WO2018000657A1 - 一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒 - Google Patents

一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒 Download PDF

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WO2018000657A1
WO2018000657A1 PCT/CN2016/101862 CN2016101862W WO2018000657A1 WO 2018000657 A1 WO2018000657 A1 WO 2018000657A1 CN 2016101862 W CN2016101862 W CN 2016101862W WO 2018000657 A1 WO2018000657 A1 WO 2018000657A1
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gene
tlr4
cell
pyrogen
cell model
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牛刚
谭焕然
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牛刚
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Priority to EP16907053.9A priority patent/EP3480302B1/en
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Definitions

  • the invention belongs to the field of biotechnology, relates to detection of pyrogens in pharmaceuticals and biological products (including genetic engineering products), specific method for constructing cell model for detecting pyrogen, cell model and method and kit for detecting pyrogen .
  • pyrogens have a more precise definition, referring to pyrogenic substances produced by microorganisms that can cause abnormal temperature rise in warm-blooded animals.
  • Seibert confirmed that pyrogens are produced by microorganisms and are characterized by heat resistance, non-volatility, water solubility, filtration and adsorption, are not easily trapped, and have high thermal stability, although they are strictly sterilized, The occurrence of a pyrogen reaction cannot be ruled out.
  • Pyrogens can be divided into two types: exogenous pyrogens and endogenous pyrogens.
  • Exogenous pyrogens are mainly endotoxin of Gram-negative bacteria and lipoic acid of Gram-positive bacteria, endogenous pyrogens. It mainly includes cytokines (such as TNF- ⁇ , INF- ⁇ , IL-1, growth factors) and hormones (such as steroids and prostaglandins) in the body.
  • cytokines such as TNF- ⁇ , INF- ⁇ , IL-1, growth factors
  • hormones such as steroids and prostaglandins
  • the "pyrogen” we usually refer to is mainly a bacterial pyrogen, which is a metabolite of certain bacteria, a bacterial corpse and an endotoxin.
  • the most heat-producing is the product of Gram-negative G - bacteria, followed by Gram-positive G + bacilli, Gram-positive G + cocci is weak, and mold, yeast and even viruses can produce pyrogens. Charles A, Dinarello. Endotoxin Research, 2004, 10: 201).
  • Endotoxin belongs to the exogenous pyrogen and is a unique structure on the outer wall layer of the cell wall of Gram-negative G - bacteria. It is a complex of phospholipids, lipopolysaccharides (LPS) and proteins. LPS is the main component and active center of the composite, and has the strongest heat-producing effect. However, its chemical composition varies from strain to strain, and the molecular weight is 5 ⁇ 10 4 to 5 ⁇ 10 5 . The stronger the molecular weight, the stronger the thermal effect. . Bacterial endotoxin is not a metabolite of bacteria or bacteria, but a biologically active toxic substance released after the death or disintegration of bacteria. It cannot be removed by ordinary sterilization methods.
  • Bacterial endotoxin entering the human body can not only directly cause toxicity to cell biofilm, but also induce synthesis and release of various cytokines and inflammatory factors through mononuclear macrophage-mediated immune inflammatory reaction, over-expressed inflammatory transmitters.
  • the receptors acting on the mononuclear-macrophage membrane in an autocrine, paracrine or endocrine manner further activate the expression of inflammatory mediators (Guha M, Mackman N. Cell Signal, 2001, 13(2): 85-94. ).
  • the uncontrolled release of these inflammatory factors affects the integrity of cellular functions, causing metabolic disorders in the body, elevated blood coagulation, insufficient blood perfusion in the tissue, and severe multiple organ failure and death (Hotchkiss RS, Karl IE.N Engl J Med) , 2003, 348 (2): 138-150.).
  • the detection of pyrogens in medicines and medical devices has become the focus of recent research due to the wide range of distribution of Gram-negative bacteria. .
  • the methods currently included in the Chinese Pharmacopoeia include the rabbit pyrogen test method and the sputum reagent endotoxin test method.
  • the seventh edition of the European Pharmacopoeia (2011 edition) also includes the third new method of pyrogen test-- Monocyte activation assay.
  • Rabbit pyrogen test method Since the reaction of rabbits to pyrogens is basically similar to that of humans, the rabbit method is a statutory method for examining pyrogens prescribed by national pharmacopoeias. A certain dose of the test sample is intravenously injected into the rabbit body, and the temperature rise of the rabbit is observed within a prescribed time to determine whether the limit of the pyrogen contained in the test product is in compliance with the regulations.
  • the rabbit law still has many limitations.
  • the rabbit method can not be quantified, can not be standardized, sensitivity and repeatability are poor, and the sensitivity of rabbits to pyrogens is individual, and the sensitivity of different individuals to pyrogens is more than 10 times.
  • certain types of drugs can interfere with test results, such as some antipyretic analgesics, cytotoxic drugs, or some drugs that easily affect the body temperature center, and cannot use the rabbit method.
  • many products that have been tested by rabbit pyrogens still have pyrogen reactions in the clinic.
  • the rabbit method can not fully represent the human body's fever reaction. Its operation is cumbersome and time-consuming, and can not be used for quality monitoring in the injection production process, and some drugs are not Be applicable.
  • the blood of the American cockroaches produces gels when they are Gram-negative, and the US, UK, Europe, Japan, and Chinese Pharmacopoeia currently include this test.
  • the sputum reagent endotoxin test method uses a guanidine reagent to detect or quantify bacterial endotoxin produced by Gram-negative bacteria to determine whether a limit of bacterial endotoxin in the test sample conforms to a prescribed method. The amount of bacterial endotoxin is expressed in endotoxin units (EU).
  • EU endotoxin units
  • the sensitivity of the sputum reagent test for endotoxin is 0.015 EU/mL, which is more sensitive than the rabbit method, the operation is simple and easy, the test cost is low, the result is rapid and reliable, and it is suitable for pyrogen control and rabbit method in the production process of injection. Certain cytotoxic pharmaceutical preparations that cannot be detected.
  • the sputum reagent method is greatly affected by environmental factors, and it is prone to false positive results. It is not possible to detect the formulation with its own color. What the sputum reagent method can actually detect is only the agglutination activity of endotoxin on sputum blood cells, not the febrile activity or chemical concept of endotoxin on the human body, and it is impossible to detect other heat sources other than endotoxin. This method can not detect some enzyme inhibitors, calcium and magnesium ion chelating agents, so the sputum test can not completely replace the rabbit pyrogen test.
  • China's Chinese cockroach, which is a cockroach reagent is a species that is earlier than dinosaurs.
  • Monocyte activation assay In addition to the rabbit and sputum reagent methods, the seventh edition of the European Pharmacopoeia (2011 edition) has included a new method of pyrogen examination, the monocyte activation test (MAT).
  • the principle is to incubate human whole blood together with the test sample, and evaluate the pyrogen in the test sample according to the amount of cytokines such as IL-6, IL-1 ⁇ and TNF- ⁇ released by monocytes in the blood. active.
  • MAT can detect pyrogen and pro-inflammatory factor contaminants, including endotoxin from Gram-negative bacteria and non-endotoxin-derived contaminants, including those from Gram-positive bacteria, Gram-negative bacteria, viruses, and fungi.
  • PAMPs Pathogen-associated molecular patterns
  • the monocyte activation test also has certain limitations.
  • the monocyte reactivity of human blood from different sources may be different, and fresh human blood is not easy to obtain in large quantities. Therefore, this method has the disadvantage of poor stability in addition to being difficult to popularize.
  • LPS has some highly conserved pathogen structures in a variety of pathogens, and these conserved pathogen structural forms are called Pathogen associated molecular patterns (PAMP).
  • PAMP Pathogen associated molecular patterns
  • Toll-like family receptors as an important component of the innate immune system, are thought to be similar to the pattern-recognition receptors (PRRs), which initiate the immune response by identifying PAMP on the surface of pathogenic microorganisms.
  • PRRs pattern-recognition receptors
  • LBP lipopolysaccharide Lipopolysaccharide binding protein
  • TLR4 Toll like receptor-4
  • MD2 Myeloid differentiation protein-2
  • MD2 leukocyte differentiation antigen 14
  • CD14 leukocyte differentiation antigen 14
  • LBP is a glycoprotein widely present in human and animal serum. Strictly speaking, LPS-binding protein is not a binding receptor for LPS, but LPS-mediated cell activation requires the involvement of proteins that bind to LPS on the plasma or cell surface. It is an important carrier for LPS to play a biological role. It has a high affinity for bacterial endotoxin and lipid A in LPS. LBP binds to LPS to form a complex and LPS is transmitted to the CD14 receptor on the cell membrane, and binds to the TLR4-MD2 complex to activate target cells and release pre-inflammatory cytokines and immunity through a series of transmembrane and intracellular signal transduction processes. Adjustment factor.
  • TLR4 belongs to the TLRs family (Toll like receptors).
  • TLR1-10 TLRs family proteins
  • TLR1-10 Lemaitre B, Nicolas E, Miehaut L et al. Cell, 1996, 86(6). : 973-983; Huang B. Zhao J. Unkeless J C et a1.
  • Oneogene, 2008, 27 (2): 218-224 Liu Xing, Feng Wenli, Kang Gefei. Foreign Medical Clinical Biochemistry and Laboratory Science, 2001 , 22(3): 134-136; T Kawai, S Akira. Cell Death and Differentiation, 2006, 13: 816-825).
  • Different TLRs transduction signals are stimulated by microorganisms from different sources.
  • TLR4 is the most important type of pattern recognition receptor in inflammatory response, and can specifically recognize some conserved sequences (such as LPS) in the evolution of microorganisms.
  • TLR4 is the most important receptor for LPS-induced cell signal transduction, and is mainly expressed on cells involved in host defense functions, such as peripheral blood leukocytes, T lymphocytes, B lymphocytes, mononuclear macrophages, mast cells, Langhans Cells, dendritic cells and the like, among which are most expressed in bone marrow mononuclear cells (Medzhitov R, Preston Hurlburt P, Janeway Jr. CA. Nature, 1997, 388: 394-79).
  • MD2 is a secreted protein. It was confirmed by immunoprecipitation experiments using transfectants expressing TLR4 and MD2 that MD2 is accompanied by TLR4 expression and secreted to the cell surface, and is anchored to the cell membrane by physical action on TLR4. MD2 helps TLR4 recognize LPS and locks LPS to the binding site on TLR4, which plays a crucial role in the transfer and expression of TLR4 to the membrane. The study found that MD2 molecule is lacking, and TLR4 is reactive to LPS. Very low (Miyake K, R. Shimazu, J. Kondo, et al. Immunol. 1998, 161:1348-1353; Pugin J, Stem Voeffray S, Daubeuf B. Blood.
  • CD14 is a protein expressed on the surface of cytoplasm and leukocytes as a phospholipid phosphoinositide connexin, which has a high affinity with LPS, but CD14 molecules lack a cytoplasmic segment and cannot directly conduct LPS signals into cells.
  • LPS-LBP-CD14 ligand and cofactor complex are first formed, and then combined with TLR4 to transmit signals into cells (Saitoh S, Akashi S, Yamada T, et al. Endotoxin Res, 2004, 10(4): 257-260; Nagai Y, Akashi S, Nagafuku M, et al. Nat Immunol, 2002, 3(7): 667-672; Zielger, Heitbrock HWL and Ulevitch RJ. Immunol Today, 1993, 14 ( 3): 121-125).
