WO2019052274A1 - 一种筛选cho细胞系高表达位点的方法 - Google Patents

一种筛选cho细胞系高表达位点的方法 Download PDF

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WO2019052274A1
WO2019052274A1 PCT/CN2018/095254 CN2018095254W WO2019052274A1 WO 2019052274 A1 WO2019052274 A1 WO 2019052274A1 CN 2018095254 W CN2018095254 W CN 2018095254W WO 2019052274 A1 WO2019052274 A1 WO 2019052274A1
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cells
lentivirus
hours
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周松涛
金坚
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江南大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the invention relates to the field of biological gene technology, in particular to a method for infecting CHO cells by using a lentivirus with a green fluorescent gene and screening for high expression sites.
  • CHO Chinese Hamster Ovary cells
  • transgenic An increase in copy number does not necessarily mean that the yield of the protein of interest is significantly increased; and even if the protein expression is increased, such expression is often unstable.
  • the currently widely used method for constructing a stable cell is time consuming and labor intensive, mainly because a large number of monoclonal screening processes need to be repeated, so it is generally expected in the field of pathway engineering that a kind of can be obtained in a short time.
  • a method of high expression and stable expression of cells, and the ability to ensure that the products so constructed have the same level of quality as traditional methods to ensure regulatory approval.
  • the traditional method for constructing a foreign protein-expressing cell line is to randomly integrate the foreign gene into the genome of the cell, and then screen through a layer of high-expression monoclonal cells to obtain a cell line with high expression of the foreign protein due to the site effect.
  • the level of expression of the recombinant cells varies, so it takes a long time to select high-expression monoclonal cells; this increases the cost of research and development of biopharmaceuticals.
  • site-specific integration techniques to obtain high-expression monoclonal cells quickly and efficiently has been discussed in academia for more than a decade and has been repeatedly mentioned in various literature reviews.
  • the site-directed integration method ensures that the foreign gene is accurately integrated into the high expression site in the CHO genome, thereby avoiding the interference of positional effects caused by random integration and saving a large amount of time for selecting highly expressed monoclonals.
  • a potential method for finding high expression sites is to extract the genomes of existing high expression cell lines and then pass Targeted Locus Amplification (TLA) was used to identify integration sites, and then CRISPR/Cas9-mediated gene-site integration technology was used to study the level of expression of the found sites.
  • TLA Targeted Locus Amplification
  • CRISPR/Cas9-mediated gene-site integration technology was used to study the level of expression of the found sites.
  • this method objectively requires the researcher to have a high expression cell line, and needs to master the difficult and expensive TLA technology to find the integration site. On the other hand, it needs to be checked one by one through complicated point integration technology. .
  • This method is used to find high expression sites with low flux (only one cell line can be studied at a time), which is technically difficult, costly, and time consuming, and the expression level of a single locus found may not be able to Achieving the desired requirements (there may be some synergistic mechanisms between multi-copy integration of exogenous genes within the cell line, making it a higher level of expression than the combined integration alone).
  • the applicant of the present invention provides a method for screening a high expression site of a CHO cell line.
  • the present invention utilizes novel screening methods to find several highly expressed sites within the CHO genome.
  • the CRISPR/Cas9-mediated site-specific integration technology is combined, the foreign gene to be expressed can be rapidly inserted into these found sites, and the exogenous protein high-expressing cell line can be obtained quickly and efficiently.
  • a method for screening a high expression site of a CHO cell line which integrates a lentivirus with a green fluorescent gene into a CHO cell genome, and collects and expands a monoclonal cell having a high fluorescence expression amount by a flow sorting method. After the cultivation, the corresponding high expression integration sites were screened by chromosome shifting technique.
  • the method includes the following specific steps:
  • plasmids of pLVX-CMV-MCS-T2A-Zsgreen, pSPAX2 and pMD2G were extracted from the endotoxin-free plasmid extraction kit and co-transfected into HEK-293T cells, which were taken twice at 48 hours and 72 hours, respectively.
