WO2020034584A1 - 先天性免疫系统重构的bhk21细胞群及其细胞克隆增毒应用 - Google Patents

先天性免疫系统重构的bhk21细胞群及其细胞克隆增毒应用 Download PDF

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WO2020034584A1
WO2020034584A1 PCT/CN2019/072274 CN2019072274W WO2020034584A1 WO 2020034584 A1 WO2020034584 A1 WO 2020034584A1 CN 2019072274 W CN2019072274 W CN 2019072274W WO 2020034584 A1 WO2020034584 A1 WO 2020034584A1
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bhk21
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virus proliferation
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冯磊
陈丽
恽君雯
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江苏省农业科学院
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Definitions

  • the invention belongs to the technical field of biotechnology and veterinary biological products engineering, and more particularly relates to a BHK21 cell population for randomly knocking out innate immune system effector protein molecules and the application of its cell clone proliferation virus.
  • BHK21 cells are currently commonly used host cells for the proliferation of livestock and poultry vaccine viruses.
  • Major research institutions and veterinary vaccine manufacturers are conducting research on cell proliferation, cell suspension culture, and virus proliferation processes for the proliferation of various viruses.
  • Research hotspots The innate immune system of the host cell is generated in the early stages of phylogeny and appears in the initial stage of the host's anti-infective response. It performs an immune defense mechanism. It acts as an anti-infection, removes harmful components in the body, autoimmune, and rejects transplant Features.
  • the host cell recognizes the pathogen's invasion through its pathogen-associated recognition receptor, and then initiates interferon and a series of cytokines to resist the invasion of the pathogen.
  • model molecules are expressed in different types of cells or in different organelle regions, such as cell membranes, endosome membranes, lysosomal membranes, cytoplasm, etc., and are mainly responsible for monitoring various component molecules of the pathogen to initiate the inflammatory response and antiviral Immunity.
  • Specific model molecules include Toll-like receptors, RIG-I-like receptors, NOD-like receptors, DAI molecules, AIM2 molecules, and the like.
  • Toll-like receptors include Toll-like receptors, RIG-I-like receptors, NOD-like receptors, DAI molecules, AIM2 molecules, and the like.
  • reducing the signal transduction pathway of the innate immune system and inhibiting the antagonistic effect of virus proliferation is conducive to the efficient amplification of the virus.
  • Using genome editing technology to randomly knock out signal molecules in the innate immune system of BHK21 cells, reduce the innate immune response caused by foreign virus infection, and provide good intracellularity for the high titer proliferation of various viruses in BHK21
  • the CRISPR / Cas9 system is often used to target viral genomic DNA to effectively inhibit viral infection.
  • a gRNA targeting the conservative UL30 gene of PRV was designed, and a PK-15 cell line capable of stably expressing sgRNA and Cas9 endonuclease was successfully constructed.
  • the results showed that after infecting the cells with PRV, the editing effect of the CRISPR / Cas9 system on the UL30 gene of PRV can result in the deletion or insertion of the base of the UL30 gene, and can significantly inhibit the proliferation of PRV in cells.
  • ERp57 is a member of the protein disulfide isomerase family in the endoplasmic reticulum, which can catalyze the synthesis of protein disulfide bonds to promote protein folding.
  • some researchers constructed a guide RNA (gRNA) knockout plasmid pGEM-erp57gRNA of erp57 gene using CRISPR / Cas9 technology and co-transfected it with pCas9-EGFP into humans.
  • gRNA guide RNA
  • A549 cell lines with erp57 gene knockout were obtained by target genome sequencing and western blot detection. There was no significant difference in growth kinetics between erp57 knockout cell line and wild type cells.
  • H1N1 subtype influenza viruses were infected with wild-type and erp57 gene knockout cell lines, respectively, to determine the effect of erp57 gene knockout on influenza virus replication levels. The results showed that the replication of influenza virus in erp57 gene knockout cells was significantly suppressed.
  • the invention provides a method for enhancing virus proliferation, characterized in that the virus is propagated in BHK21 cells undergoing innate immune system remodeling.
  • the present invention provides a method for enhancing virus proliferation, further characterized in that the signaling pathway in the innate immune system in BHK21 cells is reduced.
  • the invention provides a method for enhancing virus proliferation, further characterized in that the reduction of the signaling pathway is achieved by gene knockout of a variety of signal molecules in the innate immune system of BHK21 cells.
  • the present invention provides a method for enhancing virus proliferation, further characterized in that the reduction of the signal transduction pathway is achieved by random combination gene knockout of multiple signal molecules in the innate immune system of BHK21 cells.
  • the invention provides a method for enhancing virus proliferation, further characterized in that the plurality of signal molecules are selected from the group consisting of a recognition receptor protein, an effector protein, a stress protein, a protein kinase, an interferon and its downstream regulatory products. One or more.
  • the invention provides a method for enhancing virus proliferation, which is also characterized in that gene knockout of multiple signal molecules is realized by the CRISPR / Cas9 system.
  • the invention provides a method for enhancing virus proliferation, further characterized in that gene knockout of a plurality of signal molecules includes selecting at least two target sequences of the plurality of signal molecules, and designing a pair for each target sequence DNA primers.
  • the invention provides a method for enhancing virus proliferation, further characterized in that the target sequence is obtained by analyzing the genome sequence of a golden yellow hamster.
