WO2023093006A1 - 一种cho细胞基因nw_003614092.1内稳定表达蛋白质的位点及其应用 - Google Patents

一种cho细胞基因nw_003614092.1内稳定表达蛋白质的位点及其应用 Download PDF

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WO2023093006A1
WO2023093006A1 PCT/CN2022/099463 CN2022099463W WO2023093006A1 WO 2023093006 A1 WO2023093006 A1 WO 2023093006A1 CN 2022099463 W CN2022099463 W CN 2022099463W WO 2023093006 A1 WO2023093006 A1 WO 2023093006A1
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protein
site
gene
cho
expression
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陈蕴
金坚
丁学峰
瞿丽丽
李华钟
蔡燕飞
杨兆琪
朱景宇
鲁晨
俞琪
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江南大学
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    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the invention relates to a site for stably expressing protein in CHO cell gene NW_003614092.1 and its application, belonging to the field of gene technology.
  • CHO cells Chinese hamster ovary (CHO) cells were established in Dr. Theodore T. Puck's laboratory in 1957. They are immortalized non-secreting cells that rarely secrete endogenous proteins; Post-translational modifications closer to human natural proteins, less susceptible to human virus infection, and large-scale culture in serum-free medium with defined chemical components, etc., are widely used in the field of biopharmaceuticals, and more than 70% of proteins are produced drug.
  • mammalian cells CHO cells have a long culture cycle and high culture costs.
  • the demand for recombinant products such as monoclonal antibodies continues to increase. The continuous growth of demand means that the specific productivity still needs to be optimized.
  • the expression level can be increased by increasing the copy number of the gene, developing a new strong promoter, finding a suitable enhancer, etc.
  • the expression level of most CHO cells is unstable during the long-term culture process, which directly affects the supervision of the product by the regulatory authorities. Approval and listing issues. Therefore, it is very important to construct a CHO expression strain that stably and highly expresses the target gene for the development and marketing of protein drugs.
  • Random integration is the most mature traditional method for constructing protein expression systems, but obtaining stable high-expression cell lines through random integration requires multiple screenings, which is time-consuming and expensive.
  • the loss of expression stability of the cells in the later stage of culture is completely unpredictable: this instability may not occur at all; it may also gradually appear after numerous cell divisions; It is also possible that apparent instability occurs after only a few generations of cell division.
  • the stability issue will not only affect the time to market of the product, but will also conflict with drug regulatory management.
  • the information of random integration sites is not clear, and the site effect of exogenous gene integration will also lead to a significant decrease in the expression level of the target gene. According to existing literature reports, this instability of recombinant CHO cell lines occurs in all recombinant CHO cell lines, making the problem of unstable expression an extremely common problem.
  • the present invention provides a site for stably expressing proteins in the genome of CHO cells.
  • the screening process and time in the cell construction process reduce the cost of research and development.
  • the first object of the present invention is to provide a stable protein expression site in the CHO cell genome, and the stable protein expression site is located in the 1159367-1159567th base of the CHO cell gene NW_003614092.1.
  • nucleotide sequence of bases 1159367-1159567 of the CHO cell gene NW_003614092.1 is shown in SEQ ID NO.1.
  • the site for stably expressing the protein can be recognized by the 5'NNNNNNNNNNNNNNNNNNNGG 3' sequence of the CRISPR/Cas9 technology.
  • the 5'NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGG 3' sequence in the embodiment of the present invention selects the following nine groups of sequences: 5'-TAGGTCATGGGATTCCATGCTGG-3', 5'-TATGGCTTCATCTATGGAGTAGG-3', 5'-GTTCATACAAGTATTAGACTTGG-3', 5'-TCCATAGAAGCCCACCCTGG-3 ', 5'-CCATACCTGGACCCCTAGG-3', 5'-CTTTTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTTGTGG-3 ' Aggaagagggggaggggg-3 ', 5'-AGTGTGTGTGTGTTCCCCATGGGGG-3'.
  • the above-mentioned 9 groups of sequences are distributed within the 1159367-1159567th base of the CHO cell gene NW_003614092.1, covering most of the upper, middle and downstream sequences within the 200 bases of the present invention, indicating that the 200 bases of the present invention All bases can be used as sites for stably expressing proteins.
  • the present invention does not limit the above nine groups of sequences.
  • the above nine groups of sequences are only used as the preferred technical means to introduce the coding gene of the target protein into the stable expression site.
  • the purpose of stably expressing the protein of the present invention can be realized.
  • the protein is a protein with a molecular weight less than 160KDa.
  • the protein is one of polypeptides, functional proteins, antibodies, and fusion proteins.
  • the second object of the present invention is to provide the application of the stable protein expression sites in the CHO cell genome in the stable expression of foreign proteins in CHO cells.
  • the application specifically involves constructing a gene encoding an exogenous protein or polypeptide at the site of stably expressing the protein in the CHO cell gene NW_003614092.1.
  • the third object of the present invention is to provide an expression vector for expressing protein in CHO cells, the coding gene of the protein is located between the 5' homology arm and the 3' homology arm of the expression vector region, the 5' homology arm and the 3' homology arm are sequences with a length of 600 bp upstream and downstream of the stable protein expression site respectively.
  • the expression vector is a vector suitable for expression in CHO cells.
  • the expression vector also includes a promoter sequence located upstream of the gene encoding the protein, and the promoter controls the expression of the protein.
  • the promoters are: CMV (strong mammalian expression promoter derived from human cytomegalovirus), EF-1a (strong mammalian expression promoter derived from human elongation factor 1 ⁇ ), SV40 (simian vacuolar virus 40 derived mammalian expression promoter), PGK1 (mammalian promoter derived from phosphoglycerate kinase gene), UBC (mammalian promoter derived from human ubiquitin C gene), human beta actin ( ⁇ -actin Gene-derived mammalian promoter), CAG (strong hybrid mammalian promoter) and the like.
