CN108707626A - A kind of preparation method of the monoclonal cell system of detectable pyrogen - Google Patents

Abstract

The invention discloses a kind of preparation methods of the monoclonal cell system of detectable pyrogen.This method is mainly in such a way that slow virus builds stable cell lines, so that the luciferase gene under NF- κ B response elements or the control of IFN-β promoter stablizes expression in human epithelial cells A549 cells, then it screens to obtain monoclonal by streaming, further screening has LPS the positive clone molecule of reaction, it is finally simultaneously compared simultaneously with reagents using the stimulation of bacterial endotoxin standard items, to establish a kind of new method for detecting pyrogen using epithelial cell line.The method of the described epithelial cell line detection pyrogen need not use animal being capable of true reaction of the simulation human body to pyrogen, and can to the extremely wide LPS of distribution have stronger reaction, high sensitivity, stability is good, and commercially available be easy to get of A549 cells keeps the pyrogen detection method simple and practicable.

Description

A kind of preparation method of the monoclonal cell system of detectable pyrogen

Technical field

The invention belongs to biotechnologies, are related to the inspection of pyrogen in drug and biological products (including recombinant product) Survey technology field

Background technology

Pyrogen is a kind of isomeric types compound that can cause to generate heat and induce inflammatory reaction in host.Drug and doctor The pollution for treating instrument pyrogen is the most common reason of Systemic inflammation, or even can worse cause septic shock (Palma,L.,et al.Assay Drug Dev Technol,2017.15(2):64-76.；Gimenes,I.,et al.Regul Toxicol Pharmacolm,2015.73(1):356-360.).Most of for the pyrogen known to us is micro- Biological source, including bacterium, yeast, fungi, virus and their component and ambient particle (Daneshian, M., et al.J Immunol Methods,2008.336(1):64-70.).The component of most preferably bacteria cell wall is studied, such as is removed from office The lipopolysaccharides (LPS, also referred to as endotoxin) of Lan Shi negative bacteriums or the lipoteichoicacid (LTA) and peptide glycan of gram-positive bacteria (PGN)(Daneshian,M.,et al.J Immunol Methods,2008.336(1):64-70.；Stang,K.,et al.J Mater Sci Mater Med,2014.25(4):1065-1075.), these pyrogenic substances meeting stimulation of host is white thin Born of the same parents go release inflammatory cytokine and lead to serious reaction, such as multiple organ failure, septic shock even death (Stang, K.,et al.J Mater Sci Mater Med,2014.25(4):1065-1075.；Hasiwa,N.,M.Daneshian, and T.Hartung,2013.30(2):169-209.).Therefore, pyrogen test is the prerequisite of a medical product.Mesh Before, several different detection pyrogen methods can be obtained, and be rabbit pyrogen test method (RPT), detection of bacterial endotoxin respectively Method (BET, also referred to as limulus reagent test (LAL)) and monocyte detection method (MAT) (Stang, K., et al.J Mater Sci Mater Med,2014.25(4):1065-1075.)。

Rabbit pyrogen test method (RPT), the pyrogen test standard of a gold, it is based on to be detected in intravenous injection Rising situation (Stang, K., et al.J the Mater Sci of rabbit temperature are observed after solution or transplantation medical instrument Mater Med,2014.25(4):1065-1075.；Su,W.and X.Ding,2015.20(4):354-364.).In 20 generation Record just, due to rabbit to the sensitivity of pyrogen and the sensitivity of the mankind be it is similar (Vipond, C., et al., ALTEX, 2016.33(1):47-53.), therefore, RPT is applied to detect different types of pyrogen contamination (Palma, L., et al.Assay Drug Dev Technol,2017.15(2):64-76.).However, rabbit sensitivity be easy by it is many because Element influences, for example, rabbit strain, age and (Stang, K., et al.J the Mater Sci such as gender and rearing conditions Mater Med,2014.25(4):1065-1075.).Moreover, RPT is both expensive and time consuming.In addition to this, Rabbit viscera is only It is qualitative detection rather than quantitative (Hasiwa, N., M.Daneshian, and T.Hartung, 2013.30 (2):169- 209.)。

Detection of bacterial endotoxin method (BET) is a kind of external method of quality control of alternative rabbit, it predominantly detects leather The endotoxin of Lan Shi negative bacteriums, based on the enzyme-linked cascade reaction of gel caused by horseshoe crab ameboid cell lysate, and therefore this is It is also referred to as reagents detection method (Novitsky, T.J., Biology and Conservation of Horseshoe Crabs,2009:315-329.).It is compared with Rabbit viscera, LAL methods can carry out sxemiquantitative or quantitative detection with induced by endotoxin, very To being in winged gram level.However, the result of LAL can be influenced by protease.LAL can also show the sugar of some polymerized forms Reactivity, for example there is in the 3- glucans in fungi, algae and yeast cells.Although β-(1,3)-D- glucans are not heat The substance of originality, but it can induce gel cascade reaction, then interfere LAL to the reaction of LPS (Su, W.and X.Ding, 2015.20(4):354-364.；Ding,J.L.and B.Ho,TRENDS in Biotechnology,2001.19(8):277- 281.).In addition, this detection method is extremely different to the reaction of LPS and the immune response of people.Therefore, LAL detections can not Rabbit viscera (Hasiwa, N., M.Daneshian, and T.Hartung, 2013.30 (2) can be replaced completely:169-209.).