  • the supernatant, the collected supernatant is subjected to ultracentrifugation and titer to obtain a high titer of lentivirus;
  • the CHO cells were plated on a 24-well plate, cultured at 37 ° C, 5% CO 2 for 24 h, and the old medium was aspirated, and then the fluorescent label of the lentivirus obtained in the step (1) was used for fresh cell culture medium. Dilute, add 250 ⁇ L to the well, infect the cells, add another 250 ⁇ L of cell culture medium after infection for 4 hours at 37 °C, replace the fresh medium after 24 hours, infect 120 hours after 2 passages, and obtain the infected lentivirus.
  • CHO cells Dilute, add 250 ⁇ L to the well, infect the cells, add another 250 ⁇ L of cell culture medium after infection for 4 hours at 37 °C, replace the fresh medium after 24 hours, infect 120 hours after 2 passages, and obtain the infected lentivirus.
  • the cells with the brightest fluorescence intensity were sorted by high-throughput screening method, flow cytometry, and directly inoculated into 96-well plates. After one week, the cells grew into monoclonal colonies, and then under fluorescence microscope. Under the observation, the brightest, normal-form and normal number of cell lines were labeled and transferred to a 24-well plate to expand the culture. After the cells were full, they were transferred to a 6-well plate for culture, and finally expanded to a 10 cm culture dish. , extracting the genomic DNA of each cell line;
  • the preparation of the three plasmids in the step (1) is as follows: three plasmids are transformed into the Tenggen Escherichia coli strain DH5 ⁇ (CB101-03), and the monoclonal bacteria are selected by the Amp-resistant plate, and then inoculated into Amp. In the resistant LB medium, the culture was shaken overnight at 37 ° C and a rotation speed of 250 rpm, and then the bacteria were used to prepare three kinds of plasmids for preparing lentivirus by reusing the Tiangen plasmid mass extraction kit DP117.
  • the specific preparation method of the lentivirus in the step (1) is: inoculation of the HEK-293T cells after resuscitation into a T75 culture flask, and after 1-2 days of culture, the cell density reaches 70-80% confluence.
  • the MOI value in the infection process in the step (2) is less than 1; the specific process of the infection is: the CHO cells are plated on a 24-well plate, cultured at 37 ° C, 5% CO 2 for 24 hours, so that the cells are attached. When the wall and cell convergence are 50%, the old medium is aspirated, and the fluorescent label-linked lentivirus is diluted with fresh complete medium, and 250 ⁇ L of infected cells are taken to ensure MOI ⁇ 1, and infection is carried out at 37 °C. After 4 hours, the medium was supplemented to 500 ⁇ L, and fresh medium was replaced after 24 hours. After 120 hours of infection, CHO cells infected with lentivirus were prepared after 2 passages.
  • the specific method for screening the chromosome walking technique in step (4) is: using the kit Lenti-X The Integration Site Analysis Kit (Clontech, 631263) looked for the top six cell lines in the selected fluorescence intensity and located the integration site of the lentivirus in its genome, which is a high expression site.
  • the present invention dilutes the lentivirus to make the number of virus particles per cell corresponding to less than 1, to facilitate integration of the lentiviral single copy into the genome of the cell.
  • the present invention utilizes a random integration of a lentivirus with a reporter gene to produce cells with different expression levels, and then combines a high-throughput screening method to screen a reporter gene high expression cell line.
  • the present invention utilizes chromosome walking techniques to find all sites where lentiviruses are integrated into the genome of a cell.
  • the present invention discloses for the first time a high expression site associated with the genome of the CHO cell line.
  • Figure 1 is a graph showing the fluorescence of the six cell lines with the highest fluorescence intensity obtained in the present invention.
  • a method for screening a high expression site of a CHO cell line which integrates a lentivirus with a green fluorescent gene into a CHO cell genome, and collects and expands a monoclonal cell having a high fluorescence expression amount by a flow sorting method. After the cultivation, the corresponding high expression integration sites were screened by chromosome shifting technique.