  • LPS When bacteria invade the body, LPS is released into the blood by bacterial lysis, and can form a complex with the free lipopolysaccharide binding protein LBP in plasma to form a complex with CD14 or directly bind to the accessory protein of TLR4, the myeloid differentiation protein MD2. , LPS-MD2/TLR4 complex is formed, and TLR4 is activated with the aid of these molecules.
  • CRISPR/Cas9 The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR Associated (Cas) system
  • CRISPRs are a class of repetitive structures widely distributed in the genome of bacteria and archaea, referring to clustered, regularly spaced short palindromic repeats, which are adaptive immunity of bacteria.
  • the protective mechanism is a DNA fragment that has evolved to cope with bacteriophages.
  • Bacteria express Cas9 protein and transcribe CRISPR RNA, which is complementary to the genome of the virus. This Cas9 complex cuts the genome of the virus and inactivates the virus.
  • CRISPR-Cas9 uses a single-directed RNA (sgRNA) identical to the target sequence to direct Cas9 nucleases to recognize and cleave specific targeting DNA, resulting in double-stranded or single-strand breaks in DNA, and then the cells will have their own
  • the two repair mechanisms repair the fragmented DNA, either non-homologous end joining (NHEJ) or homologous-mediated repair (HDR).
  • Endotoxin is the main component of the pyrogen reaction caused by Gram-negative bacteria, and is the key to seriously threaten the safety of drugs. Factors, so it is particularly urgent to establish a more reasonable and more suitable pyrogen detection method.
  • Another object of the present invention is to provide a method of constructing the above-described cell model for detecting pyrogens.
  • a third object of the present invention is to provide a kit for detecting a pyrogen, which comprises the above cell model.
  • a fourth object of the present invention is to provide a method for detecting a pyrogen which is detected using the above kit.
  • the invention provides a method for constructing a cell model for detecting a pyrogen, comprising the following steps:
  • the TLR4 gene is knocked into the intron of a gene on the chromosome of the cell line to obtain a cell line stably expressing the TLR4 gene;
  • the cell line is HEK293T, HEK293 or NIH3T3.
  • the position in which the TLR4 gene is knocked into in the step 2) is the first intron of the PPR1R12C gene of the 19th chromosome.
  • the position in which the CD14 gene and the MD2 gene are knocked into in the step 4) is the first intron of the CCR5 gene of the third chromosome.
  • the present invention also provides a cell model constructed by the above-described construction method, which is a knock-in TLR4 gene in the first intron of the PPR1R12C gene of the 19th chromosome of the cell line HEK 293T, on chromosome 3.
  • the first intron of the CCR5 gene is knocked into the CD14 gene and the MD2 gene.
  • the classification name of the cell model is human embryonic kidney cell (HEK) variant 293T/TLR4/CD14/MD2, and the depositing unit is: General Microbiology Center of China Microbial Culture Collection Management Committee, address: Beichenxi, Chaoyang District, Beijing No. 3, No. 1 Road, Institute of Microbiology, Chinese Academy of Sciences, Date of deposit: May 19, 2016, deposit number: CGMCC No. 12296.
  • HEK human embryonic kidney cell
  • the present invention also provides a kit for detecting a pyrogen, comprising the following composition: 1) a cell model constructed as described above, or a cell model as described above, 2) a pyrogen standard, 3) IL6 and/or TNF ⁇ control.
  • the pyrogen standard is LPS lyophilized powder
  • IL6 reference substance is IL6 lyophilized powder
  • TNF ⁇ reference substance is TNF ⁇ lyophilized powder.
  • the invention further provides a method for detecting a pyrogen, comprising the steps of:
  • the method of detecting the content of IL-6 and/or TNF- ⁇ in step 3) is carried out by Western Blot, ELISA, gold labeling or mass spectrometry.
  • the present invention uses the CRISPR/CAS9 method to knock in three genes in a cell, two of which are knocked into one chromosome at a fixed point, and the other gene is knocked into another chromosome.
  • the knock-in TLR4 gene was traced by green fluorescent GFP; CD14-MD2, which was knocked in at the point, was traced by red fluorescent RFP.
  • the self-cleavage 2A peptide multi-gene vector construction strategy was used to separate the CD14, MD2 and red fluorescent RFP gene structures using two different 2A peptides to avoid affecting protein expression.
  • F2A and T2A Two 2A peptide structures, F2A and T2A, are used in the examples of the present invention, wherein F2A is a 2A peptide derived from foot-and-mouth disease viruses (FMDV), and T2A is derived from the Tiller's brain cerebrospinal cord.
  • FMDV foot-and-mouth disease viruses
  • T2A is derived from the Tiller's brain cerebrospinal cord.
  • the 2A peptide of the virus avoids the use of the same 2A gene sequence to cross-react when the expression vector is constructed, and it is difficult to clone into the destination site.
  • TMEV The 2A peptide of the virus
  • the release of IL-6 and/or TNF- ⁇ cytokines can be detected by ELISA, Western Blot, mass spectrometry, magnetic beading, and the like.
  • the invention adopts a method of site-directed knocking to transfer a foreign gene into a cell, instead of using a vector such as a virus or a lentivirus to perform transient transfection, thereby making the gene stable.
  • the existing CRISPR/CAS9 technology is generally applied to gene knockout or gene mutation.
  • gene knock-in the off-target effect is overcome in the existing methods, the homologous recombination efficiency is low, and the process of constructing the knock-in vector is cumbersome and difficult, especially The difficulty of knocking in multiple genes is even greater.
  • the present invention adopts gene knock-in, and successfully knocks multiple genes into cell lines, such as HEK293T cells, and solves the problem that the CRISPR/CAS9 technology gene knock-in is difficult.
  • the invention serially knocks three genes of TLR4 gene, CD14 gene and MD2 gene into two different chromosomes, and the insertion position is to ensure that the gene does not affect the physiological function of the cell itself after the knock-in, and the normal survival and stable passage of the cells are ensured.
  • Site the TLR4 gene is inserted into the first intron of the PPR1R12C gene of chromosome 19 of HEK 293T, and the CD14 gene and the MD2 gene are inserted into the first intron of the CCR5 gene of chromosome 3 of HEK 293T.
  • Each position can ensure that the gene does not affect the physiological function of the cell itself after the knock-in, ensuring normal cell survival and stable passage.
  • the cell model constructed by the present invention belongs to the tool cell, and has no expression of cytokines such as IL-6 and TNF- ⁇ in the absence of LPS stimulation.
  • cytokines such as IL-6 and TNF- ⁇ in the absence of LPS stimulation.
  • Cells from other sources, such as human monocytes will have a certain amount of background expression of IL-6, TNF- ⁇ and other cytokines in the absence of LPS stimulation, and background expression will have an effect on subsequent detection, and this cell
  • the model showed no cytokine expression in the absence of LPS stimulation and no background presence.
  • certain genetically engineered products may contain substances that stimulate cell proliferation. Different degrees of cell proliferation will have an impact on the uncertainty of detection, and the cell model of the present invention does not have such a risk.
  • the minimum detection limit of the cells of the present invention for LPS is up to 0.005 EU/mL, and the minimum detection limit of the sputum reagent method is 0.025 EU/mL, which indicates that the sensitivity of the present invention is much higher than that of the sputum reagent method.
  • ⁇ is a national second-class protected animal with limited blood sources.
  • the use of sputum reagents will affect the protection of cockroaches, which is not conducive to the sustainable development of biological resources.
  • Different methods for producing bismuth reagents from different manufacturers are different, and pyrogen detection of the same product is easy to produce. Inconsistent results.
  • the monocyte activation assay requires the use of human blood, and the reactivity of monocytes from different human blood sources is unstable, resulting in poor stability of the method.
  • the cell model provided by the invention can stably pass the test gene into the chromosome, has good repeatability to the pyrogen detection, has good reactivity, and the model is stable, and overcomes the shortcomings of the traditional method and the novel human monocyte activation test method.
  • the invention provides a cytological model which can be used for pyrogen detection of pharmaceutical biological products including genetic engineering drugs, a construction method thereof and a pyrogen detection method and a kit.
  • the cell model uses CRISPR/CAS9 to induce a double bond break at a specific position in the genome, and uses the principle of homologous recombination to knock into the TLR4 and CD14-MD2 on both chromosomes of the cell line, respectively, and green fluorescent GFP and red fluorescent RFP, respectively.
  • TLR4/CD14/MD2 fixed-point knock-in fluorescent tracer cell model LPS thorn
  • the release of IL-6 and TNF-a cytokines can be detected by ELISA, Western Blot, mass spectrometry and magnetic beads.
  • the cell model has good stability and sensitivity, and the minimum detection limit is 0.005 EU/mL. It is much lower than 0.025 EU/mL of the sputum reagent method.
  • Figure 1A is a plasmid map of TLR4 and pMD19-T linked in a preferred embodiment of the present invention.
  • Figure 1B is a plasmid map of CD14 and pMD19-T linked in a preferred embodiment of the invention.
  • Figure 1C is a plasmid map of MD2 linked to pMD19-T in a preferred embodiment of the invention.
  • 2A is a plasmid map of the transient expression vector pCAG-TLR4-GFP constructed by TLR4 and pCAG-GFP in a preferred embodiment of the present invention.
  • Figure 2B is a plasmid map of the transient expression vector pCAG-CD14-GFP constructed by CD14 and pCAG-GFP in a preferred embodiment of the present invention.
  • 2C is a plasmid map of the transient expression vector pCAG-MD2-GFP constructed by MD2 and pCAG-GFP in a preferred embodiment of the present invention.
  • FIG. 3 is a diagram showing a carrier construction strategy for TLR4 fixed-point typing in a preferred embodiment of the present invention.
  • FIG. 4 is a schematic diagram showing the principle of TLR4 spotting and knocking into cells in a preferred embodiment of the present invention.
  • Figure 5A is a photograph of a white light illuminated by TLR4 after it has been knocked into cells in a preferred embodiment of the present invention.
  • Figure 5B is a photograph showing the green fluorescence of TLR4 after being knocked into cells by a fixed point in a preferred embodiment of the present invention.
  • FIG. 6 is a diagram showing a carrier construction strategy for MD2-CD14 fixed-point knocking in a preferred embodiment of the present invention.
  • Figure 7 is a schematic diagram showing the principle of MD2-CD14 site-directed knock-in cells in a preferred embodiment of the present invention.
  • Figure 8 is a diagram showing the fluorescence detection of MD2-CD14 after it has been knocked into cells in a preferred embodiment of the present invention.
  • Figure 9 is a diagram showing the fluorescence expression of four cells in a preferred embodiment of the present invention.
  • Figure 10 is a representation of the expression of four cells observed by confocal microscopy in a preferred embodiment of the invention.
  • FIG. 11A is a schematic diagram showing the strategy of four cell PCRs for knocking in TLR4 according to a preferred embodiment of the present invention.
  • Figure 11B is an electropherogram of the PCR result of Figure 11A.
  • Figure 11C is a schematic diagram showing the strategy of four cell PCR identification knock-in CD14/MD2 in a preferred embodiment of the present invention.
  • Figure 11D is an electropherogram of the PCR results of Figure 11C.
  • Figure 12 is a graph showing the results of Western blot analysis of protein expression in four preferred cells of the present invention.
  • Figure 13A is a diagram showing the use of ELISA to detect four cell lines stimulated by LPS in a preferred embodiment of the present invention. Post-cellular IL-6 release.
  • Figure 13B is a diagram showing the release of TNF ⁇ from four cell lines stimulated by LPS using ELISA in a preferred embodiment of the present invention.