As people are to the growing interest of animal welfare issues, above-mentioned two kinds of traditional pyrogen detection methods are for product Quality control is faced with stern challenge.Recently, the alternative of RPT or LAL a kind of has gradually developed, that is, monokaryon Cell-stimulating detects (MAT) (Wunderlich, C., S.Schumacher, and M.Kietzmann, BMC Pharmacology and Toxicology,2014.).MAT is measured carefully based on pyrogen activation person monocytic cell and using enzyme-linked immunosorbent assay The release of intracellular cytokine, such as IL-1 β, IL-6, TNF-α, and also it is phase that the release of these cell factors, which is with the exothermic reaction of people, As (Gimenes, I., et al.Regul Toxicol Pharmacolm, 2015.73 (1):356-360.).Currently, although Monocyte activation detection method seems more to meet the requirements：Without using animal, reaction of the energy real simulation human body to pyrogen, and energy Quantitatively or semi-quantitatively detect the pyrogenic substances compared with multiple types, equal (Hartung, T., ALTEX, 2015.32 easy to operate (2):79-100.), but it needs to find suitable cell, at present the more popular cell such as MM6 subcloned cells of research, compared with It seldom arrives, is not suitable for extensive popularization, and THP-1 cell sensitivities are relatively low.Therefore, pyrogen test is carried out using cell line method to deposit In many urgent problems to be solved, and it is still extremely urgent to find new pyrogen detection method.

Endotoxin is the elicitor of the most powerful bacterial cell factor.In course of infection, pyrogen stimulates the monokaryon of people Cell goes to synthesize or discharge inflammatory cytokine, including IL-1 β, IL-6, TNF-α (Cavaillon, J.M., Toxicon, 2017.；J.J.LEE,et al.,Japanese Journal of Physiology,2003.53:367-375.； Nakagawa,Y.,H.Maeda,and T.Murai,Clinical and Vaccine Immunology,2002.9(3): 588-597.).And NF- κ B are the main transcription factors that regulation and control coding promotees these inflammatory cytokine genes.Moreover, Evidence suggests the available strategy (J.J.LEE, the et al., Japanese that prevent the activation of NF- κ B from may is that treatment fever Journal of Physiology,2003.53:367-375.；Barnes,P.J.and M.Karin,New England Journal of Medicine,1997.).At the same time, in normal rabbit body, the beta-interferon of people can be increased with conspicuousness Add this exothermic reaction caused by endotoxin, it be by enhance TNF rely on TNF generation come realize (Kawasaki, H.,et al.,INFECTION AND IMMUNITY,1987.55(11):2574-2578.；Kawasaki,H.,et al., INFECTION AND IMMUNITY,1989.57(10):3131-3135.；Kawasaki,H.,M.Moriyama,and H.Nariuchi.,INFECTION AND IMMUNITY,1992.60(3):933-936.), and pyrogen can also be made to be resistant to Rabbit restore normally the exothermic reaction caused by endotoxin (Kawasaki, H., M.Moriyama, and A.Tanaka., INFECTION AND IMMUNITY,1987.55(5):1121-1125.；Beeson and P.B.,J.Exp.Med.,1947: 29-38.；Greisman,S.E.,and R.B.Hornick.,J.Infect.Dis.,1973.128(SUPPLEMENT): S265-S276.).Therefore, imitating human body by detection can to the activation of NF- κ B or the β-IFN of pathogen normal physiological reaction To reflect the generation of the cell factor in its downstream.

Pervious report has shown that：Bacterial flagellum can stimulate people's alveolar epithelial cells to produce by Toll-like receptor TLR5 Raw cell factor IL-8 (Im, J., et al., Mol Immunol, 2009.47 (2-3):614-622.；Lopez-Boado, Y.S.,et al.,Infect Immun,2005.73(11):7525-7534.).This just illustrates that people's alveolar epithelial cells is also permitted Cell line to be used for pyrogen test as us.But other pyrogenic substances are how induced activation lung epithelial is thin actually Born of the same parents are illustrated clear completely not yet.Therefore, in our study, we are dedicated to exploitation one kind by using fluorescein Enzyme reporting system and people's alveolar epithelial cells system A549 in order to stabilize, it is sensitive, rapidly detect pyrogen cytology.