  • the method includes the following specific steps:
  • plasmids pLVX-CMV-MCS-T2A-Zsgreen, pSPAX2 and pMD2G were transformed into Tiangen Escherichia coli strain DH5 ⁇ (CB101-03), and monoclonal bacteria were selected by Amp-resistant plates, and then inoculated into Amp resistance.
  • the LB medium was incubated at 37 ° C, 250 rpm for 24 h, and then the bacteria were extracted through the Tiangen plasmid mass extraction kit DP117 to extract three plasmids for the preparation of lentivirus;
  • the resuscitated HEK-293T cells were inoculated into T75 flasks (the medium was containing 10% FBSDMEM complete medium), and after 1-2 days of culture, after the cell density reached 70-80% confluence, using transfection reagent Lipofiter TM (Han Heng organisms) will 10 ⁇ g pLVX-CMV-MCS-T2A -Zsgreen, 10 ⁇ g pSPAX2 and 10 ⁇ g pMD2G three plasmids were co-transfected into cells in a T75, 6 hours after transfection medium was changed, transfection The virus supernatant was collected twice at 48 h and 72 h, and the cell debris was removed by centrifugation at 4 ° C, 2000 g, 10 min; then the supernatant of the virus stock solution was collected and placed in an ultracentrifuge tube at 4 ° C, 82700 g, and centrifuged for 120 min. Finally, the lentiviral super-dissociation liquid is
  • the HEK-293T cells were digested and counted, diluted to 1 ⁇ 10 5 /mL, and added to a 96-well plate at 100 ⁇ L/well to prepare 6 wells for the virus. Place in a 5 ° CO 2 incubator at 37 ° C. On the next day, prepare 6 1.5 mL EP tubes, add 10 ⁇ L of virus solution to the first EP tube, and then do a 3-fold gradient dilution for 6 dilutions. On the fifth day, the results were observed under a fluorescence microscope. Fresh 10% FBSDMEM complete medium was replaced 6 h before the observation, 80 ⁇ L of medium was aspirated from the well, and then 80 ⁇ L of fresh 10% FBSDMEM complete medium was added.
  • the cells were cultured in a 5% CO 2 incubator at 37 ° C. After 6 h, the results were observed under a fluorescence microscope, and the virus titer was calculated in the percentage of fluorescence of 10 to 30 %. As a result, the lentivirus titer finally obtained was 10 8 .
  • the CHO cells were plated on a 24-well plate, cultured at 37 ° C, 5% CO 2 for 24 h, and the cells were adhered. When the cell concentration was 50%, the old medium was aspirated, and then the step (1) The fluorescent label-attached lentivirus was diluted with fresh cell culture medium, 250 ⁇ L was added to the well, and the cells were infected. After incubating for 4 hours at 37 ° C, another 250 ⁇ L of fresh cell culture medium was added, and fresh culture was replaced after 24 hours. Base, infected with 120 hours after 2 passages, producing CHO cells infected with lentivirus;
  • the cells with the brightest fluorescence intensity were sorted by high-throughput screening method, flow cytometry, and directly inoculated into 96-well plates. After one week, the cells grew into monoclonal colonies, and then under fluorescence microscope. Under the observation, the brightest, normal-form and normal number of cell lines were labeled and transferred to a 24-well plate to expand the culture. After the cells were full, they were transferred to a 6-well plate for culture, and finally expanded to a 10 cm culture dish. , extracting the genomic DNA of each cell line;
  • NW_006880285.1 somewhere from 0-2000000;
  • NW_003613638.1 somewhere from 0-2000000;
  • NW_006882077.1 somewhere in the 0-1000000;
  • NW_006882456.1 Somewhere from 0-2000000;
  • NW_006884764.1 somewhere in the 0-10000000.