  • an expression vector for expressing a protein in CHO cells comprising the following steps: inserting the gene encoding the protein into the region between the 5'arm and the 3'arm of the plasmid, making the protein encoding The gene is located downstream of the promoter and is regulated by the promoter to obtain the expression vector for expressing the protein in CHO cells.
  • the fourth object of the present invention is to provide a CHO recombinant cell with site-specific integration and stable expression of protein.
  • the CHO recombinant cell is obtained by using the expression vector for expressing protein in CHO cells and the sgRNA corresponding to the target sequence. Plasmids and Cas9 plasmids were transformed into CHO cells.
  • the target sequence is preferably 5'-TAGGTCATGGGATTCCATGCTGG-3', 5'-TATGGCTTCATCTATGGAGTAGG-3', 5'-GTTCATACAAGTATTTAGACTTGG-3', 5'-TCCATAGATGAAGCCATACCTGG-3', 5'-CCATACCTGGAACCCTTATCAGG-3', 5'-CTTTCCAGCCCAGTCTTTGTAGG-3', 5'-CTTGTGATCATTTTCCCCTCTGG-3', 5'-TATCAGGAAGAGTTTGGAGAGGG-3' or 5'-AGTGTCTGTGTTCTTCCATGGGG-3'.
  • a method for constructing CHO recombinant cells comprising the steps of:
  • the plasmids are respectively the expression vector for expressing protein in CHO cells, the sgRNA plasmid and the Cas9 plasmid corresponding to the target sequence;
  • the stable expression site obtained by the present invention is located within 100 bp upstream and downstream of the 1159467th base of the CHO cell gene NW_003614092.1, that is, the 1159367-1159567th base, which can integrate foreign protein genes and perform stable expression.
  • the present invention integrates the target gene into the stable expression region through site-specific integration, which solves the problem of unclear integration sites caused by random integration; the present invention uses base 1159467 of the stable expression site NW_003614092.1 in the CHO genome.
  • the site-specific integration of exogenous genes within the range of 1159367-1159567 bases upstream and downstream of the base overcomes the expression instability caused by the position effect and the repeated and tedious cell line screening process, reducing the original 6-12 months of screening time to 1-3 months, effectively shortening the R&D time for constructing stable expression cell lines and reducing costs.
  • Fig. 1 is a schematic diagram of fixed-point integration of the present invention
  • Figure 2 shows the expression of EGFP at different passages in cells constructed with different target sequences.
  • Figure 3 shows the expression of HSA in different passages of cells constructed with different target sequences.
  • the method for measuring the average fluorescence intensity of cells culture the cells until the confluence reaches about 90%, digest the cells with 0.25% trypsin, and terminate the digestion with a complete medium equal to the amount of trypsin, and collect the cells in a sterile centrifuge tube at 1000rpm/ Centrifuge for 5 min, discard the supernatant and resuspend with PBS, pass through a cell strainer and collect in a flow loading tube, and use a blank CHO-K1 cell as a control to analyze the fluorescence intensity of the cells with a flow cytometer.
  • the CHO-K1-1d2 cells expressing the Zsgreen1 reporter gene screened by high-throughput flow cytometry were cultured in an adherent culture until they were in good condition, and the CHO cells at this time were regarded as the 0th generation, and then cultured continuously for 20
  • the expression of Zsgreen1 protein in cells at passages 0, 10, and 20 was observed under an inverted fluorescence microscope, and the average fluorescence intensity of cells at passages 0, 10, and 20 was detected by BD flow cytometry.
  • CHO-K1-1d2 cells could still express 100% Zsgreen1 protein in the adherent culture state, and Zsgreen1 protein between different passages
  • the expression levels are basically the same, and there is a strong green fluorescent signal.
  • the CHO-K1-1d2 cells that passed the verification of anchorage stability were subjected to suspension acclimation, and the CHO-K1-1d2 cells that were successfully acclimated to the suspension were continuously passaged for 60 passages, and the success of the suspension acclimation was regarded as the 0th passage.
  • the expression of Zsgreen1 protein in the cells at passages 0, 10, 20, 30, 40, 50, and 60 was observed under an inverted fluorescence microscope, and at the same time, the cells at passages 0, 10, 20, 30, 40, and 50 were detected by flow cytometry. Mean fluorescence intensity of cells at passage 60.
  • CHO-K1-1d2 cells could still express 100% Zsgreen1 protein in suspension culture state, and Zsgreen1 protein expression between different passages The levels are basically the same, and there is a strong green fluorescent signal. It shows that CHO-K1-1d2 cells can stably express the Zsgreen1 reporter gene, and at the same time, it shows that the integrated site of the lentivirus carrying the Zsgreen1 reporter gene is a stable expression site.
  • Lenti-X Integration Site Analysis Kit (Clontech: 631263) related to chromosome walking technology was used to analyze the integration site of lentiviral vector in CHO-K1-1d2 cells. The specific steps are as follows:
  • Collect CHO-K1-1d2 cells use the DNA extraction kit to extract the genome, use DraI, SspI, HpaI three restriction endonucleases to digest the genome at 37°C for 16-18h, and the digestion system is as follows:
  • the digested product was purified and recovered using a PCR purification kit, and the chromosome walking adapter GenomeWalker Adaptor was connected to both ends of the purified digested fragment.
  • the connection system was as follows:
  • a round of PCR reaction system is as follows:
  • the second round of PCR reaction system is as follows:
  • the second round of PCR products were subjected to agarose gel electrophoresis and gel recovery sequencing, and the sequencing was performed according to the kit Lenti-X Integration Site Analysis Kit (Clontech: 631263).