Invention content

The present invention is low for the method sensitivity of detection pyrogen in the prior art, and stability is poor, and detection cycle is long, Yi Jijian The MM6 subcloned cells and people's whole blood of survey, it is more difficult to obtain, be not suitable for extensive the defects of promoting, provide on a kind of user The method that pyrogen detects in chrotoplast system, the detection pyrogen that the method can be quick, sensitive and stable, and A549 used The commercially available easy acquisition of cell.

Experiment of the inventor by extensive research and repeatedly, finds A549 cells right after the method for the present invention culture Pyrogenic substances are sensitive, can be used in cell line method detection pyrogen, therefore inventor has invented a kind of user's epithelial cell line inspection The method of calorimetric original.

The present invention provides a kind of method of user's epithelial cell line detection pyrogen comprising following step：

(1) NF- κ B or IFN-β gene are assembled into the Lentiviral that can express puromycin, structure contains There is the recombinant plasmid of NF- κ 1 B genes；

(2) recombinant plasmid containing NF- κ B or IFN-β gene described in step (1) is stably transfected into epithelial cell In system；

(3) epithelium for obtaining the stable transfection containing NF- κ B or IFN-β gene recombination plasmid described in step (2) Cell line is screened using puromycin, and airflow classification monoclonal is used in combination；

(4) monoclonal for obtaining the sorting described in step (3) is stimulated using the LPS of high dose, wherein having reaction to LPS Then be NF- κ B or IFN-β positive clone molecule；

(5) by described in step (4) NF- κ B or IFN-β positive clone molecule using lower dosage LPS stimulate, obtain Extremely sensitive positive colony.

(6) the NF- κ B positive clone molecules described in step (4) are reused into the stimulation of bacterial endotoxin standard items, and is tried with horseshoe crab Agent is compared.

Preferably, either IFN-β gene is located at pNF κ B-luc or pGL3-Control plasmids to the NF- κ B On.

Further, the Lentiviral described in step (1) is pCDH-CMV-MCS-EF1 serial carriers.

Further, the epithelial cell line behaviour alveolar epithelial cells A549.

Further, its a concentration of 500ng/ml of the LPS of the high dose described in step (4)；Described has reaction to LPS It is by luciferase reporting measuring.

Further, its is dense in stimulation NF- κ B or IFN-β positive clone molecule by the lower dosage LPS described in step (5) Degree is respectively 1,10,50,100ng/ml or 10,25,50,100ng/ml.

Further, the bacterial endotoxin standard items described in step (6) its a concentration of 0.1,0.25,0.5,1EU/ml； Its sensitivity of the reagents is 0.125EU/ml.

Further, the time of LPS described above stimulation is 6h.

The present invention also provides a kind of methods that above-mentioned method goes detection pyrogen, and the method for the detection pyrogen is people's lung Bubble epithelial cell A549 passes through the post-stimulatory reactions of luciferase reporting measuring 500ng/ml LPS.

The beneficial effects of the present invention are：The method of epithelial cell line detection pyrogen of the present invention need not use dynamic Object can true reaction of the simulation human body to pyrogen, and can to the extremely wide LPS of distribution have it is stronger Reaction, high sensitivity, stability is good, is swift in response, and commercially available be easy to get of A549 cells keeps the pyrogen detection method simple and practicable. The cell model minimum detection limit of the present invention is less than the 0.125EU/ml of limulus reagent test up to 0.1EU/mL simultaneously, i.e., the most frequently used Detection limit, be less than people fever threshold value 0.3EU/ml.

Description of the drawings

The plasmid map of Fig. 1 pCDH-CMV-Puro.

The plasmid map of Fig. 2 pNF- κ B-Luc.

The plasmid map of Fig. 3 pGL3-IFN- β-Luc.

Collection of illustrative plates is identified in the digestion of Fig. 4 pCDH-Puro-NF- κ B/IFN- β plasmids.

Representative result figure of Fig. 5 NF- κ B reporter cells screening.

Representative result figure of Fig. 6 IFN-β reporter cells screening.

The fluorescence results figure for NF- κ B the positive clone molecules 3#, 8#, 123#, 191# that Fig. 7 is screened several times.

The fluorescence results figure for the IFN-β positive clone molecule 33# that Fig. 8 is screened several times.

The PCR of Fig. 9 NF- κ 1 B genes schemes.

The PCR of Figure 10 IFN-β genes schemes.

Figure 11 NF- κ B reporter cells clone 3#, 8#, 123#, and 191# is in the post-stimulatory fluorescence results figures of various dose LPS.

Figure 12 IFN-β reporter cells clone 33# in the post-stimulatory fluorescence results figures of various dose LPS.

Figure 13 NF- κ B reporter cells 3#, 8#, 123#, 191# is in the post-stimulatory fluorescence results figures of high agent concentration RSE.