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Abstract

一种筛选CHO细胞系高表达位点的方法，所述方法将带有绿色荧光基因的慢病毒整合到CHO细胞基因组中，通过流式分选方法将荧光表达量高的单克隆细胞收集并扩培后，再利用染色体移步技术筛选出相应的高表达整合位点。该方法结合CRISPR/Cas9介导的定点整合技术，可以快速的在已找到的位点上精确插入所要表达的外源基因，可以快速高效获得外源蛋白高表达细胞系。

Description

一种筛选CHO细胞系高表达位点的方法 技术领域
本发明涉及生物基因技术领域，尤其是涉及一种利用带有绿色荧光基因的慢病毒感染CHO细胞，并筛选出高表达位点的方法。
 背景技术
中国仓鼠卵巢细胞(Chinese Hamster Ovary cell, CHO)作为生物制药领域的主力细胞系，已经被开发出许多不同种类的CHO细胞系，甚至包括那些可以用来扩大基因拷贝数的细胞系；然后，转基因拷贝数的增多并不一定意味着目的蛋白产率得到显著提高；且即使蛋白表达增多，此类表达也常常不稳定。此外，当前普遍使用的构建稳转细胞的方法耗时耗力，这主要是因为需要重复大量的单克隆筛选过程，所以当前在途径工程领域普遍期待可以开发出一种能在短时间内，获得高表达及稳定表达的细胞的方法，并且能够确保如此构建出来的产物与传统方法相比，有相同的质量水平，以确保监管机构的批准。
传统构建外源蛋白表达细胞系的方法是通过外源基因随机整合到细胞基因组上，再经过一层层的高表达单克隆细胞筛选，以获得外源蛋白高表达细胞系，由于位点效应的存在，随机整合产生的重组细胞表达水平各异，因此需要后期花费很长的时间用于挑选高表达单克隆细胞；这增加了生物制药的研发成本。利用定点整合技术，快速高效的获得高表达单克隆细胞已经在学术界被讨论了超过十年，并在不同的文献综述中屡次被提及。这主要是因为定点整合方法可以确保外源基因精确整合到CHO基因组内的高表达位点，从而避免因随机整合带来的位置效应的干扰，省下大量用于挑选高表达单克隆的时间，以降低生物制药领域的开发成本。而为了能实现外源基因的定点整合，至关重要的是首先寻找到CHO细胞系基因组内可以高效表达外源基因的那些位点。
 技术问题
目前尚未有已经公开的高表达位点，也并未有公开的寻找高表达位点的方法，潜在的一种寻找高表达位点的方法即提取已有的高表达细胞系的基因组，再通过靶向位点扩增技术（Targeted Locus Amplification，TLA）找出整合位点，再利用CRISPR/Cas9介导的基因定点整合技术，对找到的位点进行表达量的高低进行逐个研究。此方法一方面客观上需要研究人员已经拥有高表达量的细胞系，且需要掌握难度较大且成本昂贵的TLA技术来找到整合位点，另一方面还需要通过繁杂的定点整合技术来逐个排查。通过这套方法来寻找高表达位点，其通量低（每次只能研究一个细胞系），技术难度大，成本昂贵，且耗时长，并且找到的单个位点的表达量也不一定能达到理想的要求（细胞系内外源基因多拷贝整合之间可能存在着一些协同机制，使得其比单独的整合加起来的表达水平更高）。
 技术解决方案
针对现有技术存在的上述问题，本发明申请人提供了一种筛选CHO细胞系高表达位点的方法。本发明利用新颖的筛选方法，找到了若干个CHO基因组内高表达的位点。今后只要再结合CRISPR/Cas9介导的定点整合技术，就可以快速的在这些已找到的位点上精确插入所要表达的外源基因，可以快速高效获得外源蛋白高表达细胞系。
本发明的技术方案如下：
一种筛选CHO细胞系高表达位点的方法，所述方法将带有绿色荧光基因的慢病毒整合到CHO细胞基因组中，通过流式分选方法将荧光表达量高的单克隆细胞收集并扩培后，再利用染色体移步技术筛选出相应的高表达整合位点。
所述方法包括如下具体步骤：