  • Figure 14 is a graph showing the relationship between IL-6 release and time after stimulation with LPS in a 293T/TLR4/CD14/MD2 cell line using ELISA in a preferred embodiment of the present invention.
  • Figure 15 is a diagram showing the release of IL-6 by different cell numbers 293T/TLR4/CD14/MD2 after LPS stimulation using ELISA according to a preferred embodiment of the present invention.
  • Figure 16 is a diagram showing the sensitivity of IL-6 release in 293T/TLR4/CD14/MD2 cell lines stimulated with different amounts of LPS using ELISA in a preferred embodiment of the present invention.
  • Figure 17A is a diagram showing the reactivity of 293T/TLR4/CD14/MD2 cells to LPS-stimulated TNF- ⁇ using Western blot in a preferred embodiment of the present invention.
  • Figure 17B is a diagram showing the reactivity of 293T/TLR4/CD14/MD2 cells to LPS-stimulated IL-6 using Western blot in a preferred embodiment of the present invention.
  • Restriction enzymes were purchased from Fermentas, Canada.
  • pMD19-T purchased from Takara Bioengineering (Dalian) Co., Ltd. (Takara).
  • pCAG-GFP, pCAG-RFP, pX330, pMCS.DT-A and pAAV-CAG-RFP were purchased from Addgene.
  • the human monocyte separation solution kit was purchased from Tianjin Yuyang Biological Products Co., Ltd.
  • RPMI 1640 medium (serum free) and DMEM medium (serum free) were purchased from GIBCO, and fetal calf serum was purchased from Hyclone.
  • HEK293T cells were purchased from the Chinese Academy of Sciences (Shanghai) cell bank.
  • the cell culture solution of human peripheral blood mononuclear cells prepared in the step 1.1 was discarded, washed 3 times with a PBS solution, and 200 ⁇ L of TRIzol Reagent (Gibco BRL) was added to each well in a six-well plate, and the cells were lysed repeatedly.
  • the lysed cells were harvested in a 1.5 mL centrifuge tube and allowed to stand at room temperature for 5 min.
  • 0.1 mL of chloroform was added, vigorously shaken for 15 sec, and allowed to stand at room temperature for 2 to 3 minutes. After centrifugation at 12,000 g for 15 min at 4 ° C, the aspirate aqueous phase was transferred to another 1.5 mL centrifuge tube.
  • RNA samples were denatured in a 70 ° C water bath for 5 min and placed on ice for 5 min.
  • the reverse transcription reaction was carried out in accordance with the following reaction system, and the total reaction volume was 20 ⁇ L.
  • mRNA sequences of the human genomes TLR4, MD2 and CD14 were obtained by NCBI search.
  • RT-PCR primers were designed using Primer premier 5.0 software, and the restriction endonuclease recognition sequence EcoRI was introduced at the 5' end of the MD2 and CD14 upstream primers, respectively, at the 5' end of the downstream primer.
  • the restriction endonuclease recognition sequence KpnI and the protective base the restriction endonuclease recognition sequence KpnI was introduced at the 5' end of the TLR4 upstream primer, and restriction endonuclease recognition was introduced at the 5' end of the downstream primer.
  • the sequence SmaI and the protective base primer were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd.
  • the number of cycles is determined by a cyclic graph to allow the PCR process to be exponentially increasing.
  • the cleavage sites used therein are the cleavage sites that the corresponding genes do not contain.
  • the product size was identified by agarose gel electrophoresis.
  • the TLR4 gene was located at about 2500 bp, the CD14 gene was located at about 1100 bp, and the MD2 gene was located at about 500 bp, which was consistent with expectations.
  • the TLR4 gene, CD14 gene and MD2 gene were separately collected using an agarose gel DNA recovery kit and stored at -20 °C.
  • the recovered DNA fragments were ligated into the pMD19-T cloning vector, respectively, in which TLR4 was inserted between Kpn I and Sma I, CD14 was inserted between EcoR I and Kpn I, and MD2 was inserted between EcoR I and Kpn I to obtain Three cloning vectors were transformed into E. coli DH5 ⁇ , amplified, and plasmids were extracted. Enzyme digestion and electrophoresis were used to identify positive clones.
  • the selected positive clones were named pMD19-T-TLR4 (as shown in Figure 1A), pMD19-T-CD14 (shown in Figure 1B), pMD19-T-MD2 (shown in Figure 1C) were sequenced, and A matched gene clone was retrieved from the Pubmed gene database.
  • the correct pMD19-T-TLR4 positive clone plasmid and the pCAG-GFP eukaryotic expression vector plasmid were subjected to double digestion with SmaI and KpnI, and the fragment was subjected to electrophoresis separation, and the target fragment and the vector arm were recovered.
  • the TLR4 fragment was ligated with the pCAG-GFP fragment, transformed into E. coli DH5 ⁇ , amplified, and the plasmid was extracted, and the positive clone was identified by electrophoresis.
  • pCAG-TLR4-GFP (as shown in Fig. 2A)
  • pCAG-TLR4-GFP was sequenced, and the matched gene clone was searched from the Pubmed gene database.
  • the construction method of MD2, CD14 eukaryotic expression vector is basically the same as that of TLR4 gene expression vector, and the correctly sequenced pMD19-T-MD2, pMD19-T-CD14 positive clone plasmid and pCAG-GFP eukaryotic expression vector plasmid are used in restriction nucleic acid.
  • the dicer enzymes EcoRI and KpnI were double-digested and ligated. After transformation, the monoclonal antibody was selected for culture.
  • the plasmid was extracted in small amount and then digested by double enzyme digestion. After agarose gel electrophoresis, the target fragment was inserted and the size was correct.
  • the genomic DNA sequence of the human genome 19th chromosome PPPR1R12C was searched using the Nucleotide database in NCBI.
  • the human PPP1R12C gene is located at 19q13.42, GeneID: 54776.
  • the genomic DNA is 26688 bp in length
  • the first exon sequence is 1-378 bp
  • the first intron sequence is 378-4806 bp.
  • the first intron sequence of PPPR1R12C is input into http://crispr.mit.edu /Evaluate appropriate CRISPR knock-in targets and obtain appropriate targets for guideRNA based on the results given on the above website.
  • the sgRNA locus was located at 1849 bp using Primer premier 5.0 bio-software designed primers to amplify 1849 bp upstream 1000 bp and downstream 1000 bp long and short arms.
  • the first intron position of the genomic DNA sequence of the 19th chromosome PPPR1R12C was selected as a site-specific knock-in site of the TLR4 gene, and it was reported that the inserted foreign gene had no effect on the physiological function of the cell itself.
  • the TLR4 gene can also be inserted into other chromosomal loci that do not affect the physiological function of the cell, inserted into different genes, and the design of the target primer can be adjusted accordingly.
  • the pX330 plasmid was used as a backbone to synthesize a complementary target sgRNA sequence, and a BbsI restriction site was introduced at both ends.
  • the sgRNA synthesis sequence is shown in Table 5.
  • the complementary two-segment sgRNA fragment was annealed and phosphorylated, and the sgRNA was ligated into the enzyme-cut linearized pX330.
  • the ligation reaction product was transformed into E. coli DH5 ⁇ , amplified, and the plasmid was extracted and then digested with BbsI. After restriction enzyme digestion, the presence or absence of the insert was confirmed by electrophoresis, and a recombinant positive plasmid was obtained, which was named pPsg-Cas9, and sequenced.
  • the HEK 293T (abbreviated as 293T) genomic DNA was extracted, and a sequence of about 1000 bp was selected upstream and downstream of the sgRNA site of the PPPR1R12C gene as a long and short arm of the targeting vector.
  • Primers were designed using Primer premier 5.0 as shown in Table 6. PCR long arm, short arm, gel recovery, ligated into pMD19-T vector, transformed, identified, sequenced, and obtained pMD19-T-long arm plasmid and pMD19-T-short arm plasmid.
  • the long arm and the short arm were separately digested and ligated into the pMCS.DT-A targeting vector by the pMCS.DT-A plasmid and the correctly sequenced pMD19-T-long arm plasmid for Sal I and Hind III double enzymes.
  • the reaction was ligated, electrophoresed, recovered, and the long arm fragment and the vector fragment were ligated with T4 ligase, and the reaction product was ligated into Escherichia coli DH5 ⁇ .
  • the recombinant plasmid was identified by enzyme digestion and sequencing, and DTA-long arm was obtained.
  • the correctly sequenced DTA-long arm plasmid and pMD19-T-short arm plasmid were subjected to Sal I and Hind III double digestion reaction, electrophoresis, recovery, The short arm fragment and the DTA-long arm fragment were ligated with T4 ligase, and the reaction product was ligated into E. coli DH5 ⁇ . Identification and sequencing of recombinant plasmids by digestion and electrophoresis Identification, DTA-long arm-short arm was obtained and sequenced.
  • the correctly sequenced DTA-long arm-short arm plasmid (DONOR) and pCAG-TLR4-GFP plasmid were subjected to double digestion with Spe I and Not I, electrophoresis, recovery, and CAG-GFP-TLR4 fragment was ligated with T4 ligase.
  • the DTA-long arm-short arm was digested and the ligation reaction product was transformed into E. coli DH5 ⁇ .
  • the recombinant plasmid was identified by enzyme digestion and sequencing, and the DTA-short arm-CAG-TLR4-GFP-long arm targeting vector (TLR4-DONOR) was obtained.
  • the construction strategy is shown in Figure 3, and the principle of TLR4 typing into cells is shown. As shown in Figure 4.
  • 293T cells were inoculated with 0.5-1.0 ⁇ 10 5 cells in a 6-well plate, and the cell confluence reached 90-95% at the time of transfection; the targeting vector DTA-shortarm-CAG-TLR4-GFP-longarm was homologously recombined.
  • the 5' end of the short arm of the arm was linearized by BstBI single digestion; the cells were washed with PBS, trypsinized, centrifuged at 800 rpm for 3 min to collect the cells; 293T cells were resuspended in electroporation buffer (serum free DMEM) to adjust the concentration to 1 ⁇ 10 7 /mL.
  • a 4 mm electric shock cup was used, and each cell was shocked with 300 ⁇ L, 25 ng of targeting vector, and 25 ng of pPsg-Cas plasmid.
  • Collect well-preferred cells to make a suspension accurately count the number of cells in the cell suspension according to the cell counting method; and make the cell into a 10/mL cell suspension by gradient dilution, ie, 1 cell per 0.1 ml; cell suspension Seeded in 96-well plate, 0.1 ml per well; 96-well plate was placed in a 37 ° C, 5% CO 2 incubator, and taken out after 4 days, recorded and statistically calculated; selected monoclonal growth wells, good growth, green fluorescence positive Intensive, transferred to 24-well plate and then cloned or cultured, and after repeated passages, cell clones stably expressing green fluorescence were named as 293T/TLR4 cell clones, as can be seen from Figure 5A and Figure 5B. When 293T/TLR4 is irradiated with white light, no fluorescence is exhibited, and when irradiated with excitation light, green fluorescence is exhibited.
  • the pAAV-CAG-RFP was used as a template to amplify the RFP fragment, and the F2A sequence was introduced at the 5' end (as shown in Table 7), the ageI restriction site was introduced, and the Not I cleavage site was introduced at the 3' end. point.
  • the pCAG-CD14-GFP plasmid was digested with AgeI and Not I. The green fluorescent GFP fragment was excised and ligated into the F2A-RFP fragment.
  • the primer sequences are shown in Table 8.
  • the pCAG-CD14-F2A-RFP plasmid was identified by enzyme digestion and sequenced.