Figure 14 NF- κ B reporter cells clone 3#, and 8# is in the post-stimulatory fluorescence results figures of various concentration RSE.

The result schematic diagram of gel reaction occurs for Figure 15 various concentrations endotoxin standard and LAL.

Specific implementation mode

The technical solution further illustrated the present invention below by specific embodiment, but therefore do not limit the present invention to Among the embodiment described range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, Or it is selected according to product manual.

Material, reagent and instrument

Cell culture medium：DMEM culture mediums (being purchased from the Shanghai biotech inc Yuan Pei) 500mL, is added tire ox Serum (being purchased from Gibco) 50mL mixings, that is to say complete DMEM culture mediums.

Cell：A549 purchased from Beijing Union Medical College (Number：CCL-185^TM)。

Plasmid：PNF- κ B-Luc are purchased from green skies biotechnology company；pGL3-Control-Vector(pGL3-IFN-β- Luc it) is provided by Wuhan University teacher Shu Hongbing；PCDH-CMV-MCS-EF1-Puro is purchased from U.S. SystemBiosciences； PCDH-Puro-NF- κ B/IFN- β are voluntarily built by this laboratory.

Reagent：Puromycin (Puromycin) is purchased from AMRECO companies；From Escherichia coli 0111：The fat of B4 types is more Sugared (LPS) is all available from Sigma Co., USA；From Escherichia coli 0111：The endotoxin standard (RSE) of B4 types and 0.125EU/ml reagents LAL is available from National Institute for Food and Drugs Control；Single Fluorescence kit is public purchased from Promega Department.

Consumptive material：Cell counting board, aseptic plastic centrifuge tube (1.5mL, 50mL), disposable plastic pipette (sterile, single packet Dress, 10mL), 10cm culture dishes, 6 holes or 48 holes or 96 porocyte culture plates.

Instrument:

37 DEG C of cell constant temperature CO2 incubators：MCO-15AC, SANYO company；Centrifuge 5424R centrifuges： 5404DJ914920, Eppendorf company；GloMax Multi multi-testers：E6080GloMax, Promega company； PCR instrument：BioRad companies of the U.S..

Culture involved in the present invention to all monoclonal cells and passage are identical as A549 cells.

The structure of embodiment 1 recombinant plasmid and recombinant cell

The structure of 1 slow virus recombinant plasmid

Slow virus carrier pCDH-CMV-MCS-EF1-Puro (being abbreviated as pCDH-CMV-Puro) is as original carrier quilt Using structure reporter gene is removed, as shown in Figure 1.PCDH-CMV-Puro and pNF- κ B-Luc or pGL3-IFN- β-Luc (Fig. 2 and Shown in Fig. 3) digestion is carried out with speI and BamH I restriction enzymes respectively, it runs glue and is tapped and recovered, connected after recycling It connects, the sequence of operations such as conversion coated plate.Next day, 10 clones of each plasmid picking are bacterium colony PCR, DNA glue are run, therefrom the positive Clone shakes bacterium, waits for that pCDH-Puro-NF- κ B/IFN- β thalline grow to 16~18h, thalline it is muddy it is opaque can in upgrading grain, press According to the small specification requirement operation for putting forward middle amount kit of high-purity.After plasmid extraction is completed and has also measured concentration, respectively It is taken out 1 μ g to be used for doing digestion identification, digestion qualification figure is as shown in Figure 4.Sequencing (pCDH-Puro- is sent after identification is correct The sequence of the sequence and pCDH-Puro-IFN- β of NF- κ B), and the gene gram to match is retrieved from Pubmed gene databases It is grand.

2 cell secondary cultures and bed board

A549 cells using the cell culture medium secondary culture in 5%, 37 DEG C of carbon dioxide incubator, every 2~3 days Passage is primary, collects in sterile centrifugation tube, and 900rpm is centrifuged 5 minutes, liquid is discarded supernatant, by the fresh cell of cell precipitation Culture solution is resuspended, and takes out a part and is transferred to Tissue Culture Dish, continues to cultivate.The good cell of growth conditions is taken to carry out bed board, 900rpm is centrifuged 5 minutes, abandons supernatant, and the cell culture fluid that complete DMEM culture mediums are added is resuspended, and obtains cell suspending liquid.It adjusts thin Born of the same parents a concentration of 6 × 10⁵A/mL or 2.5 × 10⁵A/mL is added in 48 or 96 well culture plates with 100 holes μ L/, need to be mended respectively again Add in 400 μ L or 100 μ L culture mediums to culture hole.

The packaging of 3 slow virus and screening

After the success of slow virus construction of recombinant plasmid, that is to say, that the sequence of CMV promoter coding is cut from original carrier It removes, while being replaced by the sequence of the firefly luciferase coding with NF- κ B binding sites or IFN-β promoter.This Illustrate that the pCDH-Puro-NF- κ B/IFN- β slow virus plasmids built all contain luciferase gene sequence, this is advantageous In the progress of subsequent experimental.