  • the lentivirus integration site information can be obtained by comparing the sequencing results with the CHO cell genome on NCBI.
  • the lentivirus integration site in CHO-K1-1d2 cells is located at base 1159467 of the CHO cell genome NW_003614092.1.
  • CCTOP CRISPR/Cas9 online prediction system uses the CCTOP CRISPR/Cas9 online prediction system to align the sequence of 100 bp upstream and downstream of the site NW_003614092.1 at base 1159467: CCCTCTCCAAACTCTTCCTGATAAGGGTTCCAGGTATGGCTTCATCTATGGAGTAGGAGTCATATTCAATCAGAAAGTGTCTGTGTTCTTCCATGGGGTTCATACAAGTATTAGACTT GGGAGCATGTCTTTTCCAGCCCAGTCTTTGTAGGTCATGGGATTCCATGCTGGGTATGACTTGTGATCATTTTCCCCTCTGGAA (SEQ ID NO.1) was predicted and the target with higher editing efficiency was selected sequence.
  • Sequences in which the predicted editing efficiency was higher than 0.56 were selected as target sequences.
  • Target sequence 5'-TAGGTCATGGGATTCCATGCTGG-3'(SEQ ID NO.2), score 0.75
  • Target sequence 5'-TATGGCTTCATCTATGGAGTAGG-3'(SEQ ID NO.3), score 0.71
  • Target sequence 5'-GTTCATACAAGTATTAGACTTGG-3'(SEQ ID NO.4), score 0.62
  • Target sequence 5'-TCCATAGATGAAGCCATACCTGG-3'(SEQ ID NO.5), score 0.63
  • Target sequence 5'-CCATACCTGGAACCCTTATCAGG-3'(SEQ ID NO.6), score 0.63
  • Target sequence 5'-CTTTCCAGCCCAGTCTTTGTAGG-3'(SEQ ID NO.7), score 0.71
  • Target sequence 5'-CTTGTGATCATTTTCCCCTCTGG-3'(SEQ ID NO.8), score 0.76
  • Target sequence 5'-TATCAGGAAGAGTTTGGAGAGGG-3'(SEQ ID NO.9), score 0.70
  • Target sequence 5'-AGTGTCTGTGTTCTTCCATGGGG-3'(SEQ ID NO.10), score 0.78
  • CRISPR/Cas9-mediated genome editing technology Using CRISPR/Cas9-mediated genome editing technology and homologous recombination, the green fluorescent protein gene (EGFP, 26.7KDa) will be integrated at the target site.
  • CRISPR/Cas9-mediated homologous recombination technology needs to construct sgRNA plasmid and Donor Plasmid, the construction process is as follows:
  • sgRNA-R9 5'TAAAACCATGGAAGAACACAGACACT 3'(SEQ ID NO.28)
  • the PSK-u6-gRNA plasmid is digested with BBsI enzyme, and the vector after digestion is gel recovered;
  • Donor plasma The information of Donor plasma is shown in Figure 1, which was obtained by transforming the existing plasmid vector expressing EGFP. 5'arm and 3'arm are the upstream and downstream homology arms of the target sites recognized by each pair of sgRNAs, with a length of 600bp, and GOI is the integrated target gene.
  • the EGFP sequence of the target gene is carried by the original plasmid.
  • HSA human serum albumin
  • sgRNA-R1 5'TAAAACGCATGGAATCCCATGACCTA 3'
  • sgRNA-R3 5'TAAAACAGTCTAATACTTGTATGAAC 3'
  • sgRNA-R4 5'TAAAACGGTATGGCTTCATCTATGGA 3'
  • sgRNA-R5 5'TAAAACGATAAGGGTTCCAGGTATGG 3'
  • sgRNA-R9 5'TAAAACCATGGAAGAACACAGACACT 3
  • the PSK-u6-gRNA plasmid is digested with BBsI enzyme, and the vector after digestion is gel recovered;
  • Donor plasma information is shown in Figure 1. 5'arm and 3'arm are the upstream and downstream homology arms of the target site, respectively, with a length of 600bp, and GOI is the integrated target gene.
  • Detection method the cell lines obtained in Example 4 were continuously passaged for 60 passages, and the cells were collected for 15 passages per passage, and the cell fluorescence was detected with a flow cytometer and the intensity was detected, as shown in Figure 2.
  • the fluctuation range of green fluorescence intensity between the 0th passage and the 60th passage was not more than 30%.
  • Urinary microalbumin assay kit detects the expression of HSA in the cell line constructed in Example 5
  • Detection method The cell lines obtained in Example 5 were continuously passaged for 60 generations under serum-free culture conditions, and the cell fermentation supernatant of this generation was collected every 15 generations, and the urinary microalbumin assay kit was used to detect HSA content, analysis of detection results showed that the cells constructed according to different target sequences in Example 5 had stable ability to express HSA at different passages, as shown in FIG. 3 .
  • the 9 sets of target sequences screened in Example 3 of the present invention covered most of the upper, middle and downstream sequences within the 200 bp base of the present invention, and the 1159367-1159567 base range in the CHO cell gene NW_003614092.1 of the present invention was all A stable expression cell line with site-specific integration can be successfully constructed, and all of them can stably express the target protein.