  • the 293T cells were stained for fluorescence expression; the MD2 fragment was amplified by PCR, and the 3D T2A sequence (shown in Table 7) was introduced into the Age I restriction site, and the pCAG-CD14-F2A-RFP plasmid was digested with Kpn I enzyme and used.
  • the abm cloning kit was ligated into the MD2-T2A fragment by homologous recombination, and the primer sequence was as shown in Table 8.
  • the pCAG-MD2-T2A-CD14-F2A-RFP plasmid was identified by restriction endonuclease digestion.
  • the genomic DNA sequence of the human genome third chromosome CCR5 was searched using the Nucleotide database in NCBI.
  • the first intron sequence of the CCR5 gene was imported into http://crispr.mit.edu/ to evaluate the appropriate CRISPR knock-in target, and the appropriate target of guideRNA was obtained based on the results given above.
  • the first intron position of the genomic DNA sequence of the third chromosome CCR5 was selected as a site-in site for the CD14 gene and the MD2 gene, and it was reported that the inserted foreign gene had no effect on the physiological function of the cell itself.
  • the CD14 gene and MD2 gene can also be inserted into other chromosomal loci that do not affect the physiological functions of cells, inserted into different genes, and the design of target primers can be adjusted accordingly.
  • the pX330 plasmid was used as a backbone to synthesize complementary target sgRNA sequences and introduce BbsI enzyme at both ends. Cut the spot.
  • the sgRNA synthesis sequence is shown in Table 9.
  • the complementary two-segment sgRNA fragment was annealed and phosphorylated, and the sgRNA was ligated into the enzyme-cut linearized pX330.
  • the ligation reaction product was transformed into E. coli DH5 ⁇ , amplified, and the plasmid was extracted and then digested with BbsI. After restriction enzyme digestion, the presence or absence of the insert was confirmed by electrophoresis, and a recombinant positive plasmid was obtained, which was named pCsg-Cas, and sequenced.
  • a sequence of about 1000 bp was selected upstream and downstream of the sgRNA site of the CCR5 gene as a long and short arm of the targeting vector.
  • Primers were designed using Primer premier 5.0 as shown in Table 10. PCR long arm, short arm, gel recovery, ligating into pM19-T vector, transformation, identification, sequencing.
  • the long arm and the short arm are separately digested and ligated into the pMCS.DT-A targeting vector.
  • step 3.2.2 in which the short arm, Sac II and Sac are connected in the middle of the pMCS.DT-A vector Sal I and HindIII. I joined the long arm between I and finally got the DTA-long arm-short arm.
  • pCAG-MD2-T2A-CD14-F2A-RFP plasmid was digested and ligated into DTA-long arm-short arm vector.
  • step 3.2.2 refers to obtain DTA-short arm-CAG-MD2-T2A-CD14.
  • -F2A-RFP-long arm targeting vector the construction strategy is shown in Figure 6, and the schematic diagram of MD2 and CD14 knock-in cells is shown in Figure 7.
  • the targeting vector and pCsg-Cas were electroporated into 293T and 293T/TLR4 by the method of step 3.2.3, and the monoclonal was screened by the method of step 3.2.4 to obtain two red fluorescent cloned cells, 293T/CD14/MD2 cells and 293T.
  • /TLR4/CD14/MD2 cells as shown in Figure 8, it can be seen that both cell clones can emit red fluorescence.
  • 293T/CD14/MD2 cells were used as controls, 293T/TLR4/CD14/MD2 was fine The cell is the cell line of interest.
  • the primer sequences are shown in Table 11.
  • the wild type can amplify the fragment with primer 1+primer2 and primer4+primer5, while primer 1+primer 3, primer 4+primer 6 can not amplify the fragment.
  • TLR4 knock-in cells can be amplified with primer 1+primer 3
  • CD14-MD2 knock-in cells can be amplified with primer 4+primer 6.
  • the 293T/TLR4, 293T/CD14/MD2, 293T/TLR4/CD14/MD2 knock-in sites were correctly identified by PCR.
  • Total protein was extracted from 293T, 293T/TLR4, 293T/CD14/MD2, 293T/TLR4/CD14/MD2 four cell line lysing cells.
  • the expressions of TLR4, CD14 and MD2 were identified by Actin as an internal standard. As shown in Figure 12, 293T cells were free of TLR4, CD14, and MD2 expression, 293T/TLR4 had TLR4 expression, no CD14, and MD2 expression, 293T/CD14/MD2 had TLR4, CD14, and MD2 expression, and 293T/TLR4/CD14/MD2 had TLR4, CD14, MD2 expression.
  • 293T/CD14/MD2 may activate endogenous TLR4 due to the presence of CD14/MD2 such that there is a small amount of TLR4 expression.
  • Mass spectrometry was used to identify the reactivity of 293T/TLR4/CD14/MD2 cells to LPS stimulation, and to detect the expression of IL-6 and TNF ⁇ .
  • Colloidal gold test paper detection method is to use nano-scale colloidal gold as a tracer and detection agent, and the detection method of antigen-antibody reaction is applied. After the 293T/TLR4/CD14/MD2 cell line LPS is stimulated for 5 hours, the sample to be tested is tested. After the model cells were co-cultured for 5 hours (the same as the sample and culture method used by Western Blot and ELISA), 50 ⁇ L of the supernatant of the culture solution was added to the colloidal gold test strips of IL-6 and TNF ⁇ , respectively. Commercially available or custom made), based on the reaction of the test strip to determine whether the expression of IL-6 and TNF ⁇ is positive.
  • the cell model provided by the present invention can react to pyrogen stimulation, and can detect not only endotoxin of Gram-negative bacteria but also non-endotoxin-derived pollutants, including from Gram.