Next we will first pack slow virus, by taking 6cm dish packaging as an example for once specific packaging step.

(1) half an hour before transfecting, the DMEM cultures of the DMEM and complete (10%FBS) of 37 DEG C of preheating serum-free antibiotic-frees Base；

(2) 2.3 μ g slow virus recombinant plasmid pCDH-Puro-NF- κ B/IFN- β and 2.3 μ g helper plasmids, 3 auxiliary are taken Plasmid is respectively 0.75 μ g pGag-Pol, 0.23 μ g pRev, 1.38 μ g pVSVG) it is diluted in 100 μ l serum-free antibiotic-frees DMEM in, gently mixing, is stored at room temperature 5min；

(3) 9.2 μ l PEI are taken to be diluted in the DMEM of 100 μ l serum-free antibiotic-frees, gently mixing, is stored at room temperature 5min；

(4) above-mentioned two solution is mixed, gently mixing (turns upside down or gently blown with 1000 μ l pipettors Beat), it is incubated at room temperature 20~25min, to form DNA-PEI compounds；

(5) the 293T cells for carrying that the previous day completes are taken out, original culture medium is sucked, 2.5ml is added in each 6cm dish Fresh complete DMEM culture mediums；

(6) DNA-PEI compounds are added in 6cm dish, gently mixing；

(7) it is placed in 37 DEG C of incubators containing 5%CO2 and is incubated culture 48h, wherein in 6~8h again in each 6cm Dish adds the fresh complete DMEM culture mediums of 2.5ml；

(8) until after 48h, the supernatant of 293T cells in 6cm dish, as lentiviral particle liquid are collected.

The lentiviral particle of collection removes cell fragment and impurity with 0.22 μm of membrane filtration, is then helped with 10 μ g/ml Turn agent polybrene and ready A549 cells are added in the serum added together.After infecting 48h, screened with puromycin The cell screening of tolerance three days, cell survival is seldom after screening, then needs to maintain cell with the puromycin of sxemiquantitative Culture 3~5 days.

The sorting and selection of 4 monoclonals

The airflow classification monoclonal cell of the cell in 6cm culture dishes, it is placed in advance plus 96 holes of good culture medium is cultivated (contain puromycin) in plate, after the completion of sorting, 2000rpm/min centrifuges 2min, is placed on 5%CO2, is trained in 37 DEG C of incubators It supports, cell starts to grow one by one after 7~15 days.Then just according to 24 orifice plates, 12 orifice plates, 6 orifice plates, 6cm dish's is suitable Cell is transferred out by sequence.

The determination of 5 positive clone molecules

After monoclonal length is got up, had to measure its reporter gene using high-throughput single luciferase reporting experiment Do not rotate into.Specifically operating procedure is：The monoclonal that each length is got up is layered in 96 orifice plates, morning next day is used The LPS of 500ng/ml stimulates 6h, after stimulation, is set cell using the lysate in single luciferase reporter gene kit In 4 DEG C of shaking table lytic cell 15min, then 2000rpm/min winks are from cell fragment, to board bottom, supernatant is transferred to 96 micropores In plate, then operation instruction is instructed to measure fluorescent value using GLOMAX (96microplate luminometer) according to artificial. The NF- κ B that Figures 5 and 6 are shown either a separation results of all monoclonals of IFN-β and Fig. 7 and 8 is NF- κ B or IFN-β The fluorescence measurements of all positive clone molecules.

It measures obtained numerical value and illustrates that clone's reporter gene does not rotate into if it is double figures, if instead numerical value exists Three digits even four five-digit numbers then illustrate that gene has rotated into.Therefore, base in cell is detected by way of this high throughput Because expression is very convenient and efficient.

Embodiment 2 verifies the insertion of NF- κ B/IFN- β genes

1. recombinant cell RNA extractions and reversion

We mainly use Trizol reagents extraction RNA to be as follows by taking 6 orifice plates as an example：

(1) cell is handled：After removing culture medium, 1ml Trizol examinations are added into the 6 orifice plates with A549 reporter cells Cell is blown down and is put into the sterilizing RNA centrifuge tubes of a 1.5ml by agent.

(2) it is layered：200 μ l chloroforms (chloroform) are added per 1ml Trizol, acutely shake 15s, room on the oscillator Temperature places 3~5min, 12000rpm/min, 4 DEG C of centrifugation 5min.

(3) RNA precipitate：After centrifugation, upper strata aqueous phase (about 500 μ l) is transferred in new centrifuge tube, it is rather few not more, Isometric ice-cold isopropanol (being put into -20 DEG C in advance) is added, -20 DEG C are placed 20min, 12000rpm/min, 4 DEG C of centrifugations 20min abandons supernatant.