（1）构建带荧光标签的慢病毒；
通过无内毒素质粒提取试剂盒抽提出pLVX-CMV-MCS-T2A-Zsgreen，pSPAX2及pMD2G三种质粒并将其共同转染至HEK-293T细胞，分别于48小时、72小时取两次细胞上清液，收集后的上清液经过超速离心、测滴度获得高滴度的慢病毒；
（2）慢病毒感染CHO细胞；
将CHO细胞铺在24孔板上，在37℃，5% CO 2培养条件下，培养24h，吸去旧培养基，之后将步骤（1）制得的荧光标签的慢病毒用新鲜细胞培养基稀释，加入250μL到孔内，感染细胞，在37℃条件下，感染4小时后补齐另外250μL细胞培养基，24h后更换新鲜培养基，感染120小时经过2次传代后，制得感染慢病毒的CHO细胞；
（3）筛选荧光较强细胞，并进行培养；
通过高通量的筛选方法即流式细胞分选方法，分选出荧光强度最亮的细胞，并将其直接接种到96孔板内；一周后细胞长成单克隆聚落时，再在荧光显微镜下观察，将最亮的、形态正常且数量正常的细胞系标注并转移到24孔板内扩大培养，细胞长满后再转移到6孔板内培养，最后再扩大至10cm的培养皿中培养，抽提出每个细胞系的基因组DNA；
（4）将步骤（3）获得的各个细胞系的基因组DNA利用染色体步移技术筛选慢病毒所有的整合位点，从而找出CHO细胞系高表达位点。
步骤（1）中所述三种质粒的制备过程为：将三种质粒转化到Tiangen公司大肠杆菌菌株DH5α(CB101-03)，通过Amp抗性平板挑选到单克隆细菌后，将其接种到Amp抗性的LB培养基内，于37℃，转速250rpm条件下震荡过夜培养，之后将细菌通过再利用Tiangen公司质粒大量抽提试剂盒DP117，大量提取出用于制备慢病毒的三种质粒。
步骤（1）中所述慢病毒的具体制备方法为：将复苏后的HEK-293T细胞接种到T75培养瓶内，经过1-2天的培养，待其细胞密度，达到70~80%的汇合率后，即可结合汉恒生物Lipofiter TM将pLVX-CMV-MCS-T2A-Zsgreen、pSPAX2及pMD2G三质粒来转染细胞，转染6小时后换液，转染后 48 h和72 h分别两次收集病毒上清，通过4℃，2000g，10 min，离心去除细胞碎片；然后收集病毒原液上清置于超速离心管中，4℃，82700g，离心120 min，最后将慢病毒超离液分装到灭菌处理过的病毒管中。
步骤（2）中所述感染过程中MOI值小于1；所述感染具体过程为：将CHO细胞铺在24孔板上，在37℃，5%CO 2培养条件下，培养24h，使细胞贴壁，细胞汇和度为50%时，吸去旧培养基，再将带荧光标签的慢病毒用新鲜的完全培养基稀释，取250μL感染细胞，确保MOI<1，在37℃条件下，感染4小时后补齐培养基到500μL，并于24h后更换新鲜培养基，感染120小时经过2次传代后，制得感染慢病毒的CHO细胞。
步骤（4）中所述染色体步移技术筛选的具体方法为：利用试剂盒Lenti-X Integration Site Analysis Kit (Clontech, 631263)寻找筛选到的荧光强度排名前六位的细胞系，定位其基因组内慢病毒的整合位点，即为高表达位点。
 有益效果
本发明通过将慢病毒稀释，使每个细胞对应的病毒颗粒数小于1，以利于慢病毒单拷贝整合到细胞基因组内。
本发明利用带有报告基因的慢病毒随机整合产生了表达水平各异的细胞，再结合高通量筛选方法将其中报告基因高表达细胞系筛选出来。
本发明利用染色体步移技术寻找到慢病毒整合到细胞基因组上的所有位点。
本发明首次公布了CHO细胞系基因组内相关的高表达位点。
 附图说明
图1为本发明所得6个荧光强度最大的细胞系的荧光图。
 本发明的实施方式
实施例1
一种筛选CHO细胞系高表达位点的方法，所述方法将带有绿色荧光基因的慢病毒整合到CHO细胞基因组中，通过流式分选方法将荧光表达量高的单克隆细胞收集并扩培后，再利用染色体移步技术筛选出相应的高表达整合位点。