  • the present invention provides a method for enhancing virus proliferation, further characterized in that a pUC-gRNA-library is constructed using the DNA primers.
  • the invention provides a method for enhancing virus proliferation, further characterized in that the DNA primers are selected from the group consisting of sequences 1-446.
  • the invention provides a method for enhancing virus proliferation, further characterized in that the virus is selected from the group consisting of pseudorabies virus, Japanese encephalitis virus, foot-and-mouth disease virus, rabies virus, and Newcastle disease virus.
  • the present invention provides a method for enhancing virus proliferation, which is further characterized by establishing a BHK21 cell population for innate immune system reconstruction.
  • the invention provides a method for enhancing virus proliferation, which is further characterized by selecting a population of BHK21 cells reconstituted by the innate immune system to obtain the optimal BHK21 cells reconstituted by the innate immune system for virus proliferation.
  • the present invention provides a method for enhancing virus proliferation, which is further characterized in that the progeny virus proliferated from BHK21 cells with the best innate immune system reconstruction obtained still produces positive lesions when diluted to 10-8 .
  • the invention provides a method for enhancing virus proliferation, which is further characterized in that a pUC-gRNA-library is constructed using a gRNA expression vector backbone, which contains a U6 promoter, an RNA scaffold, and a polyA signal sequence.
  • the present invention provides a method for enhancing virus proliferation, which is further characterized in that, during transfection of pUC-gRNA-library into BHK21 cells, a different culture medium is used than when the BHK21 cells are initially cultured.
  • the invention provides a method for enhancing virus proliferation, further characterized in that Opti-MEM medium is used during the transfection of pUC-gRNA-library to BHK21 cells.
  • the present invention provides a method for enhancing virus proliferation, which is also characterized in that the transfection complex is mixed evenly by dropwise addition during pUC-gRNA-library transfection of BHK21 cells.
  • the invention provides a method for enhancing virus proliferation, further characterized in that the transfection complex includes a polyethyleneimine reagent.
  • the invention provides a method for enhancing virus proliferation, which is also characterized in that the medium of the initial BHK21 cells is changed before transfection, and then pre-cultured for a period of time before transfection.
  • the invention provides a method for enhancing virus proliferation, which is further characterized in that when the titer of the virus proliferation sample is determined, the obtained cell clones are subjected to at least one freeze-thaw cycle.
  • the present invention provides a method for enhancing virus proliferation, which comprises:
  • Each pair of target sequence primers was annealed separately to produce a double-stranded DNA with sticky ends; the gRNA expression vector backbone was treated with restriction enzymes to produce the same sticky ends as the primer annealing products, and the TUC DNA ligase was used to obtain pUC- gRNA-library for subsequent transfection experiments;
  • BHK21 cells with an initial cell density of 1 ⁇ 10 6 cells / ml were seeded in 6-well plates and cultured overnight. The medium was changed to Opti-MEM medium and incubated for 10 minutes before transfection; 100ul of polyethyleneimine (PEI) reagent Add to 1ml of Opti-MEM and mix. Add 1 ⁇ 10ug of pCas9-IRES-GFP and pUC-gRNA-library plasmid vector to 1ml of Opti-MEM and mix. Incubate at room temperature for 5 minutes.
  • PEI polyethyleneimine
  • the two mixed solutions were mixed by dropwise addition, and incubated at room temperature for 15 minutes to form a PEI-DNA transfection complex; the 2ml transfection complex was added to a 6-well plate by dropwise addition. Transfection was performed in BHK21 cells; 8 hours after transfection, the transfection complex was removed, and the normal medium of BHK21 cells was replaced and cultured for 24 hours;
  • BHK21 cells were digested and dispersed, recovered by centrifugation, and washed with PBS for cell sorting by flow cytometry; before sorting, cells were filtered through a sterile 600-mesh sieve and sorted by flow cytometry. , Sort the cell population with green fluorescence into the well plate; when the cell growth resumes, build a bank and save it as BHK21-KO-IM cell population;
  • the BHK21-KO-IM cell population was sorted into a 96-well plate and cultured by flow cytometry, and one cell clone per well was obtained. After the growth was restored, the cell density in each well was adjusted to approximately the same. Comparison of virus proliferation efficiency;
  • the present invention establishes a BHK21 cell population with innate immune system remodeling, and screens this cell population to obtain the optimal BHK21 cell with innate immune system reconstruction, and proliferates a variety of viruses.
  • the present invention uses randomly combined DNA primers to construct a pUC-gRNA-library, and constructs a BHK21 cell population that is reconstituted by the innate immune system, opening up new ideas for virus proliferation.
  • BHK21 cells can be used to multiply more viruses, and provide a multiplication method for multiple viruses.
  • the present invention multiplies viruses by cloning BHK21 cell clones that are reconstituted by the innate immune system, breaking the method of multiplying viruses in the prior art only by seed cell selection, cell suspension culture, and virus proliferation processes, and provides virus proliferation New methods.
  • the CRISPR / Cas9 system is used for gene knockout, which can efficiently complete gene editing, and obtain a target vector for transfecting cells.
  • the proliferative titer of the virus can be greatly increased, which is beneficial to large-scale virus proliferation, and a vaccine is obtained for immunization.