  • Pathogenic-related molecular patterns (PAMPs) of positive bacteria, Gram-negative bacteria, viruses, and fungi, and biological or chemical entities related to the preparation and processing and the minimum detection limit is lower than that of the sputum reagent, which is more sensitive. Its stability is higher than the detection of human whole blood.
  • PAMPs Pathogenic-related molecular patterns

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Abstract

一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒。利用CRISPR/CAS9诱导基因组特定位置形成双键断裂，并利用同源重组在细胞系的两个染色体分别定点敲入TLR4及CD14-MD2并以绿色荧光蛋白GFP和红色荧光蛋白RFP分别示踪，最终成功构建TLR4/CD14/MD2定点敲入荧光示踪细胞模型。

Description

一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒 技术领域
本发明属于生物技术领域，涉及药品和生物制品(包括基因工程制品)中热原的检测，具体一种用于检测热原的细胞模型的构建方法及细胞模型及检测热原的方法及试剂盒。
背景技术
1.1热原概述
众所周知，在临床治疗中使用注射类药物或医疗器材时往往导致患者出现发冷、寒战、恶心、发热、呕吐、头痛、白细胞下降、血管通透性增强、昏迷、休克等不良反应而加重病情，不仅增加了患者的痛苦，影响抢救和治疗的效果，甚至可能危及病人生命。1875年，Burdon-Sanderson(李增宏，王瑞东.佛山科学技术学院学报,2007,25(3):43-45)研究表明，在腐败的肉中分离出不带活菌的物质，其能引起上述一系列不良反应的，称其为热原(Pyrogen)，并将上述一系列症状定义为热原反应。此后，随着科学研究的深入发展，热原也有了更为确切的定义，指由微生物产生的能引起恒温动物体温异常升高的致热物质。1923年，Seibert肯定了热原由微生物所产生，具有耐热性、不挥发性、水溶性、滤过性和吸附性的特点，不易被截留，热稳定性强，虽然经过严格灭菌，但仍不能排除热原反应的发生。热原可分为外源性热原与内源性热原两类，外源性热原主要革兰氏阴性菌的内毒素和革兰氏阳性菌的脂磷壁酸，内源性热原主要包括机体内细胞因子(如TNF-α，INF-β，IL-1，生长因子)，及激素(如类固醇、前列腺素)等。而通常我们通常所指的“热原”，主要指细菌性热原，它是某些细菌的代谢产物、细菌尸体及内毒素。其中致热能力最强的是革兰阴性G-杆菌杆菌的产物，其次是革兰阳性G+杆菌类，革兰阳性G+球菌则较弱，霉菌、酵母菌甚至病毒也能产生热原(Charles A,Dinarello.Endotoxin Research,2004,10:201)。
细菌内毒素(Endotoxin)属于外源性热原，是革兰氏阴性G-菌细胞壁外壁层上的特有结构，是磷脂、脂多糖(Lipopolysaceharide,LPS)与蛋白质结合而成的复合物。LPS是复合物的主要成分及活性中心，致热作用最强，但其化学组成因菌种不同而有所差异，分子量为5×104～5×105，分子量越大致热作用则越强。细菌内毒素不是细菌本身或细菌的代谢产物，而是细菌死亡或解体后才释放出来的一种具有生物活性的毒性物质，并且不能用普通灭菌方法清除，是迄今发现生物活性最为广泛的物质之一(刘宣亚.明胶科学与技术,2013,33(1):45-48)。当细菌内 毒素通过消化系统进入人体时，并不产生危害，但细菌内毒素通过注射等方式进入血液时，则会引起不同的疾病，最为典型的是热原反应。进入人机体的细菌内毒素不仅可直接对细胞生物膜产生毒性，而且可通过单核巨噬细胞介导的免疫炎症反应，诱导多种细胞因子及炎症因子合成和释放，过度表达的炎症递质以自分泌、旁分泌或内分泌方式作用于单核-巨噬细胞膜上的受体，进一步激活炎症递质的表达(Guha M,Mackman N.Cell Signal,2001,13(2):85-94.)。这些炎症因子的失控性释放影响细胞功能的完整性，造成机体代谢紊乱，血液凝固性升高，组织血液灌注不足，严重时造成多器官功能衰竭而死亡(Hotchkiss RS,Karl IE.N Engl J Med,2003,348(2):138-150.)。随着药品与医疗管理部门对临床热原反应的重视程度日益加强，由于革兰氏阴性菌的分布范围极为广泛，故对药品及医疗器械中致热原的检测也成为了人们近期研究的焦点。
1.2现有主要热原检测方法及其局限性
对于热原的检测，目前我国药典收录的方法有家兔热原试验法和鲎试剂内毒素检测法，第七版欧洲药典(2011版)还收录了热原检查的第三种新方法--单核细胞激活检查法。
家兔热原试验法。由于家兔对热原的反应与人基本相似，家兔法为各国药典规定的检查热原的法定方法。将一定剂量的供试品，静脉注入家兔体内，在规定时间内，观察家兔体温升高的情况，以判定供试品中所含热原的限度是否符合规定。
家兔法仍然存在着许多局限性。首先，家兔法不能定量，无法标准化，灵敏性和重复性差，而且家兔对热原的敏感性存在着个体差异，不同个体对热原的敏感性相差10倍以上。其次，某些类型的药品会干扰检测结果，如一些解热镇痛药、细胞毒性药物或一些容易影响体温中枢的药品，不能够使用家兔法。另外，许多已经通过家兔热原检测的制品在临床上仍然会发生热原反应。在细胞工程技术、新生物材料以及中药制品发展迅猛、日新月异的今天，家兔法已不能完全代表人体的发热反应，其操作繁琐费时，不能用于注射剂生产过程中的质量监控，且有些药物不适用。
鲎试剂内毒素检测法。美洲鲎血液遇革兰氏阴性菌时会产生凝胶，美国、英国、欧洲、日本和中国药典目前均收载了这一检查法。鲎试剂内毒素检测法系利用鲎试剂来检测或量化由革兰氏阴性菌产生的细菌内毒素，以判断供试品中细菌内毒素的限量是否符合规定的一种方法。细菌内毒素的量用内毒素单位(EU)表示。鲎试剂法检查内毒素的灵敏度为0.015EU/mL，比家兔法灵敏，操作简单易行，试验费用低，结果迅速可靠，适用于注射剂生产过程中的热原控制和家兔法 不能检测的某些细胞毒性药物制剂。
但鲎试剂法受环境因素影响较大，容易出现假阳性结果。且对于本身带有颜色的制剂无法进行检测。鲎试剂法实际能够检测到的仅仅是内毒素对鲎血细胞的凝集活性，而不是内毒素对人体的发热活性或化学概念上的含量，而且无法检测除内毒素以外的其他致热源。该方法对一些酶抑制剂、钙镁离子鳌合剂也无法检测，因此鲎试验还无法完全取代家兔热原试验。我国制作鲎试剂的中华鲎是一种比恐龙还早的物种，近年由于填海造地，渔业活动，中华鲎面临灭绝的危险。故根据实验动物学的"3Rs原则"即减少(Reduction)、优化(Refinement)和替代(Replacement)，对鲎试剂替代方法的研究迫在眉睫。
单核细胞激活检查法。除了家兔法和鲎试剂法，第七版欧洲药典(2011版)已收录了热原检查的第三种新方法--单核细胞激活检查法(monocyte activation test，MAT)。其原理是将人的全血与供试品一同孵育培养，根据血液中单核细胞释放出的IL-6、IL-1β、TNF-α等细胞因子的量来评价供试品中热原的活性。MAT可以检测出热原和促炎症因子污染物，包括革兰氏阴性细菌的内毒素及非内毒素来源的污染物，包括来自于革兰氏阳性细菌、革兰氏阴性细菌、病毒、真菌的病原相关分子模式(PAMPs)和制品相关的以及加工工艺相关的生物或者化学实体。而单核细胞激活检查法也存在一定的局限性，不同来源的人血分离出的单核细胞反应性可能不太一样，另外新鲜的人血不易大批量获得。所以这个方法除了不容易普及外还具有稳定性差的缺陷。
由此可见应用新的基因工程制备的体外热原检测方法代替传统方法是该领域发展的国际大趋势。对于热原检测的研究，正向着细胞和分子水平层面发展。我国对于这方面的研究尚处于初级阶段，还没有用于热原检测的细胞模型收录入药典。但可以预见的是应用细胞模型检测热原已经成为研究者的主要关注焦点，方法的简易性与高效性成为研究的必然趋势。
1.3热原相关受体及信号通路
近年来，对革兰氏阴性菌G-菌引起内毒素反应的的机制已有更为深入的了解，同时也认识到LPS在激活机体免疫反应应答及导致热原反应过程中起主导用。LPS在多种病原体存在某些高度保守的病原结构，这些保守的病原结构形式被称作病原相关分子模式(Pathogen associated molecular patterns,PAMP)。Toll样家族受体作为先天性免疫系统中的重要组成部分，其作用被认为与结构识别受体(Pattern-recognition receptors,PRRs)相似，主要通过识别病原微生物表面的PAMP来启动免疫反应，进而清除外来抗原。
在LPS所致的炎症信号传导通路中，有以下四种起关键作用的受体：脂多糖 结合蛋白(Lipopolysaccharide binding protein,LBP)、Toll样受体4(Toll like receptor-4,TLR4)、髓样分化蛋白2(Myeloid differentiation protein-2,MD2)及白细胞分化抗原14(Monocyte differentiation antigen 14,CD14)。
LBP是一种广泛存在于人和动物血清中的糖蛋白，严格来说，LPS结合蛋白并不是LPS的结合受体，但LPS介导细胞激活需要血浆或细胞表面能够和LPS结合的蛋白参与，是LPS发挥生物学作用的重要载体。它对于细菌内毒素及LPS中的类脂A具有很高的亲和力。LBP与LPS结合，形成复合物并LPS传递给细胞膜上的CD14受体，结合TLR4-MD2复合物，通过一系列跨膜、胞内信号转导过程使靶细胞活化并释放炎症前细胞因子和免疫调节因子。
TLR4属于TLRs家族(Toll like receptors)成员，目前，人类相继发现了10种TLRs家族的蛋白，分别命名为TLR1-10(Lemaitre B,Nicolas E,Miehaut L et a1.Cell,1996,86(6):973-983；HuangB.Zhao J.Unkeless J C et a1.Oneogene，2008,27(2)：218-224；刘兴，冯文莉，康格非.国外医学临床生物化学与检验学分册,2001,22(3):134-136；T Kawai,S Akira.Cell Death and Differentiation,2006,13:816-825)。不同的TLRs转导信号由不同来源的微生物所刺激。所以不同TLR可以在一定程度上识别并区分入侵机体的病原体类型。在TLRs家族成员中，目前研究得最多的为TLR4、TLR2在机体先天免疫中的作用，其中又以TLR4在LPS信号转导中的机制研究的最为广泛深入(秦玉新，张庆柱.中国药理学通报,2003,19(12):1336-1339；Li-Yun Huang,James L.DuMontelle,et al.Clinlcal Microbiology,2009,47:3427-3434)。TLR4是炎症反应中最重要的一类模式识别受体，能够特异性识别微生物进化过程中的一些保守序列(如LPS)。TLR4是LPS诱导细胞信号转导的最主要受体，主要表达在参与宿主防御功能的细胞上，如外周血白细胞、T淋巴细胞、B淋巴细胞、单核巨噬细胞、肥大细胞、朗罕氏细胞、树突状细胞等，其中以在骨髓单核细胞中表达最多(Medzhitov R,Preston Hurlburt P,Janeway Jr.CA.Nature,1997,388:394-79)。
MD2是一种分泌蛋白，利用表达TLR4和MD2的转染子做免疫沉淀实验证实，MD2伴随TLR4表达，并分泌至细胞表面，通过物理作用于TLR4锚定在细胞膜上。MD2有助于TLR4识别LPS，并将LPS锁定在TLR4上的结合位点，对TLR4的向胞膜转移和表达起到至关重要的作用；研究发现缺乏了MD2分子，TLR4对LPS的反应性极低(Miyake K，R.Shimazu,J.Kondo,et al.Immunol.1998,161:1348-1353；Pugin J,Stem Voeffray S,Daubeuf B.Blood.2004,104(13):4071-4079；刘亚伟，刘靖华，唐靖，等.南方医科大学学报,2006,26(8):1101-1105；钟田雨，刘靖华，姜勇.生物化学与生物物理进展,2007,34(5): 460-464)。