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Abstract

提供了一种CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点及其应用，该位点位于CHO细胞基因NW_003614092.1的第1159467碱基处上下游100bp范围内碱基，即第1159367-1159567碱基处，可整合外源蛋白质基因并进行稳定表达，克服了位置效应所带来的表达不稳定性以及反复繁琐的细胞株筛选过程。

Description

一种CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点及其应用 技术领域
本发明涉及一种CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点及其应用，属于基因技术领域。
背景技术
中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞是1957年在Dr.Theodore T.Puck的实验室中建立的，是一种永生化的非分泌型细胞，很少分泌内源蛋白；同时因其具有与人类天然蛋白更接近的翻译后修饰、不易受人类病毒感染、能在化学成分明确的无血清培养基中大规模培养等优点，被广泛应用于生物制药领域，生产了超过70％的蛋白类药物。然而CHO细胞作为哺乳动物细胞，培养周期长，培养成本高，而同时单克隆抗体等重组产品的需求不断增加，持续增长的需求意味着比生产率仍需要优化。虽然通过增加基因的拷贝数、发展新的强启动子、寻找合适的增强子等方式可以提高表达量，但多数CHO细胞在长期培养过程中的表达水平不稳定，直接影响到监管部门对产品的审批与上市问题。因此构建稳定且高表达目的基因的CHO表达株对于蛋白类药物的研发与上市非常重要。
目前有两种构建稳定且高表达细胞株的策略。一种是传统的随机整合与高通量筛选相结合的方法，另一种是利用基因编辑技术与同源定向修复相结合的方法将目的基因定点整合到预定的染色体位点。但是，随机整合由于整合位点的不确定性，以及位置效应的存在，构建的细胞基因型差异巨大，且在长期传代的过程中容易发生表达不稳定的问题，导致后期的筛选过程非常漫长，在工业环境中，通过随机整合方式产生重组细胞系的整个过程通常需要6-12个月；与随机整合相比，定点整合利用基因编辑技术，尤其是近年来应用广泛的CRISPR/Cas9介导的基因组定点编辑技术，大大减少了研发时间和成本，且由于序列已知，定点整合后的信息比随机整合后的信息更加清晰明确。
随机整合是构建蛋白表达系统的最为成熟的传统方法，但通过随机整合方式获得稳定高表达细胞株需要经过多重筛选，耗时长，成本高。同时，对于随机整合获得的细胞系，在培养后期，对于细胞丧失表达稳定性完全无法预测：这种不稳定性可能完全不会发生；也可能在细胞出现了无数次的分裂之后，逐步显现；也可能在细胞仅仅分裂几代后，就出现了明显 的不稳定性。而稳定性问题不仅仅只是会影响到产品上市时间，更会与药品法规管理产生一定冲突。另外随机整合位点的信息不明确，外源基因整合的位点效应也会导致目的基因的表达水平显著下降。根据已有的文献报道，这种重组CHO细胞系的不稳定性出现在了所有的重组CHO细胞系中，使不稳定表达的问题成为了一个极其普遍的问题。
发明内容
为解决上述技术问题，本发明提供一种CHO细胞基因组内稳定表达蛋白质的位点，该位点信息明确，在该位点能够实现蛋白基因的定点整合，且能稳定表达蛋白质，并能大量缩短细胞构建过程中的筛选流程与时间，降低研发成本。
本发明的第一个目的是提供一种CHO细胞基因组内稳定表达蛋白质的位点，所述的稳定表达蛋白质的位点位于CHO细胞基因NW_003614092.1的第1159367-1159567碱基内。
进一步地，所述的CHO细胞基因NW_003614092.1的第1159367-1159567碱基的核苷酸序列如SEQ ID NO.1所示。
进一步地，在采用CRISPR/Cas9技术定点转入目的蛋白质的编码基因时，所述的稳定表达蛋白质的位点可被CRISPR/Cas9技术的5'NNNNNNNNNNNNNNNNNNNNNGG 3'序列识别。
进一步地，所述的5'NNNNNNNNNNNNNNNNNNNNNGG 3'序列在本发明实施例中，选取了以下9组序列：5’-TAGGTCATGGGATTCCATGCTGG-3’、5’-TATGGCTTCATCTATGGAGTAGG-3’、5’-GTTCATACAAGTATTAGACTTGG-3’、5’-TCCATAGATGAAGCCATACCTGG-3’、5’-CCATACCTGGAACCCTTATCAGG-3’、5’-CTTTCCAGCCCAGTCTTTGTAGG-3’、5’-CTTGTGATCATTTTCCCCTCTGG-3’、5’-TATCAGGAAGAGTTTGGAGAGGG-3’、5’-AGTGTCTGTGTTCTTCCATGGGG-3’。
在本发明中，上述9组序列在CHO细胞基因NW_003614092.1的第1159367-1159567碱基内分布覆盖了本发明200个碱基内的上、中、下游大部分序列，说明本发明的200个碱基都可以作为稳定表达蛋白质的位点。
本发明并不限定上述9组序列，上述9组序列只是作为其中优选的技术手段来将目的蛋白质的编码基因导入到稳定表达位点，在采用其他序列，甚至采用其他导入目的基因的手段，仍然可以实现本发明稳定表达蛋白质的目的。
进一步地，所述的蛋白质为分子量小于160KDa的蛋白质。
进一步地，所述的蛋白质为多肽、功能性蛋白质、抗体、融合蛋白质中的一种。
本发明的第二个目的是提供所述的CHO细胞基因组内稳定表达蛋白质的位点在 CHO细胞稳定表达外源蛋白质中的应用。
进一步地，所述的应用具体是将外源蛋白质或多肽的编码基因构建在所述的CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点上。