  • FIG. 1 is a schematic diagram showing the comparison of the efficiency of the proliferation of pseudorabies virus (PRV) between BHK21 mother cells and 6 cell clones selected according to the present invention.
  • PRV pseudorabies virus
  • FIG. 2 is a schematic diagram showing the comparison of the efficiency of the proliferation of Japanese encephalitis virus (JEV) by BHK21 mother cells and 9 cell clones selected according to the present invention.
  • JEV Japanese encephalitis virus
  • FIG. 3 is a schematic diagram showing the comparison of the efficiency of the proliferation of Newcastle disease virus (NDV) between BHK21 mother cells and 9 cell clones selected according to the present invention.
  • NDV Newcastle disease virus
  • Example 1 Obtaining BHK21-KO-IM cell population and formation of single cell clones
  • Target sequences For each innate immune system recognition receptor protein, effector protein, stress protein, protein kinase, interferon and its downstream regulatory products and other respective gene sequences, according to the CRISPR / Cas9 system Sequence editing for gene editing requires target sequences to be selected. Generally, at least two target sequences are selected for each gene sequence. For each target sequence, a pair of DNA primers were designed. The primers used were those having SEQ ID Nos. 1-446.
  • Each pair of target sequence primers is annealed separately to produce a double-stranded DNA with sticky ends.
  • Restriction enzyme BbsI purchased from NEB Biotechnology
  • gRNA expression vector backbone containing the U6 promoter, RNA scaffold, and polyA signal sequence
  • TUC DNA ligase purchased from TAKARA Biotechnology
  • BHK21 cells with an initial cell density of 1 ⁇ 10 6 cells / ml were seeded in 6-well plates and cultured overnight. Before transfection, the medium was changed to Opti-MEM medium (purchased from Invitrogen) and incubated for 10 minutes. Add 100ul of PEI reagent (purchased from Sigma) to 1ml of Opti-MEM and mix. Add 1 ⁇ 10ug of pCas9-IRES-GFP and pUC-gRNA-library plasmid vector to 1ml of Opti-MEM and mix. Incubate for 5 minutes at room temperature. After the incubation is completed, the two mixed solutions are added dropwise, and incubated at room temperature for 15 minutes to form a PEI-DNA transfection complex.
  • the above 2 ml transfection complex was added to BHK21 cells in a 6-well plate by dropwise addition for transfection. Eight hours after transfection, the transfection complex was removed, and the normal medium of BHK21 cells was replaced, and the culture was continued for 24 hours.
  • BHK21 cells were digested and dispersed, recovered by centrifugation, and washed with PBS for cell sorting by flow cytometry. Before sorting, the cells were filtered through a sterile 600-mesh sieve and sorted by an upstream cytometer. The cells with green fluorescence were sorted into well plates. After the cell growth resumed, the bank was established and stored as BHK21-KO-IM cell population.
  • the BHK21-KO-IM cell population was sorted into 96-well plates by flow cytometry, and one cell clone per well was cultured. After the growth of the cell clones is restored, the cell clones are subjected to the re-operation for cell preservation and virus proliferation comparison experiments.
  • the BHK21-KO-IM cell population was sorted by flow cytometry to form a single cell clone in a 96-well plate. After the growth was restored, 177 actual cell clones were obtained. The cell density was adjusted to approximately the same in each well, and the cells expanded. A duplicate plate was made for comparison of virus proliferation efficiency.
  • virus proliferation maintenance solution containing the same amount of porcine pseudorabies virus (PRV). Pay attention to observe the cytopathy every day, complete the virus proliferation within 72 hours, and freeze and thaw three times to determine the titer of virus proliferation samples in each well. Specifically, each virus sample was diluted 10 -6 , 10 -7 , and 10 -8 , and then inoculated on ordinary monolayer BHK21 cells. After 72 hours, the cytopathic condition was observed. A typical cytopathic condition was judged as positive, otherwise, Is negative. The virus proliferation ability of different cell clones was compared, and the results are shown in Tables 1 and 2.
  • the six cell clones selected above were expanded and compared for suspension growth and domestication, high-density cell culture, and PRV suspension proliferation comparison. The results are shown in Figure 1.
  • the virus proliferation ability of the six cell clones was significantly higher than that of the BHK21 mother.
  • the virus proliferation titer of cell clones 1-E2, 1-G2, and 2-D8 all exceeded 9.0 lgTCID 50 / ml, while the virus proliferation titer of BHK21 mother cells was only 7.45 lgTCID 50 / ml.
  • the BHK21-KO-IM cell population was sorted by flow cytometry to form a single cell clone in a 96-well plate. After the growth was restored, 177 actual cell clones were obtained. The cell density was adjusted to approximately the same in each well, and the cells expanded. A duplicate plate was made for comparison of virus proliferation efficiency.
  • virus proliferation maintenance solution containing the same amount of porcine Japanese encephalitis virus (JEV). Pay attention to observe the cytopathy every day, complete the virus proliferation in 96 hours, and measure the titer of virus proliferation samples in each well after 3 freeze-thaw cycles.
  • Viral titer detection method According to the quality standards of veterinary biological products of the Ministry of Agriculture, the virus proliferation samples were diluted 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 and then inoculated into ordinary monolayer BHK21 cells. Above, after 90 minutes of adsorption, 1% methylcellulose cover was added to continue the culture. After 5 days, the number of plaques was counted and the plaque formation unit was calculated, which was expressed as lgPFU / ml.