所述方法包括如下具体步骤：
（1）构建带荧光标签的慢病毒；
将pLVX-CMV-MCS-T2A-Zsgreen、pSPAX2及pMD2G三种质粒转化到Tiangen公司大肠杆菌菌株DH5α(CB101-03)，通过Amp抗性平板挑选到单克隆细菌后，将其接种到Amp抗性的LB培养基内，于37℃，转速250rpm条件下震荡培养24h，之后将细菌通过Tiangen公司质粒大量抽提试剂盒DP117，提取出用于制备慢病毒的三种质粒；
将复苏后的HEK-293T细胞接种到T75培养瓶内（培养基为含10%FBSDMEM完全培养基），经过1-2天的培养，待其细胞密度，达到70~80%的汇合率后，利用转染试剂 Lipofiter TM（汉恒生物），将10μg pLVX-CMV-MCS-T2A-Zsgreen、10μg pSPAX2及10μg pMD2G三质粒共同转染至T75内的细胞，转染6小时后换液，转染后 48 h和72 h分别两次收集病毒上清，通过4 ℃，2000g，10 min，离心去除细胞碎片；然后收集病毒原液上清置于超速离心管中，4 ℃，82700g，离心120 min，最后将慢病毒超离液分装到灭菌处理过的病毒管中。对获得的病毒进行滴度确定。
将HEK-293T细胞消化计数后稀释至1×10 5/mL,加入96孔板，100 μL/孔，为病毒准备6个孔。放入37 ℃，5 %CO 2培养箱中培养；第二天，准备6个1.5 mL EP管，第一个EP管中加入10 μL病毒液，然后做3倍梯度稀释，共6个稀释度；第五天，在荧光显微镜下观察结果，在观察结果前6 h需更换新鲜10%FBSDMEM完全培养基，从孔中吸出80 μL培养基，然后加入80 μL新鲜10%FBSDMEM完全培养基，放入37℃，5%CO 2培养箱中培养，6 h后荧光显微镜下观察结果，荧光百分比在10~30 %的孔计算病毒滴度。结果最终获得的慢病毒滴度为10 8。
以下是滴度计算的具体公式：
滴度（TU/mL）=细胞数*荧光百分比*MOI(1)*病毒稀释倍数*10^3
（2）慢病毒感染CHO细胞；
将CHO细胞铺在24孔板上，在37℃，5% CO 2培养条件下，培养24h，使细胞贴壁，细胞汇和度为50%时，吸去旧培养基，之后将步骤（1）制得的带荧光标签的慢病毒用新鲜细胞培养基稀释，加入250μL到孔内，感染细胞，在37℃条件下，感染4小时后补齐另外250μL新鲜细胞培养基，24h后更换新鲜培养基，感染120小时经过2次传代后，制得感染慢病毒的CHO细胞；
（3）筛选荧光较强细胞，并进行培养；
通过高通量的筛选方法即流式细胞分选方法，分选出荧光强度最亮的细胞，并将其直接接种到96孔板内；一周后细胞长成单克隆聚落时，再在荧光显微镜下观察，将最亮的、形态正常且数量正常的细胞系标注并转移到24孔板内扩大培养，细胞长满后再转移到6孔板内培养，最后再扩大至10cm的培养皿中培养，抽提出每个细胞系的基因组DNA；
（4）将步骤（3）获得的各个细胞系的基因组DNA利用染色体步移技术（试剂盒Lenti-X Integration Site Analysis Kit (Clontech, 631263)）筛选慢病毒所有的整合位点，从而找出CHO细胞系高表达位点。具体高荧光强度的细胞系如图1所示，所得高表达位点分别为：
NW_006883358.1，3000000-7000000的某处位点；
NW_006880285.1，0-2000000的某处位点；
NW_003613638.1，0-2000000的某处位点；
NW_006882077.1，0-1000000的某处位点；
NW_006882456.1,  0-2000000的某处位点；
NW_006884764.1，0-100000的某处位点。

Claims (6)