本发明的第三个目的是提供一种用于在CHO细胞中表达蛋白质的表达载体，所述的蛋白质的编码基因位于所述的表达载体上5’同源臂和3’同源臂中间的区域，所述的5’同源臂和3’同源臂分别为所述的稳定表达蛋白质的位点上下游长度为600bp的序列。
在本发明中，所述的表达载体为适用于CHO细胞表达的载体。
进一步地，所述的表达载体上还包括位于所述的蛋白质的编码基因上游的启动子序列，所述的启动子控制所述的蛋白质的表达。
进一步地，所述的启动子为：CMV(人类巨细胞病毒来源的强哺乳动物表达启动子)、EF-1a(人延长因子1α来源的强哺乳动物表达启动子)、SV40(猿猴空泡病毒40来源的哺乳动物表达启动子)、PGK1(磷酸甘油酸酯激酶基因来源的哺乳动物启动子)、UBC(人类泛素C基因来源的哺乳动物启动子)、human beta actin(β-肌动蛋白基因来源的哺乳动物启动子)、CAG(强杂交哺乳动物启动子)等中的一种。
在本发明中，还包括用于在CHO细胞中表达蛋白质的表达载体的构建方法，包括如下步骤：将蛋白质的编码基因插入到质粒5’arm和3’arm中间的区域，使该蛋白质的编码基因位于启动子的下游并受启动子调控，得到所述的用于在CHO细胞中表达蛋白质的表达载体。
本发明的第四个目的是提供一种定点整合、稳定表达蛋白质的CHO重组细胞，所述的CHO重组细胞通过将所述的用于在CHO细胞中表达蛋白质的表达载体、靶序列对应的sgRNA质粒以及Cas9质粒转入CHO细胞得到。
进一步地，所述的靶序列优选为5’-TAGGTCATGGGATTCCATGCTGG-3’、5’-TATGGCTTCATCTATGGAGTAGG-3’、5’-GTTCATACAAGTATTAGACTTGG-3’、5’-TCCATAGATGAAGCCATACCTGG-3’、5’-CCATACCTGGAACCCTTATCAGG-3’、5’-CTTTCCAGCCCAGTCTTTGTAGG-3’、5’-CTTGTGATCATTTTCCCCTCTGG-3’、5’-TATCAGGAAGAGTTTGGAGAGGG-3’或5’-AGTGTCTGTGTTCTTCCATGGGG-3’。
在本发明中，还提供利用构建CHO重组细胞的方法，包括如下步骤：
(1)利用脂质体转染的方式将质粒载体转染进CHO细胞，得重组CHO细胞池；
其中，所述的质粒分别为所述的用于在CHO细胞中表达蛋白质的表达载体、所述的靶序列对应的sgRNA质粒以及Cas9质粒；
(2)筛选重组细胞池，得到表达外源蛋白质的CHO重组细胞；
(3)贴壁培养CHO重组细胞，检测蛋白质的表达水平，以及对高表达贴壁CHO 重组细胞进行悬浮驯化；
(4)悬浮驯化的CHO重组细胞进行培养及稳定性验证，并检测蛋白质的表达水平。
本发明的有益效果是：
本发明获得的稳定表达位点位于CHO细胞基因NW_003614092.1的第1159467碱基处上下游100bp范围内，即第1159367-1159567碱基处，可整合外源蛋白质基因并进行稳定表达。本发明通过定点整合的方式将目的基因定点整合到稳定表达区域，解决了随机整合所带来的整合位点不明确的问题；本发明通过CHO基因组内稳定表达位点NW_003614092.1的第1159467碱基处上下游1159367-1159567碱基范围内定点整合外源基因克服了位置效应所带来的表达不稳定性以及反复繁琐的细胞株筛选过程，将原本的6-12个月的筛选时间减少至1-3个月，有效的缩短构建稳定表达细胞系的研发时间，降低成本。
附图说明：
图1为本发明的定点整合示意图；
图2为不同靶序列所构建细胞在不同代次时表达EGFP的情况。
图3为不同靶序列所构建细胞在不同代次时表达HSA的情况。
具体实施方式
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
涉及的检测方法：
细胞平均荧光强度测定方法：将细胞培养至汇合度达90％左右，用0.25％胰蛋白酶消化细胞，并用与胰蛋白酶等量的完全培养基终止消化，将细胞收集于无菌离心管中1000rpm/min离心5min，弃上清并用PBS重悬，过细胞滤网收集于流式上样管，以空白CHO-K1细胞为对照用流式细胞仪分析细胞的荧光强度。
实施例1：稳定表达位点的筛选
将利用流式细胞仪高通量筛选出的表达Zsgreen1报告基因的CHO-K1-1d2细胞在贴壁培养状态下培养至状态良好，将此时的CHO细胞视为第0代，再连续培养20个代次，并在倒置荧光显微镜下观察第0、10、20代时细胞表达Zsgreen1蛋白的情况，同时利用BD流式细胞仪检测第0、10、20代时细胞的平均荧光强度。
通过倒置荧光显微镜观察以及流式细胞仪检测，在经过连续传代20个代次后，CHO-K1-1d2细胞在贴壁培养状态下仍然能够百分百表达Zsgreen1蛋白，且不同代次间Zsgreen1蛋白表达水平基本一致，并有较强绿色荧光信号。
对通过贴壁稳定性验证的CHO-K1-1d2细胞进行悬浮驯化，并对悬浮驯化成功的CHO-K1-1d2细胞进行连续传代60个代次，以悬浮驯化成功时作为第0代。并在倒置荧光显微镜下观察第0、10、20、30、40、50、60代时细胞表达Zsgreen1蛋白的情况，同时利用流式细胞仪检测第0、10、20、30、40、50、60代时细胞的平均荧光强度。
通过倒置荧光显微镜观察以及流式细胞仪检测，在经过连续传代60个代次后，CHO-K1-1d2细胞在悬浮培养状态下仍然能够百分百表达Zsgreen1蛋白，且不同代次间Zsgreen1蛋白表达水平基本一致，并有较强绿色荧光信号。表明CHO-K1-1d2细胞能够稳定表达Zsgreen1报告基因，同时表明携带Zsgreen1报告基因的慢病毒所整合的位点为稳定表达位点。
实施例2：慢病毒整合位点分析
利用染色体步移技术相关Lenti-X Integration Site Analysis Kit(Clontech:631263)分析慢病毒载体在CHO-K1-1d2细胞中的整合位点，具体步骤如下：