  • the nine cell clones selected were compared for suspension growth and domestication, high-density cell culture, and suspension proliferation of JEV. The results are shown in Figure 2.
  • the virus proliferation ability of the nine cell clones was significantly higher than that of BHK21 mother cells.
  • the virus proliferation titers of cell clones 1-G9 and 2-D2 all exceeded 6.5 lgPFU / ml, while the virus proliferation titers of BHK21 mother cells were only 4.75 lgPFU / ml.
  • the BHK21-KO-IM cell population was sorted by flow cytometry to form a single cell clone in a 96-well plate. After the growth was restored, 177 actual cell clones were obtained. The cell density was adjusted to approximately the same in each well, and the cells expanded. A duplicate plate was made for comparison of virus proliferation efficiency.
  • virus proliferation maintenance solution containing the same amount of chicken Newcastle disease virus (NDV). Pay attention to observe the cytopathy every day, complete the virus proliferation within 72 hours, and freeze and thaw three times to determine the titer of virus proliferation samples in each well.
  • Viral titer detection method According to the quality standards of veterinary biological products of the Ministry of Agriculture, the virus proliferation samples were diluted 10 -5 , 10 -6 , 10 -7 , 10 -8 and then inoculated on monolayer chicken embryo fibroblasts to continue After 3 days of culture, the virus titer was calculated according to the condition of the disease and expressed as lgTCID 50 / ml.
  • the nine cell clones selected were compared for suspension growth and domestication, high-density cell culture, and suspension proliferation comparison of NDV. The results are shown in Figure 3.
  • the virus proliferation ability of the nine cell clones was significantly higher than that of BHK21 mother cells.
  • the virus proliferation titers of cell clones 1-C2, 1-H3, 2-D9, and 2-G8 all exceeded 9.0 lgTCID 50 / ml, while the virus proliferation titers of BHK21 mother cells were only 6.75 lgTCID 50 / ml.
  • BHK21-KO-IM cells are used to clone and proliferate pseudorabies virus (PRV), Japanese encephalitis virus (JEV), and Newcastle disease virus (NDV) viruses, but not limited to PRV, JEV, and NDV viruses.