(4) RNA is rinsed：75% ethyl alcohol of 1ml is added in RNA precipitate, flicking makes precipitation suspend, 12000rpm/

Min, 4 DEG C of centrifugation 5min.Supernatant is removed, the repetition step is primary, and then room temperature or exhaust outlet place 1~2min and dry in the air Dry precipitation, is careful not to too dry, is otherwise difficult dissolving.

(5) RNA is dissolved：20 μ l DEPC water are added in RNA precipitate, flick tube wall, 37 DEG C, 3min or 4 DEG C, 30~60min Dissolving.

(6) RNA concentration is measured, is then stored in -80 DEG C.Whole process can also can outside be carried out in super-clean bench, But certain should be noted that prevents RNA degradations and other pollutions, so to wear masks, often sprays alcohol.

Next it is exactly that RNA is inverted to cDNA after RNA extractions are completed.Reversion is broadly divided into the progress of three steps：First, The first step takes PCR pipe, and the RNA of 1 μ g is added into pipe, adds 1 μ l Oligo dT or Random primer, finally mends Add DEPC water to 5 μ l systems.It is then placed in 70 DEG C of operation 5min of PCR instrument.

PCR pipe taking-up is placed in 1min on ice from PCR instrument, then sequentially adds 45 × Reaction of μ l by second step Buffer, 2 μ l 2.5mM MgCl2,1 μ l 10mM dNTPs, 1 μ l Reverse Transcriptase add DEPC water extremely 15 μ l systems.

Third walks, according to program：25 DEG C, 5min, 42 DEG C, 60min, 70 DEG C, 15min, 4 DEG C, 90min is put into PCR instrument fortune Row.That wait for that end of run obtains is cDNA.

The acquisition of 2.NF- κ B/IFN- β genes

CDNA obtained above is done into PCR to identify whether these reporter cells contain NF- κ B/ using the primer in table 1 IFN-β gene.As shown in Figures 9 and 10, the NF- κ B/IFN- β reporter cells monoclonal screened all contains NF- κ B/IFN- β Gene, this just illustrates that the cell that we build is correct, the insertion of really purposeful gene.

Table 1.PCR primer sequences and PCR product length

Embodiment 3 verifies A549 positive colonies and can be used for detecting pyrogen

1.LPS or endotoxin standard (RSE) stimulation

First, the NF- κ B/IFN- β reporter cells screened are further used the LPS of various dose.The storage of LPS Concentration is 1mg/ml, and the original concentration of RSE is 10EU/ml.The dosage that LPS is used mainly has 1ng/ml, 10ng/ml, 25ng/ Ml, 100ng/ml, 250ng/ml, 500ng/ml, 1000ng/ml store concentration dilution, as a result such as the institutes of Figure 11 and 12 with LPS Show.

By the result in embodiment 1 it is found that the more difficult acquisition of IFN-β clone can not compare only there are one clone.Therefore, It is next experiment we the verification of endotoxin standard mainly has been carried out to NF- κ 1 B genes.RSE pairs of 1EU/ml is used first Four plants of NF- κ B positive clone molecules that above-mentioned screening obtains are stimulated, and result is as shown in figure 13.Then.We use RSE's Dosage is 0.1EU/ml, 0.25EU/ml, 0.5EU/ml, and 1EU/ml goes stimulation cell, result as shown in figure 14.

2. reagents (LAL) is tested

It is required according to artificial teachings, is measured using the LAL (National Institute for Food and Drugs Control) of 0.125EU/ml The endotoxin standard of various concentration (0,0.1,0.125,0.25,0.5,10EU/ml), according to reagents solidification and muddiness State measures endotoxic content.To form a striking contrast with the reporter cell of the present invention, to also illustrate me Reporter cell have reached and measure endotoxic sensitivity, just as shown in FIG. 14 and 15.

From above-described embodiment as can be seen that the method for epithelial cell line provided by the invention detection pyrogen, need not use Animal can true reaction of the simulation human body to pyrogen, and can to the extremely wide LPS of distribution have it is relatively strong Reaction, high sensitivity, commercially available be easy to get of A549 cells keeps the pyrogen detection method simple and practicable, and its minimum detection limit is less than Reagents, sensibility is stronger, stability and Billy's employment whole blood test higher.

Sequence table

<110>Institute of Microorganism, Academia Sinica

University of Anhui

Beijing CoSci Med-Tech Co., Ltd.