(1)、慢病毒整合文库的构建
收集CHO-K1-1d2细胞，利用DNA提取试剂盒抽提基因组，使用DraI、SspI、HpaI三种限制性内切酶分别对基因组于37℃酶切16-18h，酶切体系如下：
Figure PCTCN2022099463-appb-000001
将酶切后产物利用PCR纯化试剂盒进行纯化回收，并将染色体步移接头GenomeWalker Adaptor连接至纯化后酶切片段两端，连接体系如下：
Figure PCTCN2022099463-appb-000002
在16℃下孵育过夜，70℃孵育5分钟，终止反应后，向体系中加入32μl TE(10/1，pH 7.5)，获得三个慢病毒整合文库。
(2)、慢病毒整合文库的PCR扩增
对实施例2中(1)步骤所得三个慢病毒整合文库分别进行两轮巢式PCR。利用实施例2中(1)步骤中所连步移接头的接头引物AP1、AP2，慢病毒序列特异引物LSP1、LSP2将LTR区域同临近的CHO-K1细胞基因组区域扩增出来
一轮PCR反应体系如下：
Figure PCTCN2022099463-appb-000003
反应程序如下：
Figure PCTCN2022099463-appb-000004
Figure PCTCN2022099463-appb-000005
取1μl一轮PCR产物用deionized H2O稀释至50μl。
二轮PCR反应体系如下：
Figure PCTCN2022099463-appb-000006
Figure PCTCN2022099463-appb-000007
反应程序如下：
Figure PCTCN2022099463-appb-000008
Figure PCTCN2022099463-appb-000009
(3)、测序与分析
对二轮PCR产物进行琼脂糖凝胶电泳并进行胶回收测序，测序按照试剂盒Lenti-X Integration Site Analysis Kit(Clontech:631263)进行操作。将测序结果在NCBI上与CHO细胞基因组进行比对，即可获得慢病毒整合位点信息，CHO-K1-1d2细胞中慢病毒整合位点位于CHO细胞基因组NW_003614092.1第1159467碱基处。
实施例3：靶序列选择
根据就近原则，使用CCTOP CRISPR/Cas9在线预测系统对位点NW_003614092.1第1159467碱基处上下游各100bp的序列：CCCTCTCCAAACTCTTCCTGATAAGGGTTCCAGGTATGGCTTCATCTATGGAGTAGGAGTCATATTCAATCAGAAAGTGTCTGTGTTCTTCCATGGGGTTCATACAAGTATTAGACTTGGGAGCATGTCTTTCCAGCCCAGTCTTTGTAGGTCATGGGATTCCATGCTGGGTATGACTTGTGATCATTTTCCCCTCTGGAA(SEQ ID NO.1)进行预测，并挑选出编辑效率较高的靶序列。
相关参数设置如下：
1)、NGG后20bp序列内允许前13bp的最大错配碱基数为1；
2)、NGG后所有20bp的错配碱基数为4。
CCTOP CRISPR/Cas9在线预测系统会对识别出的5'NNNNNNNNNNNNNNNNNNNNNGG 3'靶序列进行编辑效率打分，LOW efficacy(score<0.56)；MEDIUM efficacy(0.56<＝score<＝0.74)；HIGH efficacy(score>0.74)。
选择其中预测编辑效率高于0.56的序列作为靶序列。
靶序列5’-TAGGTCATGGGATTCCATGCTGG-3’(SEQ ID NO.2)，score＝0.75
靶序列5’-TATGGCTTCATCTATGGAGTAGG-3’(SEQ ID NO.3)，score＝0.71
靶序列5’-GTTCATACAAGTATTAGACTTGG-3’(SEQ ID NO.4)，score＝0.62
靶序列5’-TCCATAGATGAAGCCATACCTGG-3’(SEQ ID NO.5)，score＝0.63
靶序列5’-CCATACCTGGAACCCTTATCAGG-3’(SEQ ID NO.6)，score＝0.63
靶序列5’-CTTTCCAGCCCAGTCTTTGTAGG-3’(SEQ ID NO.7)，score＝0.71
靶序列5’-CTTGTGATCATTTTCCCCTCTGG-3’(SEQ ID NO.8)，score＝0.76
靶序列5’-TATCAGGAAGAGTTTGGAGAGGG-3’(SEQ ID NO.9)，score＝0.70
靶序列5’-AGTGTCTGTGTTCTTCCATGGGG-3’(SEQ ID NO.10)，score＝0.78
实施例4：定点整合EGFP
利用CRISPR/Cas9介导的基因组定点编辑技术以及同源重组将将绿色荧光蛋白基因(EGFP，26.7KDa)定点整合在靶位点。CRISPR/Cas9介导的同源重组技术需要构建sgRNA质粒以及Donor Plasmid，构建过程如下：
1、sgRNA质粒构建
1)、根据实施例3选择的靶序列，合成寡核苷酸链
sgRNA-F1 5'TTTGTAGGTCATGGGATTCCATGCGT 3'(SEQ ID NO.11)
sgRNA-R1 5'TAAAACGCATGGAATCCCATGACCTA 3'(SEQ ID NO.12)
sgRNA-F2 5'TTTGTATGGCTTCATCTATGGAGTGT 3'(SEQ ID NO.13)
sgRNA-R2 5'TAAAACACTCCATAGATGAAGCCATA 3'(SEQ ID NO.14)
sgRNA-F3 5'TTTGGTTCATACAAGTATTAGACTGT 3'(SEQ ID NO.15)