  • PRV pseudorabies virus
  • JEV Japanese encephalitis virus
  • NDV Newcastle disease virus
  • JEV, NDV virus as examples, the virus proliferation application of the present disclosure also includes foot-and-mouth disease virus (FMDV), rabies virus (RV), and the like.

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Abstract

提供了一种增强病毒增殖的方法，并构建了先天性免疫系统重构的BHK21细胞群，用于增殖病毒。该方法为病毒增殖打开了新思路，扩大了BHK21细胞的应用范围，为能够防治病毒的疫苗的研制打下了坚实的基础。

Description

先天性免疫系统重构的BHK21细胞群及其细胞克隆增毒应用 技术领域
本发明属于生物技术及兽用生物制品工程技术领域，更具体涉及一种随机敲除先天性免疫系统效应蛋白分子的BHK21细胞群及其细胞克隆增殖病毒的应用。
背景技术
BHK21细胞是目前畜禽疫苗病毒增殖的常用宿主细胞，各大研究机构以及兽用疫苗生产企业都在进行该细胞增殖各种病毒的种细胞筛选、细胞悬浮培养、病毒增殖工艺等研究，是目前的研究热点。宿主细胞的先天性免疫系统是产生于系统发育的早期和出现在宿主抗感染应答的初始阶段，执行免疫功能的机体防御机制，它起到了抗感染，清除体内有害成分，自身免疫以及移植排斥的功能。宿主细胞通过其病原相关模式识别受体感知病原体的入侵，进而启动干扰素和一系列的细胞因子抵抗病原的入侵。这些模式分子在不同类型的细胞中或不同的细胞器区域内表达，如细胞膜、内涵体膜、溶酶体膜、细胞质等等，主要负责监测病原体的各种成分分子，以启动炎症反应和抗病毒免疫。具体的模式分子包括Toll样受体、RIG-I样受体、NOD样受体、DAI分子、AIM2分子等等。对于病毒的规模化增殖而言，降低先天性免疫系统的信号传导通路，抑制病毒增殖拮抗作用有利于病毒的高效扩增。利用基因组编辑技术对BHK21细胞先天性免疫系统中各信号分子进行随机组合敲除，减少因外源病毒感染造成的先天性免疫应答，为多种病毒在BHK21细胞中高滴度增殖提供良好的胞内生物环境。
现有技术中常利用CRISPR/Cas9系统靶向病毒基因组DNA有效抑制病 毒感染。根据CRISPR/Cas9技术原理，设计靶向PRV保守的UL30基因的gRNA，并成功构建能够稳定表达sgRNA和Cas9核酸内切酶的PK-15细胞系。结果显示，用PRV感染该细胞后，CRISPR/Cas9系统对PRV UL30基因的编辑作用可以导致UL30基因的碱基缺失或者插入，且能够显著抑制PRV在细胞中的增殖。
ERp57是细胞内质网内的蛋白二硫键异构酶家族成员，可以催化蛋白二硫键的合成以促进蛋白折叠。为研究erp57基因敲除对流感病毒复制的影响，有研究人员通过CRISPR/Cas9技术构建了erp57基因的向导RNA(gRNA)敲除质粒pGEM-erp57gRNA，并将其与pCas9-EGFP共转染至人肺腺癌细胞A549中，采用流式分选及限制性稀释的方法筛选单细胞克隆株，通过靶基因组测序及western blot检测，筛选获得了erp57基因敲除的A549细胞系。erp57基因敲除细胞系与野生型细胞生长动力学无显著差异。此外，将H1N1亚型流感病毒分别感染野生型和erp57基因敲除细胞系以测定erp57基因敲除对流感病毒复制水平的影响。结果显示流感病毒在erp57基因敲除细胞中的复制显著地受到了抑制。
纵观现有技术，可以发现，本领域技术人员多用基因敲除的方法抑制病毒的增殖，发明人未曾发现先天性免疫系统重构的BHK21细胞群及其细胞克隆在病毒增殖方面的应用。
发明内容
本发明提供一种增强病毒增殖的方法，其特征在于，所述病毒在进行了先天性免疫系统重构的BHK21细胞中进行增殖。
本发明提供一种增强病毒增殖的方法，其特征还在于，BHK21细胞中的先天性免疫系统中的信号传导通路被降低。
本发明提供一种增强病毒增殖的方法，其特征还在于，所述信号传导通路的降低通过对BHK21细胞先天性免疫系统中多种信号分子进行基因敲除而实现。
本发明提供一种增强病毒增殖的方法，其特征还在于，所述信号传导通路的降低通过对BHK21细胞先天性免疫系统中多种信号分子进行随机组合基因敲除而实现。
本发明提供一种增强病毒增殖的方法，其特征还在于，所述多种信号分子选自由识别受体蛋白、效应蛋白、应激蛋白、蛋白激酶、干扰素及其下游调节产物组成的组中的一种或多种。
本发明提供一种增强病毒增殖的方法，其特征还在于，对多种信号分子的基因敲除通过CRISPR/Cas9系统实现。
本发明提供一种增强病毒增殖的方法，其特征还在于，对多种信号分子的基因敲除包括选取所述多种信号分子的至少两条靶序列，并针对每条靶序列，设计一对DNA oligo引物。
本发明提供一种增强病毒增殖的方法，其特征还在于，所述靶序列通过分析金黄色地仓鼠的基因组序列获得。
本发明提供一种增强病毒增殖的方法，其特征还在于，使用所述DNA oligo引物构建了pUC-gRNA-文库。
本发明提供一种增强病毒增殖的方法，其特征还在于，所述DNA oligo引物选自由序列1-446组成的组中。