CD14是一种在胞浆和白细胞表面作为葡萄糖磷脂酞肌醇连接蛋白表达的蛋白质，与LPS具有较高的亲和力，但CD14分子缺乏胞浆区段，不能直接向细胞内传导LPS信号。LPS与CD14结合后，首先形成LPS-LBP-CD14配体和辅助因子复合物，然后再与TLR4结合，将信号传递进入细胞(Saitoh S,Akashi S,Yamada T,et al.Endotoxin Res,2004,10(4):257-260；Nagai Y,Akashi S,Nagafuku M,et al.Nat Immunol,2002,3(7):667-672；Zielger,Heitbrock HWL and Ulevitch RJ.Immunol Today,1993,14(3):121-125)。
当细菌侵入机体后，LPS由细菌裂解释放入血，可以与血浆中游离的脂多糖结合蛋白LBP结合形成复合物，与CD14分子结合或直接与TLR4的附属蛋白即髓样分化蛋白MD2相结合，形成LPS-MD2/TLR4复合物，在这些分子的辅助下激活TLR4。
1.4CRISPR/CAS9系统的基本原理
CRISPR/Cas9(The Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)and CRISPR Associated(Cas)system)是一种能快速、方便有效地靶向人类基因组任何基因的新方法。CRISPRs是一类广泛分布于细菌和古菌基因组中的重复结构，指的是成簇的、规律间隔的短回文重复序列(clustered regularly inter-spaced short palindromic repeats)，这是细菌的适应性免疫保护机制，是为了对付噬菌体(bacteriophages)病毒而进化出的一种DNA片段。细菌表达Cas9蛋白，同时会转录出CRISPR RNA，这种RNA能够与病毒的基因组互补配对。这种Cas9复合物就可以切割病毒的基因组，使病毒失活。研究表明，CRISPR与一系列相关蛋白、前导序列一起，能为原核生物提供对抗噬菌体等外源基因的获得性免疫能力。CRISPR-Cas9技术是利用一段与靶序列相同的单导向RNA(sgRNA)来引导Cas9核酸酶对特异靶向DNA进行识别和切割，造成DNA的双链或单链断裂，然后，细胞会利用自身具备的两种修复机制对断裂的DNA进行修复，即非同源性末端接合(NHEJ)或同源介导修复(HDR)。
CRISPR/Cas系统的出现为基因工程提供了一个强有力的应用新工具，它将给基因组定向编辑的研究和应用领域带来突破性的技术革命，特别是在基因功能解析、人类疾病靶向治疗等应用中有巨大的潜力和广阔的前景。更令人鼓舞的是，其操作简单、实验周期短、节约成本，有利于在普通实验室推广这一技术，因此，CRISPR/Cas系统的广泛应用将对生物学研究产生深远的影响。利用CRISPR/Cas系统对基因组进行定点编辑，将有助于人们更好地了解基因的功能。
内毒素是革兰阴性菌引起热原反应的主要成分，是严重威胁药品安全的关键 因素，因此建立一种更合理、更适宜的热原检测方法就显得尤为迫切。
发明内容
本发明的目的在于提供一种用于检测热原的细胞模型，该细胞模型可用于药品生物制品包括基因工程药品的热原检测。
本发明的另一目的在于提供一种上述用于检测热原的细胞模型的构建方法。
本发明的第三个目的在于提供一种用于检测热原的试剂盒，该试剂盒中含有上述细胞模型。
本发明的第四个目的在于提供一种用于检测热原的方法，该方法使用上述试剂盒进行检测。
为了实现上述目的，本发明采用如下技术方案：
本发明提供一种用于检测热原的细胞模型的构建方法，包括如下步骤：
1)将TLR4基因组装到可表达绿色荧光的表达载体中，构建含有TLR4基因的重组质粒；
2)采用CRISPR/CAS9法将TLR4基因定点敲入细胞系一染色体上的一基因的内含子中，获得一稳定表达TLR4基因的细胞系；
3)使用两个不同的2A肽将CD14、MD2和红色荧光RFP基因结构隔开构建含有CD14基因和MD2基因并可表达红色荧光的重组质粒；
4)采用CRISPR/CAS9法将CD14基因和MD2基因定点敲入步骤2)所获得的细胞系的敲入该TLR4基因的染色体以外的另一染色体上的一基因的内含子中，获得一可稳定表达TLR4基因、CD14基因和MD2基因，并可同时表达绿色荧光和红色荧光的细胞株，即为所述的细胞模型。
优选地，所述细胞系为HEK293T、HEK293或NIH3T3。
更进一步地，所述步骤2)中敲入TLR4基因的位置为第十九号染色体PPR1R12C基因第一位内含子。
更进一步地，所述步骤4)中敲入CD14基因和MD2基因的位置为第三号染色体CCR5基因第一位内含子。
本发明还提供一种采用上述的构建方法构建的细胞模型，所述细胞模型是在细胞系HEK 293T的十九号染色体PPR1R12C基因第一位内含子内定点敲入TLR4基因，在三号染色体CCR5基因第一位内含子内定点敲入CD14基因和MD2基因。
其中，所述细胞模型的分类名称为人源胚胎肾细胞(HEK)变种293T/TLR4/CD14/MD2，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所， 保藏日期：2016年05月19日，保藏编号：CGMCC No.12296。
本发明还提供一种用于检测热原的试剂盒，包括如下组成：1)如上述的构建方法构建的细胞模型，或上述的细胞模型，2)热原标准品，3)IL6和/或TNFα对照品。
优选地，所述热原标准品为LPS冻干粉，IL6对照品为IL6冻干粉，TNFα对照品为TNFα冻干粉。
本发明进一步提供一种用于检测热原的方法，包括如下步骤：
1)制备待测样品溶液和热原标准品溶液；
2)将上述待测样品溶液加入到含有采用上述的构建方法构建的细胞模型，或上述的细胞模型的培养液中37℃培养5h；
3)收集培养后的上清液检测其中IL-6和/或TNF-α的含量。
优选地：步骤3)所述检测IL-6和/或TNF-α的含量的方法采用Western Blot、ELISA、金标法或质谱法。
本发明采用CRISPR/CAS9方法在细胞中敲入3个基因，其中有两个基因定点敲入到一条染色体上，另外一个基因敲入另外一条染色体。敲入的TLR4基因以绿色荧光GFP示踪；定点敲入的CD14-MD2以红色荧光RFP示踪。应用自我剪切2A肽多基因载体构建策略使用两个不同的2A肽将CD14、MD2和红色荧光RFP基因结构隔开，避免对蛋白表达产生影响。
在本发明的实施例中使用了F2A和T2A两种2A肽结构，其中F2A为来源于口蹄疫病毒(foot-and-mouth disease viruses，FMDV)的2A肽，T2A为来源于蒂勒氏鼠脑脊髓病毒(Theiler’s murine encephalitis virus，TMEV)的2A肽，避免了使用同一2A的基因序列在表达载体构建时出现交叉反应难以克隆进目的位点。
热源刺激该模型细胞后，可以通过ELISA、Western Blot、质谱、磁珠法等方法检测到IL-6和/或TNF-α细胞因子的释放。
本发明的特点在于：
1.使用CRISPR/CAS9技术定点敲入特定基因
本发明采用定点敲入的方式将外源基因转入细胞，而非采用病毒、慢病毒等载体形式进行瞬时转染，使得遗传稳定。现有CRISPR/CAS9技术一般多应用于基因敲除或基因突变，而对于基因敲入，现有方法中要克服脱靶效应，同源重组效率低，敲入载体构建过程繁琐等重重困难，尤其进行多个基因的敲入困难就更大。而本发明采用基因敲入，将多基因成功敲入细胞系，如HEK293T细胞，解决了CRISPR/CAS9技术基因敲入困难的问题。
2.将基因敲入不同染色体
本发明将TLR4基因、CD14基因和MD2基因这三个基因定点敲入两条不同染色体，插入的位置为确保基因敲入后不会影响细胞本身的生理功能，保证细胞的正常存活和稳定传代的位点。如本发明实施例中TLR4基因插入HEK 293T的十九号染色体PPR1R12C基因第一位内含子内，CD14基因和MD2基因插入HEK 293T的三号染色体CCR5基因第一位内含子内，这两个位置都是可以确保基因敲入后不会影响细胞本身的生理功能，保证细胞的正常存活和稳定传代。同时，不将三个基因插入到同一染色体是由于插入同一染色体，会由于位阻效应影响表达，同时三个基因的融合蛋白过大，会影响染色体上基因表达。在插入染色体的基因后面加入绿色荧光蛋白或红色荧光蛋白，可以示踪蛋白是否表达。在细胞传代中也可以示踪蛋白的表达。
3.检测灵敏度高
本发明构建的细胞模型，属于工具细胞，在没有LPS刺激的情况下无IL-6、TNF-α等细胞因子的表达。其他来源的细胞，如人单核细胞，在没有LPS刺激时会有一定量的IL-6、TNF-α等细胞因子的本底表达，而本底表达会对后续的检测产生影响，而本细胞模型在没有LPS刺激时没有细胞因子的表达，没有本底的存在。同时，某些基因工程产品可能含有刺激细胞增殖的物质，不同程度的细胞增殖，将对检测的不确定性造成影响，而本发明的细胞模型不存在这种风险。本发明细胞对于LPS的最低检测限可达0.005EU/mL，而鲎试剂法的最低检测限为0.025EU/mL，可以表明本发明的灵敏度远高于鲎试剂法。
4.解决现有热原检测中的不足
鲎是国家二级保护动物，其血液来源有限，使用鲎试剂会影响鲎的保护，不利于生物资源的可持续发展，并且不同厂家生产鲎试剂的方法不同，对同一产品的热原检测易产生不一致的结果。单核细胞激活检查法由于需要使用人血，而不同人血来源的单核细胞反应性不稳定，造成该方法的稳定性差。本发明提供的细胞模型通过将检测基因插入染色体，使之可以稳定传代，对热原检测重复性好，反应性好，模型稳定，克服了传统方法和新型人单核细胞激活检查法的缺点。
本发明的有益效果在于：
本发明提供一种可以用于药品生物制品包括基因工程药品的热原检测的细胞学模型，其构建方法及热原检测方法和试剂盒。该细胞模型利用CRISPR/CAS9诱导基因组特定位置形成双键断裂，并利用同源重组修复的原理在细胞系的两个染色体分别定点敲入TLR4及CD14-MD2并以绿色荧光GFP和红色荧光RFP分别示踪最终我们成功构建TLR4/CD14/MD2定点敲入荧光示踪细胞模型，LPS刺 激细胞模型可通过ELISA、Western Blot、质谱及磁珠法检测到IL-6、TNF-a细胞因子的释放，该细胞模型稳定性好，灵敏性高，最低检测限可达0.005EU/mL，远远低于鲎试剂法的0.025EU/mL。
附图说明
图1A为本发明一优选实施例中TLR4与pMD19-T连接的质粒图谱。
图1B为本发明一优选实施例中CD14与pMD19-T连接的质粒图谱。
图1C为本发明一优选实施例中MD2与pMD19-T连接的质粒图谱。
图2A为本发明一优选实施例中TLR4与pCAG-GFP构建的瞬时表达载体pCAG-TLR4-GFP的质粒图谱。
图2B为本发明一优选实施例中CD14与pCAG-GFP构建的瞬时表达载体pCAG-CD14-GFP的质粒图谱。
图2C为本发明一优选实施例中MD2与pCAG-GFP构建的瞬时表达载体pCAG-MD2-GFP的质粒图谱。
图3为本发明一优选实施例中TLR4定点敲入的载体构建策略。
图4为本发明一优选实施例中TLR4定点敲入细胞的原理示意图。
图5A为本发明一优选实施例中TLR4定点敲入细胞后照射白光的照片。
图5B为本发明一优选实施例中TLR4定点敲入细胞后经激发显示绿色荧光的照片。
图6为本发明一优选实施例中MD2-CD14定点敲入的载体构建策略。
图7为本发明一优选实施例中MD2-CD14定点敲入细胞的原理示意图。
图8为本发明一优选实施例中MD2-CD14定点敲入细胞后的荧光检测图。
图9为本发明一优选实施例中四种细胞荧光表达情况。
图10为本发明一优选实施例中四种细胞通过共聚焦显微镜观察到的表达情况。
图11A为本发明一优选实施例中四种细胞PCR鉴定敲入TLR4的策略示意图。
图11B为图11A的PCR结果电泳图。
图11C为本发明一优选实施例中四种细胞PCR鉴定敲入CD14/MD2的策略示意图。
图11D为图11C的PCR结果电泳图。
图12为本发明一优选实施例中四种细胞Western blot检测蛋白表达情况结果图。
图13A为本发明一优选实施例中使用ELISA检测四种细胞系经LPS刺激细 胞后IL-6释放情况。
图13B为本发明一优选实施例中使用ELISA检测四种细胞系经LPS刺激细胞后TNFα释放情况。
图14为本发明一优选实施例中使用ELISA检测293T/TLR4/CD14/MD2细胞系经LPS刺激后IL-6释放情况与时间的关系图。
图15为本发明一优选实施例中使用ELISA检测不同细胞数293T/TLR4/CD14/MD2经LPS刺激后IL-6释放量。
