
SEQ ID NO：34
引物JJ-EA-F:GAGCAAAAACAGGAAGGCAAAA
SEQ ID NO：35
引物JJ-EA-R:CATCGCGAAGCAGCGCAAAACG
SEQ ID NO：36
引物JJ-BC-F:GACGTTGGAGTCCACGTTCTTT
SEQ ID NO：37
引物JJ-BC-R:CTTGCATCTCAGGCCCTCGCCG
SEQ ID NO：38
引物JJ-CD-F:GTACCACATCCGGGCCTACGAT
SEQ ID NO：39
引物JJ-CD-R:GCTCTAGCTAGAGGTCGACGGT

Claims (8)

  1. 一种谷氨酰胺合成酶基因，其具有如SEQ ID NO：8所示的核苷酸序列。
  2. 一种真核表达载体，其含有权利要求1所述的谷氨酰胺合成酶基因。
  3. 如权利要求2所述的真核表达载体，其是通过将初始载体pcDNA3.1(+)位点2136-2931bp间序列替换为所述谷氨酰胺合成酶基因而得到。
  4. 如权利要求2或3所述的真核表达载体，其是通过以下步骤制备：
    1)将初始载体pcDNA3.1(+)位点2136-2931bp间序列替换为mGS基因以获得中间载体pcDNA3.1-mGS，其中所述mGS基因具有如SEQ ID NO.7所示的核苷酸序列；
    2)将初始载体pcDNA3.1(+)位点232-819bp间序列克隆进所述中间载体pcDNA3.1-mGS的1252-1253bp位点之间，以获得载体pcDNA3.1-mGS-DGV；以及
    3)将所述载体pcDNA3.1-mGS-DGV上mGS基因替换为所述谷氨酰胺合成酶基因。
  5. 一种在宿主细胞中表达目的蛋白的方法，包括将所述目的蛋白的编码基因和权利要求1所述的谷氨酰胺合成酶基因可操作地置于同一真核表达载体中并将所述真核表达载体导入所述宿主细胞。
  6. 如权利要求5所述的方法，其中还包括在缺乏外源谷氨酰胺并存在甲硫氨酸砜亚胺的条件下培养所述宿主细胞，以便让所述目的蛋白的编码基因在所述宿主细胞中得到扩增。
  7. 如权利要求5或6所述的方法，其中所述宿主细胞为CHOK1细胞。
  8. 如权利要求5所述的方法，其中所述目的蛋白为抗体，所述目的蛋白的编码基因包括所述抗体的重链编码基因和轻链编码基因。
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