本发明提供一种增强病毒增殖的方法，其特征还在于，所述病毒选自由伪狂犬病毒、日本脑炎病毒、口蹄疫病毒、狂犬病病毒、新城疫病病毒组成的组中。
本发明提供一种增强病毒增殖的方法，其特征还在于，建立了先天性免疫系统重构的BHK21细胞群。
本发明提供一种增强病毒增殖的方法，其特征还在于，对先天性免疫系统重构的BHK21细胞群进行筛选，以获得最佳先天性免疫系统重构的BHK21细胞进行病毒增殖。
本发明提供一种增强病毒增殖的方法，其特征还在于，所获得的最佳先 天性免疫系统重构的BHK21细胞增殖出的子代病毒在稀释至10 -8依然产生阳性病变。
本发明提供一种增强病毒增殖的方法，其特征还在于，采用gRNA表达载体骨架构建pUC-gRNA-文库，所述gRNA表达载体骨架内含U6启动子、RNA支架、polyA信号序列。
本发明提供一种增强病毒增殖的方法，其特征还在于，pUC-gRNA-文库转染BHK21细胞期间采用与初始培养BHK21细胞时不同的培养基。
本发明提供一种增强病毒增殖的方法，其特征还在于，pUC-gRNA-文库转染BHK21细胞期间采用Opti-MEM培养基。
本发明提供一种增强病毒增殖的方法，其特征还在于，pUC-gRNA-文库转染BHK21细胞期间采用逐滴加入的方式混匀转染复合物。
本发明提供一种增强病毒增殖的方法，其特征还在于，转染复合物包括聚乙烯亚胺试剂。
本发明提供一种增强病毒增殖的方法，其特征还在于，转染前先更换初始BHK21细胞的培养基，然后预培养一段时间后，再进行转染。
本发明提供一种增强病毒增殖的方法，其特征还在于，测定病毒增殖样品的效价时，所获得的细胞克隆经受了至少一次冻融。
本发明提供一种增强病毒增殖的方法，其特征在于，包括：
首先，分析金黄色地仓鼠的基因组序列，针对先天性免疫系统的各个识别受体蛋白、效应蛋白、应激蛋白、蛋白激酶、干扰素及其下游调节产物各自的基因序列，根据CRISPR/Cas9系统中基因编辑的序列识别要求，选取靶序列；每个基因序列挑选至少两条靶序列；针对每条靶序列，设计一对DNA oligo引物；
每对靶序列引物分别进行退火处理，产生具有粘性末端的DNA双链；采用限制性内切酶处理gRNA表达载体骨架，产生与引物退火产物相同的粘性末端，经T4DNA连接酶作用，获得pUC-gRNA-文库，用于后续转染实 验；
接种初始细胞密度为1×10 6细胞/ml的BHK21细胞于6孔板中，培养过夜，转染前培养基更换为Opti-MEM培养基孵育10分钟；将100ul聚乙烯亚胺(PEI)试剂加入至1ml的Opti-MEM中混匀，将1～10ug的pCas9-IRES-GFP、pUC-gRNA-文库质粒载体加入至1ml的Opti-MEM中混匀，室温下孵育5分钟；孵育完成后将上述两个混合液采用逐滴加入的方式混匀，在室温下孵育15分钟，形成PEI-DNA转染复合物；采用逐滴加入的方式将上述2ml转染复合物加入至6孔板中的BHK21细胞中进行转染；转染后8小时，去除转染复合物，更换成BHK21细胞的正常培养基继续培养24小时；
转染后24小时的BHK21细胞经消化分散、离心回收以及PBS清洗后用于流式细胞仪的细胞分选；分选前细胞经无菌600目筛网过滤后上流式细胞仪进行分选操作，将带有绿色荧光的细胞群分选至孔板中；待细胞生长恢复，建库保存为BHK21-KO-IM细胞群；
经流式细胞仪将BHK21-KO-IM细胞群分选至96孔板中培养，每孔一个细胞克隆；恢复生长后，调整每孔细胞密度至大致相同，细胞扩增后制作复板用于病毒增殖效率比较；
去除培养上清，分别接种含有相同的病毒增殖维持液200ul/孔；每天注意观察细胞病变，72小时完成病毒增殖，经3次冻融，测定每孔病毒增殖样品的效价；有细胞病变即判定为阳性，否则即为阴性，比较不同细胞克隆的病毒增殖能力，获得增殖能力提高的细胞克隆。
一种先天性免疫系统重构的BHK21细胞群，其通过上述方法构建。
一种用上述方法构建的先天性免疫系统重构的BHK21细胞群用于增殖病毒的用途。
本发明取得了以下有益效果：
(1)本发明建立了先天性免疫系统重构的BHK21细胞群，并对该细胞 群进行筛选，获得了最佳先天性免疫系统重构的BHK21细胞，并对多种病毒进行增殖。
(2)本发明采用随机组合的DNA oligo引物来构建pUC-gRNA-文库，并构建先天性免疫系统重构的BHK21细胞群，为病毒增殖打开了新思路。
(3)通过本发明，可使得BHK21细胞用于增殖更多种病毒，为多种病毒提供增殖方式。
(4)pUC-gRNA-文库建立，为构建先天性免疫系统重构的BHK21细胞提供了新的思路。
(5)本发明通过先天性免疫系统重构的BHK21细胞克隆来增殖病毒，打破了现有技术中仅通过种细胞筛选、细胞悬浮培养、病毒增殖工艺来增殖病毒的方法，为病毒增殖提供了新的方法。
(6)本发明采用CRISPR/Cas9系统进行基因敲除，可高效完成对基因的编辑，获得目的载体用于转染细胞。
(7)本发明获得的先天性免疫系统重构的BHK21细胞群，在用于增殖病毒时，可大大提高病毒的增殖效价，有利于病毒增殖的大规模进行，并获得疫苗进行免疫。
附图说明
从以下结合附图的详细描述中可以看出本发明的其他特征和优点，其中：
图1示出了BHK21母细胞与根据本发明筛选出的6个细胞克隆增殖伪狂犬病毒(PRV)的效率的比较示意图。