  1. 一种筛选CHO细胞系高表达位点的方法，其特征在于所述方法将带有绿色荧光基因的慢病毒整合到CHO细胞基因组中，通过流式分选方法将荧光表达量高的单克隆细胞收集并扩培后，再利用染色体移步技术筛选出相应的高表达整合位点。
  2. 根据权利要求1所述的方法，其特征在于所述方法包括如下具体步骤：（1）构建带荧光标签的慢病毒；
    通过无内毒素质粒提取试剂盒抽提出pLVX-CMV-MCS-T2A-Zsgreen，pSPAX2及pMD2G三种质粒并将其共同转染至HEK-293T细胞，分别于48小时、72小时取两次细胞上清液，收集后的上清液经过超速离心、测滴度获得高滴度的慢病毒；
    （2）慢病毒感染CHO细胞；
    将CHO细胞铺在24孔板上，在37℃，5% CO 2培养条件下，培养24h，吸去旧培养基，之后将步骤（1）制得的荧光标签的慢病毒用新鲜细胞培养基稀释，加入250μL到孔内，感染细胞，在37℃条件下，感染4小时后补齐另外250μL细胞培养基，24h后更换新鲜培养基，感染120小时经过2次传代后，制得感染慢病毒的CHO细胞；
    （3）筛选荧光较强细胞，并进行培养；
    通过高通量的筛选方法即流式细胞分选方法，分选出荧光强度最亮的细胞，并将其直接接种到96孔板内；一周后细胞长成单克隆聚落时，再在荧光显微镜下观察，将最亮的、形态正常且数量正常的细胞系标注并转移到24孔板内扩大培养，细胞长满后再转移到6孔板内培养，最后再扩大至10cm的培养皿中培养，抽提出每个细胞系的基因组DNA；
    （4）将步骤（3）获得的各个细胞系的基因组DNA利用染色体步移技术筛选慢病毒所有的整合位点，从而找出CHO细胞系高表达位点。
  3. 根据权利要求2所述的方法，其特征在于步骤（1）中所述三种质粒的制备过程为：将三种质粒转化到Tiangen公司大肠杆菌菌株DH5α(CB101-03)，通过Amp抗性平板挑选到单克隆细菌后，将其接种到Amp抗性的LB培养基内，于37℃，转速250rpm条件下震荡过夜培养，之后将细菌通过再利用Tiangen公司质粒大量抽提试剂盒DP117，大量提取出用于制备慢病毒的三种质粒。
  4. 根据权利要求2所述的方法，其特征在于步骤（1）中所述慢病毒的具体制备方法为：将复苏后的HEK-293T细胞接种到T75培养瓶内，经过1-2天的培养，待其细胞密度，达到70~80%的汇合率后，即可结合汉恒生物Lipofiter TM将pLVX-CMV-MCS-T2A-Zsgreen、pSPAX2及pMD2G三质粒来转染细胞，转染6小时后换液，转染后 48 h和72 h分别两次收集病毒上清，通过4℃，2000g，10 min，离心去除细胞碎片；然后收集病毒原液上清置于超速离心管中，4℃，82700g，离心120 min，最后将慢病毒超离液分装到灭菌处理过的病毒管中。
  5. 根据权利要求2所述的方法，其特征在于步骤（2）中所述感染过程中MOI值小于1；所述感染具体过程为：将CHO细胞铺在24孔板上，在37℃，5%CO 2培养条件下，培养24h，使细胞贴壁，细胞汇和度为50%时，吸去旧培养基，再将带荧光标签的慢病毒用新鲜的完全培养基稀释，取250μL感染细胞，确保MOI<1，在37℃条件下，感染4小时后补齐培养基到500μL，并于24h后更换新鲜培养基，感染120小时经过2次传代后，制得感染慢病毒的CHO细胞。
  6. 根据权利要求2所述的方法，其特征在于步骤（4）中所述染色体步移技术筛选的具体方法为：利用试剂盒Lenti-X Integration Site Analysis Kit (Clontech, 631263)寻找筛选到的荧光强度排名前六位的细胞系，定位其基因组内慢病毒的整合位点，即为高表达位点。
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