图16为本发明一优选实施例中使用ELISA检测293T/TLR4/CD14/MD2细胞系经不同量LPS刺激后IL-6释放敏感性。
图17A为本发明一优选实施例中使用Western blot法鉴定293T/TLR4/CD14/MD2细胞对LPS刺激的TNF-α的反应性。
图17B为本发明一优选实施例中使用Western blot法鉴定293T/TLR4/CD14/MD2细胞对LPS刺激的IL-6的反应性。
具体实施方式
下面通过具体实施例进一步说明本发明的技术方案，但并不因此将本发明限制在所述的实施例范围之中。
仪器和材料：
1.限制性内切酶均购自加拿大Fermentas公司。
2.pMD19-T购买自宝生物工程(大连)有限公司(Takara)。
3.pCAG-GFP、pCAG-RFP、pX330、pMCS.DT-A和pAAV-CAG-RFP均购自Addgene公司。
4.人单核细胞分离液试剂盒购自天津灏洋生物制品公司。
5.RPMI1640培养基(无血清)和DMEM培养基(无血清)购自GIBCO公司，胎牛血清购自Hyclone公司。
6.HEK293T细胞购自中国科学院(上海)细胞库。
7.测序：采用通用或特异引物，由北京奥科鼎盛生物科技有限公司测序。
需要说明的是，本发明中未提供详细步骤的部分均采用常规分子生物学的方法实现或按照相应试剂说明书实现。
实施例1细胞模型的构建
1.人外周血单核细胞cDNA的制备
1.1人外周血单核细胞的分离和培养
取5mL正常人新鲜抗凝全血，按照人单核细胞分离液试剂盒自带说明书进行操作，提取人外周血单核细胞，再加入RPMI1640培养基洗涤离心1次， 1500r/min离心10min，弃上清。按每毫升血液标本加3～4mL含10％胎牛血清的RPMI1640培养液重新混悬细胞，细胞计数，调整细胞密度达2×107/mL培养于10cm2培养皿中，放置于37℃、5％CO2细胞培养箱中培养2～3h后去除上清，得到贴壁的人单个核细胞。然后吸取10μL按需要稀释比例稀释后的细胞悬液与10μL台盼蓝染液(0.4％)混合，进行活细胞计数，将细胞浓度控制在2×107/mL左右。
1.2人外周血单核细胞cDNA的制备
将步骤1.1制备的人外周血单核细胞的细胞培养液弃去，用PBS溶液冲洗3遍，在六孔板中每孔加入200μL TRIzol Reagent(Gibco BRL)，反复吹打裂解细胞。收获裂解后的细胞于1.5mL离心管中，室温放置5min。加入0.1mL氯仿，剧烈振荡15sec，室温放置2～3min。经12,000g，4℃离心15min后，吸出水相转移至另一1.5mL离心管中。加入250μL异丙醇，混匀，室温放置10min。4℃，12,000g离心10min沉淀RNA，加入1mL 75％乙醇洗涤沉淀，空气中干燥5 10min。加入适量不含RNase的DEPC水溶解RNA，55～60℃水浴10min以使RNA充分溶解。用紫外分光光度计测定RNA样品的浓度(OD260)和纯度(OD260/OD280)，然后将RNA浓度调整至1μg/μL待用。
RNA样品70℃水浴变性5min后，于冰上放置5min。按照下列反应体系进行逆转录反应，总反应体积为20μL。
表1 RT反应体系
Figure PCTCN2016101862-appb-000001
混匀，37℃60min，70℃10min灭活，加水至100μL，-20℃储存待用。
2.TLR4，CD14，MD2基因的载体构建和鉴定
2.1TLR4，CD14，MD2原核表达载体的构建及鉴定
2.1.1TLR4，CD14，MD2基因DNA片段的获得
利用NCBI检索得到人类基因组TLR4、MD2和CD14的mRNA序列。使用Primer premier5.0软件，参照相关文献设计RT-PCR引物，分别在MD2和CD14上游引物的5’端引入限制性核酸内切酶识别序列EcoRI，在下游引物的5’端引 入限制性核酸内切酶识别序列KpnI和及保护碱基，在TLR4上游引物的5’端引入限制性核酸内切酶识别序列KpnI，在下游引物的5’端引入限制性核酸内切酶识别序列SmaI及保护碱基引物由北京奥科鼎盛生物科技有限公司合成。循环数则通过循环曲线图来确定，以使PCR过程处于指数增长期。其中所使用的酶切位点均为相应基因不含的酶切位点。
表2.PCR引物序列和PCR产物长度
Figure PCTCN2016101862-appb-000002
表3.PCR反应体系
Figure PCTCN2016101862-appb-000003
表4 PCR反应条件
Figure PCTCN2016101862-appb-000004
反应后进行琼脂糖凝胶电泳鉴定产物大小，经鉴定TLR4基因位于2500bp左右，CD14基因位于1100bp左右，MD2基因位于500bp左右，均与预期相符。将TLR4基因、CD14基因和MD2基因分别使用琼脂糖凝胶DNA回收试剂盒或回收，于-20℃保存。
2.1.2TLR4，CD14，MD2基因原核表达载体的构建和鉴定
将回收的DNA片段分别连接到pMD19-T克隆载体中，其中TLR4插入到Kpn I和Sma I之间，CD14插入到EcoR I和Kpn I之间，MD2插入到EcoR I和Kpn I之间，获得三种克隆载体，并转化到大肠杆菌DH5α中，扩增，提取质粒， 酶切，电泳鉴定阳性克隆。
将挑选出的阳性克隆，命名为pMD19-T-TLR4(如图1A所示)，pMD19-T-CD14(如图1B所示)、pMD19-T-MD2(如图1C所示)测序，并从Pubmed基因数据库中检索相匹配的基因克隆。
2.2TLR4，CD14，MD2真核表达载体的构建及鉴定
2.2.1TLR4，CD14，MD2真核表达载体的构建及鉴定
将测序正确的pMD19-T-TLR4阳性克隆质粒和pCAG-GFP真核表达载体质粒进行SmaI和KpnI双酶切反应，酶切片段进行电泳分离，并回收目的片段和载体臂。
将TLR4片段与pCAG-GFP片段进行连接，转化到大肠杆菌DH5α中，扩增，提取质粒，酶切，电泳鉴定阳性克隆。
将获得的重组阳性质粒，命名为pCAG-TLR4-GFP(如图2A所示)，测序，并从Pubmed基因数据库中检索相匹配的基因克隆。
MD2，CD14真核表达载体的构建方法基本同于TLR4基因表达载体，将测序正确的pMD19-T-MD2、pMD19-T-CD14阳性克隆质粒和pCAG-GFP真核表达载体质粒应用限制性核酸内切酶EcoRI和KpnI进行双酶切并连接，转化后挑单克隆进行培养，质粒小量提取后再次进行双酶切鉴定，经琼脂糖凝胶电泳后观察，选出目的片段插入且大小正确的阳性克隆，分别命名为pCAG-MD2-GFP(如图2B所示)，pCAG-CD14-GFP(如图2C所示)，测序，并在Pubmed基因数据库中Blast，查看基因克隆是否相匹配。
3.CRISPR/Cas9技术构建TLR4定点敲入细胞模型
3.1敲入靶点的确定
利用NCBI中的Nucleotide数据库搜索得到人类基因组第十九号染色体PPPR1R12C的基因组DNA序列。人PPP1R12C基因位于19q13.42，GeneID：54776。基因组DNA全长26688bp，第一位外显子序列为1-378bp，第一位内含子序列为378-4806bp，将PPPR1R12C第一位内含子序列输入到http://crispr.mit.edu/评估合适的CRISPR敲入靶点，根据上述网站给出的结果得到guideRNA合适靶点。sgRNA位点位于1849bp使用Primer premier5.0生物软件设计引物扩增1849bp上游1000bp左右，下游1000bp左右的长短臂。
本实施例中选择第十九号染色体PPPR1R12C的基因组DNA序列的第一内含子位置作为TLR4基因定点敲入部位，有报道证实位置插入的外源基因对于细胞本身的生理功能没有影响。TLR4基因也可以插入到其他不影响细胞生理功能的染色体位点上，插入到不同基因中，其靶点引物的设计可进行相应调整变化。
3.2CRISPR/Cas 9质粒的设计及构建
3.2.1CRISPR/Cas 9质粒的构建
以pX330质粒为骨架，合成互补的靶点sgRNA序列，并在两端引入BbsI酶切位点。sgRNA合成序列如表5。
表5 PPPR1R12C靶向sgRNA合成序列
Figure PCTCN2016101862-appb-000005
Bbs I酶切消化pX330，酶切片段的电泳分离，并回pX330单酶切片段。互补的两段sgRNA片段退火并磷酸化，将sgRNA连入酶切好的线性化pX330中。连接反应产物转化入大肠杆菌DH5α，扩增，提取质粒后用BbsI单酶切进行反应，酶切鉴定后经电泳检查插入片段是否存在，获得重组阳性质粒，命名为pPsg-Cas9，测序。
3.2.2打靶载体的构建
提取HEK 293T(简称293T)基因组DNA，在PPPR1R12C基因sgRNA位点上游、下游各选择1000bp左右的序列，作为打靶载体的长短臂。用Primer premier5.0设计引物，如表6所示。PCR长臂、短臂、凝胶回收、连入pMD19-T载体，转化、鉴定、测序，获得pMD19-T-long arm质粒和pMD19-T-short arm质粒。
表6 PPPR1R12C基因长短臂引物及产物长度
Figure PCTCN2016101862-appb-000006
将长臂、短臂分别酶切并连入pMCS.DT-A打靶载体，具体方法为：将pMCS.DT-A质粒和测序正确的pMD19-T-long arm质粒进行Sal I和Hind III双酶切反应，电泳、回收、用T4连接酶连接长臂片段和载体片段，连接反应产物转化入大肠杆菌DH5α。进行重组质粒的酶切电泳鉴定和测序鉴定，得到DTA-long arm；将测序正确的DTA-long arm质粒和pMD19-T-short arm质粒进行Sal I和Hind III双酶切反应，电泳、回收、用T4连接酶连接短臂片段和DTA-long arm酶切片段，连接反应产物转化入大肠杆菌DH5α。进行重组质粒的酶切电泳鉴定和测序 鉴定，得到DTA-long arm-short arm，测序。
将测序正确的DTA-long arm-short arm质粒(DONOR)和pCAG-TLR4-GFP质粒按进行Spe I和Not I双酶切反应，电泳、回收、用T4连接酶连接CAG-GFP-TLR4片段和DTA-long arm-short arm酶切片段，连接反应产物转化入大肠杆菌DH5α。进行重组质粒的酶切电泳鉴定和测序鉴定，得到DTA-short arm-CAG-TLR4-GFP-long arm打靶载体(TLR4-DONOR)，构建策略如图3所示，TLR4定点敲入细胞的原理示意图如图4所示。
3.2.3电转打靶载体和CRISPR/Cas9质粒
电转前24hr，在6孔板中接种293T细胞0.5-1.0×105细胞，至转染时细胞汇合度达到90-95％；打靶载体DTA-shortarm-CAG-TLR4-GFP-longarm在同源重组臂的短臂5’端用BstBI单酶切线性化；用PBS清洗细胞，胰酶消化，800rpm离心3min收集细胞；用电转缓冲液(无血清DMEM)重悬293T细胞，调整浓度为1×107/mL。选用4mm的电击杯，每次电击细胞300μL，25ng打靶载体，25ng pPsg-Cas质粒。室温操作，电击，260v、30ms，后将细胞种与6孔板内，补加2mL培养液于37℃培养；细胞电转后48hr，观察荧光。
3.2.4有限稀释法挑选绿色荧光单克隆
收集长势良好的细胞，制成悬液；按细胞计数方法准确计得细胞悬液的细胞数；梯度稀释法将细胞制成10/mL细胞悬液，即每0.1毫升1个细胞；细胞悬液种于96孔板，每孔0.1毫升；96孔板置于37℃、5％CO2培养箱，4天后取出观察，做好记录并统计结果；选择单克隆生长孔，生长良好，绿色荧光阳性强者，转移到24孔板再做克隆经培养或扩大培养，经多次传代，可稳定表达绿色荧光的细胞克隆，命名为293T/TLR4细胞克隆，从图5A和图5B中可以看出，当293T/TLR4用白光照射时不显示荧光，当用激发光照射时显出绿色荧光。
4.CRISPR/Cas9技术构建CD14-MD2定点敲入细胞模型
4.1构建pCAG-MD2-2A-CD14-2A-RFP质粒
以pAAV-CAG-RFP为模板，PCR扩增RFP片段，并在5’端设计引物时引入F2A序列(如表7所示)，ageI酶切位点，在3’端引入Not I酶切位点。用AgeI和Not I酶消化pCAG-CD14-GFP质粒切掉绿色荧光GFP片段，并连入F2A-RFP片段，引物序列如表8；酶切测序鉴定得到pCAG-CD14-F2A-RFP质粒，瞬时转染入293T细胞看荧光表达；PCR扩增MD2片段，在3’端T2A序列(如表7所示)引入Age I酶切位点，用Kpn I酶消化pCAG-CD14-F2A-RFP质粒，并用abm必克隆试剂盒使用同源重组的方法连入MD2-T2A片段，引物序列如表8；酶切测序鉴定得到pCAG-MD2-T2A-CD14-F2A-RFP质粒。