图2示出了BHK21母细胞与根据本发明筛选出的9个细胞克隆增殖日本脑炎病毒(JEV)的效率的比较示意图。
图3示出了BHK21母细胞与根据本发明筛选出的9个细胞克隆增殖新城疫病毒(NDV)的效率的比较示意图。
具体实施方式
下面结合具体实施例对本发明做进一步的详细说明。
实施例1 BHK21-KO-IM细胞群的获得及单细胞克隆的形成
1、pUC-gRNA-文库的构建
分析金黄色地仓鼠的基因组序列，针对先天性免疫系统的各个识别受体蛋白、效应蛋白、应激蛋白、蛋白激酶、干扰素及其下游调节产物等各自的基因序列，根据CRISPR/Cas9系统中基因编辑的序列识别要求，选取靶序列。一般每个基因序列挑选至少两条靶序列。针对每条靶序列，设计一对DNA oligo引物。所用引物为序列号为1-446的引物。
每对靶序列引物分别进行退火处理，产生具有粘性末端的DNA双链。采用限制性内切酶BbsI(购自NEB生物技术公司)处理gRNA表达载体骨架(内含U6启动子、RNA支架、polyA信号序列)，产生与引物退火产物相同的粘性末端。经T4DNA连接酶(购自TAKARA生物技术公司)作用，获得pUC-gRNA-文库，用于后续转染实验。
2、BHK21细胞转染
接种初始细胞密度为1×10 6细胞/ml的BHK21细胞于6孔板中，培养过夜。转染前培养基更换为Opti-MEM培养基(购自Invitrogen公司)孵育10分钟。将100ul PEI试剂(购自Sigma公司)加入至1ml的Opti-MEM中混匀，将1～10ug的pCas9-IRES-GFP、pUC-gRNA-文库质粒载体加入至1ml的Opti-MEM中混匀，室温下孵育5分钟。孵育完成后将上述两个混合液采用逐滴加入的方式混匀，在室温下孵育15分钟，形成PEI-DNA转染复合物。采用逐滴加入的方式将上述2ml转染复合物加入至6孔板中的BHK21细胞中进行转染。转染后8小时，去除转染复合物，更换成BHK21细胞的正常培养基继续培养24小时。
3、BHK-KO-IM细胞群的筛选
转染后24小时的BHK21细胞经消化分散、离心回收以及PBS清洗后用于流式细胞仪的细胞分选。分选前细胞经无菌600目筛网过滤后上流式细 胞仪进行分选操作，将带有绿色荧光的细胞群分选至孔板中。待细胞生长恢复，建库保存为BHK21-KO-IM细胞群。
4、BHK21-KO-IM细胞克隆的获得
经流式细胞仪将BHK21-KO-IM细胞群分选至96孔板中培养，每孔一个细胞克隆。待细胞克隆生长恢复后，对细胞克隆进行复板操作，用于细胞保存以及病毒增殖比较实验。
实施例2筛选PRV高产的BHK21细胞克隆及其病毒增殖应用
1、BHK21-KO-IM细胞群经流式细胞仪分选在96孔板中形成单细胞克隆，恢复生长后，获得实际细胞克隆为177个，调整每孔细胞密度至大致相同，细胞扩增后制作复板用于病毒增殖效率比较。
2、去除培养上清，分别接种含有相同量猪伪狂犬病毒(PRV)的病毒增殖维持液200ul/孔。每天注意观察细胞病变，72小时完成病毒增殖，经3次冻融，测定每孔病毒增殖样品的效价。具体是对每个病毒样品作10 -6、10 -7、10 -8稀释后，接种于普通单层BHK21细胞上，72小时后观察细胞病变情况，具有典型细胞病变即判定为阳性，否则即为阴性。比较不同细胞克隆的病毒增殖能力，结果如表1和表2所示。结果可见，细胞克隆1-C9、1-E2、1-E11、1-G2、2-B10、2-D8增殖出的PRV子代病毒在稀释至10 -8依然可以产生典型阳性病变，说明这几个细胞克隆的病毒增殖能力高于其他细胞克隆。
3、扩增上述筛选出的6个细胞克隆，进行悬浮生长驯化、细胞高密度培养以及PRV的悬浮增殖比较，结果如图1所示，该6株细胞克隆的病毒增殖能力显著高于BHK21母细胞，其中细胞克隆1-E2、1-G2、2-D8的病毒增殖效价均超过9.0lgTCID 50/ml，而BHK21母细胞的病毒增殖效价仅为7.45lgTCID 50/ml。
实施例3筛选JEV高产的BHK21细胞克隆及其病毒增殖应用
1、BHK21-KO-IM细胞群经流式细胞仪分选在96孔板中形成单细胞克隆，恢复生长后，获得实际细胞克隆为177个，调整每孔细胞密度至大致相 同，细胞扩增后制作复板用于病毒增殖效率比较。
2、去除培养上清，分别接种含有相同量猪日本脑炎病毒(JEV)的病毒增殖维持液200ul/孔。每天注意观察细胞病变，96小时完成病毒增殖，经3次冻融，测定每孔病毒增殖样品的效价。病毒效价检测方法根据农业部兽用生物制品质量标准要求，对病毒增殖样本进行10 -4、10 -5、10 -6、10 -7、10 -8稀释后，接种于普通单层BHK21细胞上，吸附90分钟后加入1％甲基纤维素覆盖物继续培养，5天后染色计蚀斑数，计算蚀斑形成单位，以lgPFU/ml表示。
3、筛选出的9个细胞克隆，进行悬浮生长驯化、细胞高密度培养以及JEV的悬浮增殖比较，结果如图2所示，该9株细胞克隆的病毒增殖能力显著高于BHK21母细胞，其中细胞克隆1-G9、2-D2的病毒增殖效价均超过6.5lgPFU/ml，而BHK21母细胞的病毒增殖效价仅为4.75lgPFU/ml。
实施例4筛选NDV高产的BHK21细胞克隆及其病毒增殖应用
1、BHK21-KO-IM细胞群经流式细胞仪分选在96孔板中形成单细胞克隆，恢复生长后，获得实际细胞克隆为177个，调整每孔细胞密度至大致相同，细胞扩增后制作复板用于病毒增殖效率比较。