sgRNA-R3 5'TAAAACAGTCTAATACTTGTATGAAC 3'(SEQ ID NO.16)
sgRNA-F4 5'TTTGTCCATAGATGAAGCCATACCGT 3'(SEQ ID NO.17)
sgRNA-R4 5'TAAAACGGTATGGCTTCATCTATGGA 3'(SEQ ID NO.18)
sgRNA-F5 5'TTTGCCATACCTGGAACCCTTATCGT 3'(SEQ ID NO.19)
sgRNA-R5 5'TAAAACGATAAGGGTTCCAGGTATGG 3'(SEQ ID NO.20)
sgRNA-F6 5'TTTGCTTTCCAGCCCAGTCTTTGTGT 3'(SEQ ID NO.21)
sgRNA-R6 5'TAAAACACAAAGACTGGGCTGGAAAG 3'(SEQ ID NO.22)
sgRNA-F7 5'TTTGCTTGTGATCATTTTCCCCTCGT 3'(SEQ ID NO.23)
sgRNA-R7 5'TAAAACGAGGGGAAAATGATCACAAG 3'(SEQ ID NO.24)
sgRNA-F8 5'TTTGTATCAGGAAGAGTTTGGAGAGT 3'(SEQ ID NO.25)
sgRNA-R8 5'TAAAACTCTCCAAACTCTTCCTGATA 3'(SEQ ID NO.26)
sgRNA-F9 5'TTTGAGTGTCTGTGTTCTTCCATGGT 3'(SEQ ID NO.27)
sgRNA-R9 5'TAAAACCATGGAAGAACACAGACACT 3'(SEQ ID NO.28)
2)、将合成的寡核苷酸链(1-9对)分别进行退火连接
Figure PCTCN2022099463-appb-000010
金属浴95℃5min，自然降至室温；
3)、用BBsI酶对PSK-u6-gRNA质粒进行酶切，对酶切后的载体进行胶回收；
4)、将回收后的质粒载体与退火后的寡核苷酸链进行连接
Figure PCTCN2022099463-appb-000011
22℃连接1h或4℃过夜连接；
5)、转化至DH5α感受态；
6)、挑选阳性克隆利用通用引物M13fwd测序；
7)、扩培阳性克隆菌株并提质粒。
2、Donor plasmid构建：Donor plasmid信息如图1所示，在已有表达EGFP的质粒载体上进行改造所得。5’arm与3’arm分别为每对sgRNA所识别的靶位点的上下游同源臂，长度为600bp，GOI为整合的目的基因。
1)、通过引物设计与PCR扩增获得带有质粒同源片段的位点上下游600bp长度的5’arm与3’arm；
2)、分别利用双酶切与胶回收切除Donor plasmid原有同源臂；
3)、通过同源重组的方法分别连接靶位点对应的5’arm与3’arm；
4)、目的基因EGFP序列为原有质粒自带。
3、将所构建的sgRNA质粒、Donor plasmid与Cas9-DTU质粒(丹麦科技大学Dr.Helene F Kildegaard捐赠)通过Lipofectamine 3000转染试剂以1.8：1.8:1的质量比共转染37℃、5％CO2条件下培养的CHO-K1细胞，同时设置空白对照组。转染24h后，使用10μg/ml 嘌呤霉素进行压力筛选至对照组细胞全部死亡，扩培筛选后细胞池，并利用BD流式细胞仪分选出只发绿色荧光且不发红色荧光的单克隆细胞。
4、将克隆细胞株扩培后，取一部分提取基因组，通过5’junction PCR，3’Junction PCR及out-out PCR进行鉴定，如图1所示。
5、保留阳性克隆细胞株。
实施例5：定点整合HSA
利用CRISPR/Cas9介导的基因组定点编辑技术以及同源同组将表达人血清白蛋白的基因(HSA，68KDa)定点整合在靶位点。CRISPR/Cas9介导的同源重组技术需要构建sgRNA质粒以及Donor Plasmid，构建过程如下：
1、sgRNA质粒构建：
1)、根据实施例3选择的靶序列，合成寡核苷酸链
sgRNA-F1 5'TTTGTAGGTCATGGGATTCCATGCGT 3'
sgRNA-R1 5'TAAAACGCATGGAATCCCATGACCTA 3'
sgRNA-F2 5'TTTGTATGGCTTCATCTATGGAGTGT 3'
sgRNA-R2 5'TAAAACACTCCATAGATGAAGCCATA 3'
sgRNA-F3 5'TTTGGTTCATACAAGTATTAGACTGT 3'
sgRNA-R3 5'TAAAACAGTCTAATACTTGTATGAAC 3'
sgRNA-F4 5'TTTGTCCATAGATGAAGCCATACCGT 3'
sgRNA-R4 5'TAAAACGGTATGGCTTCATCTATGGA 3'
sgRNA-F5 5'TTTGCCATACCTGGAACCCTTATCGT 3'
sgRNA-R5 5'TAAAACGATAAGGGTTCCAGGTATGG 3'
sgRNA-F6 5'TTTGCTTTCCAGCCCAGTCTTTGTGT 3'
sgRNA-R6 5'TAAAACACAAAGACTGGGCTGGAAAG 3'
sgRNA-F7 5'TTTGCTTGTGATCATTTTCCCCTCGT 3'
sgRNA-R7 5'TAAAACGAGGGGAAAATGATCACAAG 3'
sgRNA-F8 5'TTTGTATCAGGAAGAGTTTGGAGAGT 3'
sgRNA-R8 5'TAAAACTCTCCAAACTCTTCCTGATA 3'
sgRNA-F9 5'TTTGAGTGTCTGTGTTCTTCCATGGT 3'
sgRNA-R9 5'TAAAACCATGGAAGAACACAGACACT 3
2)、将合成的寡核苷酸链(1-9对)分别进行退火连接
Figure PCTCN2022099463-appb-000012