表7 F2A和T2A的序列
Figure PCTCN2016101862-appb-000007
表8 F2A-RFP和MD2-T2A引物及产物长度
Figure PCTCN2016101862-appb-000008
4.2敲入靶点的确定
利用NCBI中的Nucleotide数据库搜索得到人类基因组第三号染色体CCR5的基因组DNA序列。将CCR5基因第一位内含子序列输入到http://crispr.mit.edu/评估合适的CRISPR敲入靶点，根据上述网站给出的结果得到guideRNA合适靶点。
本实施例中选择第三号染色体CCR5的基因组DNA序列的第一内含子位置作为CD14基因和MD2基因定点敲入部位，有报道证实该位置插入的外源基因对于细胞本身的生理功能没有影响。CD14基因和MD2基因也可以插入到其他不影响细胞生理功能的染色体位点上，插入到不同基因中，其靶点引物的设计可进行相应调整变化。
4.3CRISPR/Cas 9质粒的设计及构建
4.3.1CRISPR/Cas 9质粒的构建
以pX330质粒为骨架，合成互补的靶点sgRNA序列，并在两端引入BbsI酶 切位点。sgRNA合成序列如表9。
表9 CCR5靶向sgRNA合成序列
Figure PCTCN2016101862-appb-000009
Bbs I酶切消化pX330，酶切片段的电泳分离，并回pX330单酶切片段。互补的两段sgRNA片段退火并磷酸化，将sgRNA连入酶切好的线性化pX330中。连接反应产物转化入大肠杆菌DH5α，扩增，提取质粒后用BbsI单酶切进行反应，酶切鉴定后经电泳检查插入片段是否存在，获得重组阳性质粒，命名为pCsg-Cas，测序。
4.3.2CCR5基因打靶载体的构建
在CCR5基因sgRNA位点上游、下游各选择1000bp左右的序列，作为打靶载体的长短臂。用Primer premier5.0设计引物，如表10所示。PCR长臂、短臂、凝胶回收、连入pM19-T载体，转化、鉴定、测序。
表10 PCR长短臂序列
Figure PCTCN2016101862-appb-000010
将长臂、短臂分别酶切并连入pMCS.DT-A打靶载体，具体方法参照步骤3.2.2，其中在pMCS.DT-A载体Sal I和HindIII中间连入短臂，Sac II和Sac I之间连入长臂，最终得到DTA-long arm-short arm。
然后将pCAG-MD2-T2A-CD14-F2A-RFP质粒酶切并连入DTA-long arm-short arm载体，具体方法参照步骤3.2.2，最终获得DTA-short arm-CAG-MD2-T2A-CD14-F2A-RFP-long arm打靶载体，构建策略如图6所示，MD2和CD14定点敲入细胞的原理示意图如图7所示。
4.3.3电转打靶载体和CRISPR/Cas9质粒
参考步骤3.2.3的方法将打靶载体和pCsg-Cas电转到293T和293T/TLR4中，并用步骤3.2.4的方法筛选单克隆，得到两种红色荧光克隆细胞，293T/CD14/MD2细胞和293T/TLR4/CD14/MD2细胞，如图8所示，可以看出两个细胞克隆都可以发出红色荧光。其中293T/CD14/MD2细胞作为对照，293T/TLR4/CD14/MD2细 胞为目的细胞系。
5细胞模型鉴定
5.1荧光鉴定和共聚焦显微镜鉴定
5.1.1荧光鉴定
将293T，293T/TLR4，293T/CD14/MD2，293T/TLR4/CD14/MD2四种细胞系都传代3次后照荧光，如图9所示。结果显示293T无绿色荧光及红色荧光表达，293T/TLR4有稳定绿色荧光表达，293T/CD14/MD2有稳定红色荧光表达，293T/TLR4/CD14/MD2既有稳定绿色荧光，又有稳定红色荧光表达。
5.1.2共聚焦显微镜鉴定
将上述四种细胞系进一步进行共聚焦显微镜鉴定，如图10所示，可以看出293T无绿色荧光及红色荧光表达，293T/TLR4有稳定绿色荧光表达，293T/CD14/MD2有稳定红色荧光表达，293T/TLR4/CD14/MD2既有稳定绿色荧光，又有稳定红色荧光表达。将上述两图重合时293T/TLR4/CD14/MD2(Merge模式)可显现出绿色荧光和红色荧光的重合，而293T/TLR4和293T/CD14/MD2在重合时分别显现绿色荧光和红色荧光，其结果与荧光鉴定一致。并且可以看出，TLR4的绿光聚集于细胞膜和胞浆，而CD14-MD2红光聚集于胞浆。
5.2PCR鉴定
对293T，293T/TLR4，293T/CD14/MD2，293T/TLR4/CD14/MD2四种细胞系提取基因组DNA，在基因组不同位点设计引物，看敲入是否正确，引物序列如表11所示。其中，野生型用primer 1+primer2以及primer4+primer5可以扩增出片段，而用primer 1+primer 3，primer 4+primer 6不能扩增片段。TLR4敲入的细胞用primer 1+primer 3可扩增出片段，CD14-MD2敲入的细胞用primer 4+primer 6可扩增出片段。如图11A至图11D所示，通过PCR鉴定，293T/TLR4，293T/CD14/MD2，293T/TLR4/CD14/MD2敲入位点正确。
表11 PCR鉴定的引物序列
Figure PCTCN2016101862-appb-000011
5.3Western blot鉴定蛋白表达情况
对293T，293T/TLR4，293T/CD14/MD2，293T/TLR4/CD14/MD2四种细胞系裂解细胞提取总蛋白。以Actin作为内标Western blot鉴定TLR4、CD14、MD2三种蛋白表达情况。如图12所示，293T细胞无TLR4、CD14、MD2表达，293T/TLR4有TLR4表达、无CD14、MD2表达，293T/CD14/MD2有TLR4、CD14、MD2表达，293T/TLR4/CD14/MD2有TLR4、CD14、MD2表达。
根据分析，293T/CD14/MD2由于CD14/MD2的存在可能激活了细胞内源性的TLR4使得有少量的有TLR4表达。
实施例2使用细胞模型检测热原标志物LPS
1ELISA鉴定细胞对LPS刺激的反应性，检测IL-6和TNFα的表达
对293T，293T/TLR4，293T/CD14/MD2，293T/TLR4/CD14/MD2四种细胞系用LPS进行刺激，ELISA检测上清中IL-6和TNFα细胞因子的释放情况，293T，293T/TLR4对LPS刺激无反应，293T/CD14/MD2，293T/TLR4/CD14/MD2经LPS刺激后释放IL-6或TNFα但293T/CD14/MD2释放的量不如293T/TLR4/CD14/MD2多，如图13A和图13B所示。
继续选用293T/TLR4/CD14/MD2细胞系进行后续试验。检测LPS刺激后不同时间点IL-6的释放情况。发现5～6h左右IL-6释放达到高峰，如图14所示。检测不同细胞数293T/TLR4/CD14/MD2用5EU/mL LPS刺激后6h IL-6释放量，如图15所示，可以看出，104的细胞量即可对5EU/mL LPS产生反应释放IL-6。检测293T/TLR4/CD14/MD2细胞对LPS刺激的检测最低限，从图16可以看出，0.005EU/mL LPS即可刺激293T/TLR4/CD14/MD2细胞释放IL-6，优于鲎试剂的最低检测限。
2.Western blot法鉴定293T/TLR4/CD14/MD2细胞对LPS刺激的反应性，检测IL-6和TNFα的表达
使用高浓度(4EU/mL)，中浓度(1EU/mL)，低浓度(0.5EU/mL)的LPS刺激1×106个293T/TLR4/CD14/MD2细胞，6h后取1mL培养上清，浓缩至200μL，Western blot法鉴定结果表明培养上清中IL-6，TNF-a有释放，如图17A和图17B所示。
3.质谱法鉴定293T/TLR4/CD14/MD2细胞对LPS刺激的反应性，检测IL-6和TNFα的表达
293T/TLR4/CD14/MD2细胞系LPS刺激5小时后，取1000μL培养液上清，纯化后，采用基质辅助激光解析电离飞行时间质谱(MALDI.TOF-MS)分析、校正，将获得每个蛋白质点的肽质量指纹，图谱用Mascot Distiller软件识别单同位素信号峰，将获得的肽片段质荷比(m/z)数值导入Mas—cot查询系统，检索IPI. HUMAN.v3.53蛋白质数据库，Mascot分数>61分(P<0.05)判定为阳性。
4.胶体金试纸法(金标法)鉴定293T/TLR4/CD14/MD2细胞对LPS刺激的反应性，检测IL-6和TNFα的表达
胶体金试纸检测法是利用纳米级的胶体金作为示踪物和检测剂，应用抗原抗体反应的检测方法，检测时将293T/TLR4/CD14/MD2细胞系LPS刺激5小时后，待测样品与模型细胞共培养5小时后(与Western Blot，和ELISA法使用的样品及培养方法相同)，取培养液的上清50μL分别加入IL-6和TNFα的胶体金试纸条(该试纸条为市售或定制)，根据检测带的反应判定是否为IL-6和TNFα的表达阳性。
从上述实施例可以看出，本发明提供的细胞模型，对热原刺激可实现反应，不仅可以检出革兰氏阴性细菌的内毒素及非内毒素来源的污染物，包括来自于革兰氏阳性细菌、革兰氏阴性细菌、病毒、真菌的病原相关分子模式(PAMPs)和制品相关的以及加工工艺相关的生物或者化学实体，并且其最低检测限低于鲎试剂，其敏感性更强，其稳定性又比利用人全血检测更高。

Claims (10)

  1. 一种用于检测热原的细胞模型的构建方法，其特征在于，包括如下步骤：
    1)将TLR4基因组装到可表达绿色荧光的表达载体中，构建含有TLR4基因的重组质粒；
    2)采用CRISPR/CAS9法将TLR4基因定点敲入细胞系一染色体上的一基因的内含子中，获得一稳定表达TLR4基因的细胞系；
    3)使用两个不同的2A肽将CD14、MD2和红色荧光RFP基因结构隔开构建含有CD14基因和MD2基因并可表达红色荧光的重组质粒；
    4)采用CRISPR/CAS9法将CD14基因和MD2基因定点敲入步骤2)所获得的细胞系的敲入该TLR4基因的染色体以外的另一染色体上的一基因的内含子中，获得一可稳定表达TLR4基因、CD14基因和MD2基因，并可同时表达绿色荧光和红色荧光的细胞株，即为所述的细胞模型。
  2. 如权利要求1所述的构建方法，其特征在于，所述细胞系为HEK293T、HEK293或NIH3T3。
  3. 如权利要求1所述的构建方法，其特征在于，所述步骤2)中敲入TLR4基因的位置为第十九号染色体PPR1R12C基因第一位内含子。
  4. 如权利要求1所述的构建方法，其特征在于，所述步骤4)中敲入CD14基因和MD2基因的位置为第三号染色体CCR5基因第一位内含子。
  5. 采用权利要求1-4任一项所述的构建方法构建的细胞模型，其特征在于：所述细胞模型是在细胞系HEK293T的十九号染色体PPR1R12C基因第一位内含子内定点敲入TLR4基因，在三号染色体CCR5基因第一位内含子内定点敲入CD14基因和MD2基因。
  6. 如权利要求5所述的细胞模型，其特征在于，所述细胞模型的分类名称为人源胚胎肾细胞(HEK)变种293T/TLR4/CD14/MD2，保藏单位为：中国微生物菌种保藏管理委员会普通微生物中心，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏日期：2016年05月19日，保藏编号：CGMCC No.12296。
  7. 一种用于检测热原的试剂盒，其特征在于：包括如下组成：1)如采用权利要求1-4任一项所述的构建方法构建的细胞模型，或如权利要求5或6所述的细胞模型，2)热原标准品，3)IL6和/或TNFα对照品。
  8. 如权利要求7所述的用于检测热原的试剂盒，其特征在于：所述热原标准品为LPS冻干粉，IL6对照品为IL6冻干粉，TNFα对照品为TNFα冻干粉。
  9. 一种用于检测热原的方法，其特征在于：包括如下步骤：
    1)制备待测样品溶液和热原标准品溶液；
    2)将上述待测样品溶液加入到含有采用权利要求1-4任一项所述的构建方法构建的细胞模型，或如权利要求5或6所述的细胞模型的培养液中37℃培养5h；
    3)收集培养后的上清液检测其中IL-6和/或TNF-α的含量。
  10. 如权利要求9所述的用于检测热原的方法，其特征在于：步骤3)所述检测IL-6和/或TNF-α的含量的方法采用Western Blot、ELISA、金标法或质谱法。
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