2、去除培养上清，分别接种含有相同量鸡新城疫病毒(NDV)的病毒增殖维持液200ul/孔。每天注意观察细胞病变，72小时完成病毒增殖，经3次冻融，测定每孔病毒增殖样品的效价。病毒效价检测方法根据农业部兽用生物制品质量标准要求，对病毒增殖样本进行10 -5、10 -6、10 -7、10 -8稀释后，接种于单层鸡胚成纤维细胞上继续培养，3天后根据病变情况，计算病毒效价，以lgTCID 50/ml表示。
3、筛选出的9个细胞克隆，进行悬浮生长驯化、细胞高密度培养以及NDV的悬浮增殖比较，结果如图3所示，该9株细胞克隆的病毒增殖能力显著高于BHK21母细胞，其中细胞克隆1-C2、1-H3、2-D9、2-G8的病毒增殖效价均超过9.0lgTCID 50/ml，而BHK21母细胞的病毒增殖效价仅为 6.75lgTCID 50/ml。
本发明中使用BHK21--KO-IM细胞克隆增殖伪狂犬病毒(PRV)、日本脑炎病毒(JEV)、新城疫病毒(NDV)病毒，但不局限于PRV、JEV、NDV病毒，仅以PRV、JEV、NDV病毒作为示例，本公开的病毒增殖应用还包含口蹄疫病毒(FMDV)、狂犬病病毒(RV)等。
应当强调，本发明的上述实施例仅仅是可能的示例实施方式，其仅仅是为了清楚地理解本公开的原理而提出的。在不脱离本公开的精神和原理的情况下，可以对本公开的上述实施例进行许多变化和修改。所有这些修改和变化旨在被包括在本发明的范围内并由所附权利要求保护。
Figure PCTCN2019072274-appb-000001

Claims (10)

  1. 一种增强病毒增殖的方法，其特征在于，所述病毒在进行了先天性免疫系统重构的BHK21细胞中进行增殖。
  2. 根据权利要求1所述的增强病毒增殖的方法，其特征还在于，BHK21细胞中的先天性免疫系统中的信号传导通路被降低。
  3. 根据权利要求2所述的增强病毒增殖的方法，其特征还在于，所述信号传导通路的降低通过对BHK21细胞先天性免疫系统中多种信号分子进行随机组合基因敲除而实现。
  4. 根据权利要求3所述的增强病毒增殖的方法，其特征还在于，所述信号传导通路中的多种信号分子选自由识别受体蛋白、效应蛋白、应激蛋白、蛋白激酶、干扰素及其下游调节产物组成的组中的一种或多种。
  5. 根据权利要求1所述的增强病毒增殖的方法，其特征还在于，构建了pUC-gRNA-文库用于转染BHK21细胞，且转染所述BHK21细胞期间采用与初始培养BHK21细胞时不同的培养基。
  6. 根据权利要求5所述的增强病毒增殖的方法，其特征还在于，构建pUC-gRNA-文库所用的DNA oligo引物选自由序列1-446组成的组中。
  7. 一种增强病毒增殖的方法，其特征在于，包括：
    首先，分析金黄色地仓鼠的基因组序列，针对先天性免疫系统的各个识别受体蛋白、效应蛋白、应激蛋白、蛋白激酶、干扰素及其下游调节产物各自的基因序列，根据CRISPR/Cas9系统中基因编辑的序列识别要求，选取靶序列；每个基因序列挑选至少两条靶序列；针对每条靶序列，设计一对DNA oligo引物；
    每对靶序列引物分别进行退火处理，产生具有粘性末端的DNA双链；采用限制性内切酶处理gRNA表达载体骨架，产生与引物退火产物相同的粘性末端，经T4DNA连接酶作用，获得pUC-gRNA-文库，用于后续转染实验；
    接种初始细胞密度为1×10 6细胞/ml的BHK21细胞于6孔板中，培养过夜，转染前培养基更换为Opti-MEM培养基孵育10分钟；将100ul聚乙烯亚胺(PEI)试剂加入至1ml的Opti-MEM中混匀，将1～10ug的pCas9-IRES-GFP、pUC-gRNA-文库质粒载体加入至1ml的Opti-MEM中混匀，室温下孵育5分钟；孵育完成后将 上述两个混合液采用逐滴加入的方式混匀，在室温下孵育15分钟，形成PEI-DNA转染复合物；采用逐滴加入的方式将上述2ml转染复合物加入至6孔板中的BHK21细胞中进行转染；转染后8小时，去除转染复合物，更换成BHK21细胞的正常培养基继续培养24小时；
    转染后24小时的BHK21细胞经消化分散、离心回收以及PBS清洗后用于流式细胞仪的细胞分选；分选前细胞经无菌600目筛网过滤后上流式细胞仪进行分选操作，将带有绿色荧光的细胞群分选至孔板中；待细胞生长恢复，建库保存为BHK21-KO-IM细胞群；
    经流式细胞仪将BHK21-KO-IM细胞群分选至96孔板中培养，每孔一个细胞克隆；恢复生长后，调整每孔细胞密度至大致相同，细胞扩增后制作复板用于病毒增殖效率比较；
    去除培养上清，分别接种含有相同的病毒增殖维持液200ul/孔；每天注意观察细胞病变，72小时完成病毒增殖，经3次冻融，测定每孔病毒增殖样品的效价；有细胞病变即判定为阳性，否则即为阴性，比较不同细胞克隆的病毒增殖能力，获得增殖能力提高的细胞克隆。
  8. 根据上述权利要求中任一项所述的方法，其特征还在于所述病毒选自由伪狂犬病毒、日本脑炎病毒、口蹄疫病毒、狂犬病病毒、新城疫病病毒组成的组中。
  9. 一种先天性免疫系统重构的BHK21细胞群，其通过根据权利要求1-7中任一项所述的方法构建。
  10. 一种通过根据权利要求1-7中任一项所述的方法构建的先天性免疫系统重构的BHK21细胞群用于增殖病毒的用途。
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