Figure PCTCN2022099463-appb-000013
金属浴95℃5min，自然降至室温；
3)、用BBsI酶对PSK-u6-gRNA质粒进行酶切，对酶切后的载体进行胶回收；
4)、将回收后的质粒载体与退火后的寡核苷酸链进行连接
Figure PCTCN2022099463-appb-000014
22℃连接1h或4℃过夜连接；
5)、转化至DH5α感受态；
6)、挑选阳性克隆利用通用引物M13fwd测序；
7)、扩培阳性克隆菌株并提质粒；
2、Donor Plasmid构建：Donor plasmid信息如图1所示。5’arm与3’arm分别为靶位点的上下游同源臂，长度为600bp，GOI为整合的目的基因。
1)、通过引物设计与PCR扩增获得带有质粒同源片段的位点上下游600bp长度的5’arm与3’arm；
2)、分别利用双酶切与胶回收切除Donor plasmid原有同源臂；
3)、通过同源重组的方法分别连接靶位点对应的5’arm与3’arm；
4)、通过PCR扩增获得目的基因HSA，利用酶切链接连接至质粒载体。
3、将所构建的sgRNA质粒、Donor plasmid与Cas9-DTU质粒(丹麦科技大学Dr.Helene F Kildegaard捐赠)通过Lipofectamine 3000转染试剂以1.8：1.8:1的质量比共转染37℃、5％CO 2条件下培养的CHO-K1细胞，同时设置空白对照组。转染24h后，使用10μg/ml嘌呤霉素进行压力筛选至对照组细胞全部死亡，扩培筛选后细胞池，并利用BD流式细胞仪分选出不发任何荧光的单克隆细胞。
4、将克隆细胞株扩培后，取一部分提取基因组，通过5’junction PCR，3’Junction PCR及out-out PCR进行鉴定，如图1所示。
5、保留阳性克隆细胞株。
测试例：
1、用BD流式细胞仪检测实施例4所构建细胞株的绿色荧光强度
检测方法：将实施例4所得细胞株均连续传代60个代次，每传代15个代次收集细胞，用流式细胞仪检测细胞荧光并检测强度，如图2所示，检测结果显示实施例4中根据不同靶序列所构建细胞在连续传代60个代次后均仍有98％以上细胞表达绿色荧光蛋白，且第0代与第60代间绿色荧光强度波动幅度均不超过30％。
2、尿微量白蛋白测定试剂盒检测实施例5所构建细胞株中HSA的表达情况
检测方法：将实施例5所得细胞株在无血清培养条件下均连续传代60个代次，每隔15代收集该代次下细胞发酵上清，利用尿微量白蛋白测定试剂盒检测发酵液中HSA含量，检测结果分析显示，实施例5中根据不同靶序列所构建细胞在不同代次时表达HSA的能力稳定，如图3所示。
在本发明实施例3中筛选得到的9组靶序列覆盖了本发明200bp碱基内的上、中、下游大部分序列，本发明CHO细胞基因NW_003614092.1内的第1159367-1159567碱基范围均能成功构建出定点整合的稳定表达细胞系，且均能稳定表达出目的蛋白。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (10)

  1. 一种CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点，其特征在于，所述的稳定表达蛋白质的位点位于CHO细胞基因NW_003614092.1的第1159367-1159567碱基内。
  2. 根据权利要求1所述的CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点，其特征在于，所述的CHO细胞基因NW_003614092.1的第1159367-1159567碱基的核苷酸序列如SEQ ID NO.1所示。
  3. 根据权利要求1所述的CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点，其特征在于，所述的稳定表达蛋白质的位点可被CRISPR/Cas9技术以5'NNNNNNNNNNNNNNNNNNNNNGG 3'作为靶序列识别。
  4. 根据权利要求1所述的CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点，其特征在于，所述的蛋白质为分子量小于160KDa的蛋白质或多肽。
  5. 权利要求1～4任一项所述的CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点在CHO细胞稳定表达外源蛋白质或多肽中的应用。
  6. 根据权利要求5所述的应用，其特征在于，所述的应用具体是将外源蛋白质或多肽的编码基因构建在所述的CHO细胞基因NW_003614092.1内稳定表达蛋白质的位点上。
  7. 一种用于在CHO细胞中表达蛋白质的表达载体，其特征在于，所述的蛋白质的编码基因位于所述的表达载体上5’同源臂和3’同源臂中间的区域，所述的5’同源臂和3’同源臂分别为权利要求1所述的稳定表达蛋白质的位点上下游长度为600bp的序列。
  8. 根据权利要求7所述的表达载体，其特征在于，所述的表达载体上还包括位于所述的蛋白质的编码基因上游的启动子序列，所述的启动子控制所述的蛋白质的表达。
  9. 根据权利要求8所述的表达载体，其特征在于，所述的启动子为：人类巨细胞病毒来源的强哺乳动物表达启动子、人延长因子1α来源的强哺乳动物表达启动子、猿猴空泡病毒40来源的哺乳动物表达启动子、磷酸甘油酸酯激酶基因来源的哺乳动物启动子、人类泛素C基因来源的哺乳动物启动子、β-肌动蛋白基因来源的哺乳动物启动子、强杂交哺乳动物启动子中的一种。
  10. 一种定点整合表达蛋白质的CHO重组细胞，其特征在于，所述的CHO重组细胞通过将权利要求7所述的用于在CHO细胞中表达蛋白质的表达载体、靶序列对应的sgRNA质粒以及Cas9质粒转入CHO细胞得到。
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