<120>A kind of preparation method of the monoclonal cell system of detectable pyrogen

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9249

<212> DNA

<213>The sequence (artificial sequence) of pCDH-Puro-NF- κ B

<400> 1

acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240

tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300

agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360

ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420

cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480

tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540

gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600

aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660

aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720

agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780

atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840

gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900

taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960

acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020

cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080

ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140

tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200

gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260

aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320

gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380

aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800

aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860

aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt 1920

atcgatacta gtcgtctctc aaggataagt aagtaatatt aaggtacggg aggtattgga 1980

caggccgcaa taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat 2040

cgatagtact aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat 2100

aggctgtccc cagtgcaagt gcaggtgcca gaacatttct ctggcctaac tggccggtac 2160

cgctagcggg aatttccggg aatttccggg aatttccggg aatttccaga tctgcagaag 2220

cttggcaatc cggtactgtt ggtaaagcca ccatggaaga tgccaaaaac attaagaagg 2280

gcccagcgcc attctaccca ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca 2340

tgaagcgcta cgccctggtg cccggcacca tcgcctttac cgacgcacat atcgaggtgg 2400

acattaccta cgccgagtac ttcgagatga gcgttcggct ggcagaagct atgaagcgct 2460

atgggctgaa tacaaaccat cggatcgtgg tgtgcagcga gaatagcttg cagttcttca 2520

tgcccgtgtt gggtgccctg ttcatcggtg tggctgtggc cccagctaac gacatctaca 2580

acgagcgcga gctgctgaac agcatgggca tcagccagcc caccgtcgta ttcgtgagca 2640

agaaagggct gcaaaagatc ctcaacgtgc aaaagaagct accgatcata caaaagatca 2700

tcatcatgga tagcaagacc gactaccagg gcttccaaag catgtacacc ttcgtgactt 2760

cccatttgcc acccggcttc aacgagtacg acttcgtgcc cgagagcttc gaccgggaca 2820

aaaccatcgc cctgatcatg aacagtagtg gcagtaccgg attgcccaag ggcgtagccc 2880

taccgcaccg caccgcttgt gtccgattca gtcatgcccg cgaccccatc ttcggcaacc 2940

agatcatccc cgacaccgct atcctcagcg tggtgccatt tcaccacggc ttcggcatgt 3000

tcaccacgct gggctacttg atctgcggct ttcgggtcgt gctcatgtac cgcttcgagg 3060

aggagctatt cttgcgcagc ttgcaagact ataagattca atctgccctg ctggtgccca 3120

cactatttag cttcttcgct aagagcactc tcatcgacaa gtacgaccta agcaacttgc 3180

acgagatcgc cagcggcggg gcgccgctca gcaaggaggt aggtgaggcc gtggccaaac 3240

gcttccacct accaggcatc cgccagggct acggcctgac agaaacaacc agcgccattc 3300

tgatcacccc cgaaggggac gacaagcctg gcgcagtagg caaggtggtg cccttcttcg 3360

aggctaaggt ggtggacttg gacaccggta agacactggg tgtgaaccag cgcggcgagc 3420

tgtgcgtccg tggccccatg atcatgagcg gctacgttaa caaccccgag gctacaaacg 3480

ctctcatcga caaggacggc tggctgcaca gcggcgacat cgcctactgg gacgaggacg 3540

agcacttctt catcgtggac cggctgaaga gcctgatcaa atacaagggc taccaggtag 3600

ccccagccga actggagagc atcctgctgc aacaccccaa catcttcgac gccggggtcg 3660

ccggcctgcc cgacgacgat gccggcgagc tgcccgccgc agtcgtcgtg ctggaacacg 3720

gtaaaaccat gaccgagaag gagatcgtgg actatgtggc cagccaggtt acaaccgcca 3780

agaagctgcg cggtggtgtt gtgttcgtgg acgaggtgcc taaaggactg accggcaagt 3840

tggacgcccg caagatccgc gagattctca ttaaggccaa gaagggcggc aagatcgccg 3900

tgtaataatt ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg 3960

atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt 4020

gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca 4080

attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt 4140

aaaacctcta caaatgtggt aaaatcgata aggatccgcg gccgcgaagg atctgcgatc 4200

gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 4260

gaggggtcgg caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 4320

atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 4380

tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc 4440

gaggggctcg catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc 4500

ggttgagtcg cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct 4560

aggtaagttt aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac 4620

ctagactcag ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg 4680

tttcgttttc tgttctgcgc cgttacagat ccaagctgtg accggcgcct acgctagatg 4740

accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc 4800

accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc 4860

cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc 4920

ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc 4980

gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc 5040

cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc 5100

gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc 5160

gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag 5220

acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac 5280

gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgagtc 5340

gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 5400

gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 5460

cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag 5520

ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc 5580

actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc 5640

cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg 5700

ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg 5760

ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc 5820

ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt 5880

cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcctggtacc 5940

tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg 6000

gggactggaa gggctaattc actcccaacg aaaataagat ctgctttttg cttgtactgg 6060

gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact 6120

gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg 6180

tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag 6240

tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga 6300

gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 6360

aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 6420

caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc 6480

cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 6540

gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctaga cttttgcaga 6600

gacggcccaa attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 6660

acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 6720

gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 6780

tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 6840

cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 6900

gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 6960

aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 7020

gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 7080

aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 7140

gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 7200

ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 7260

cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 7320

ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 7380

actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 7440

tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 7500

gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 7560

ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 7620

cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 7680

ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 7740

tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 7800

agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 7860

gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 7920

ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 7980

gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 8040

cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 8100

acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 8160

cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 8220

cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 8280

ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 8340

tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 8400

atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 8460

tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 8520

actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 8580

aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 8640

ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 8700

ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc 8760

cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat 8820

aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga 8880

cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 8940

gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca 9000

tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta 9060

aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg 9120

cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg 9180

cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt 9240

gccaagctg 9249

<210> 2

<211> 9661

<212> DNA

<213>The sequence (artificial sequence) of pCDH-Puro-IFN- β

<400> 2

acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60

acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120

cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180

attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240

tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300

agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360

ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420

cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480

tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540

gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600

aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660

aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720

agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780

atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840

gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900

taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960

acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020

cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080

ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140

tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200

gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260

aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320

gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380

aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440

ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500

acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560

ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620

agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680

tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740

tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800

aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860

aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt 1920

atcgatacta gtacgtctct caaggataag taagtaatat taaggtacgg gaggtattgg 1980

acaggccgca ataaaatatc tttattttca ttacatctgt gtgttggttt tttgtgtgaa 2040

tcgatagtac taacatacgc tctccatcaa aacaaaacga aacaaaacaa actagcaaaa 2100

taggctgtcc ccagtgcaag tgcaggtgcc agaacatttc tctggcctaa ctggccggta 2160

ccgctagccc gggctcgaga tctgcgatct gcatctcaat tagtcagcaa ccatagtccc 2220

gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 2280

tcgctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 2340

ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc ttggcattcc 2400

ggtactgttg gtaaagccac catggaagac gccaaaaaca taaagaaagg cccggcgcca 2460

ttctatccgc tggaagatgg aaccgctgga gagcaactgc ataaggctat gaagagatac 2520

gccctggttc ctggaacaat tgcttttaca gatgcacata tcgaggtgga catcacttac 2580

gctgagtact tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata tgggctgaat 2640

acaaatcaca gaatcgtcgt atgcagtgaa aactctcttc aattctttat gccggtgttg 2700

ggcgcgttat ttatcggagt tgcagttgcg cccgcgaacg acatttataa tgaacgtgaa 2760

ttgctcaaca gtatgggcat ttcgcagcct accgtggtgt tcgtttccaa aaaggggttg 2820

caaaaaattt tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat tatcatggat 2880

tctaaaacgg attaccaggg atttcagtcg atgtacacgt tcgtcacatc tcatctacct 2940

cccggtttta atgaatacga ttttgtgcca gagtccttcg atagggacaa gacaattgca 3000

ctgatcatga actcctctgg atctactggt ctgcctaaag gtgtcgctct gcctcataga 3060

actgcctgcg tgagattctc gcatgccaga gatcctattt ttggcaatca aatcattccg 3120

gatactgcga ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc 3180

ggatatttga tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt 3240

ctgaggagcc ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc 3300

ttcttcgcca aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct 3360

tctggtggcg ctcccctctc taaggaagtc ggggaagcgg ttgccaagag gttccatctg 3420

ccaggtatca ggcaaggata tgggctcact gagactacat cagctattct gattacaccc 3480

gagggggatg ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt 3540

gtggatctgg ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga 3600

ggtcctatga ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac 3660

aaggatggat ggctacattc tggagacata gcttactggg acgaagacga acacttcttc 3720

atcgttgacc gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa 3780

ttggaatcca tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc 3840

gacgatgacg ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg 3900

acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc 3960

ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga 4020

aaaatcagag agatcctcat aaaggccaag aagggcggaa agatcgccgt gtaattctag 4080

agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa 4140

accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 4200

ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 4260

atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 4320

tgtggtaaaa tcgataagga tctgaacgat ggagcggaga atgggcggaa ctgggcggag 4380

ttaggggcgg gatgggcgga gttaggggcg ggactatggt tgctgactaa ttgagatgca 4440

tgctttgcat acttctgcct gctggggagc ctggggactt tccacacctg gttgctgact 4500

aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc 4560

ctaactgaca cacattccac agcggatccg cggccgcgaa ggatctgcga tcgctccggt 4620

gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc 4680

ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg 4740

tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc 4800

gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac agctgaagct tcgaggggct 4860

cgcatctctc cttcacgcgc ccgccgccct acctgaggcc gccatccacg ccggttgagt 4920

cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg cgtccgccgt ctaggtaagt 4980

ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc cttggagcct acctagactc 5040

agccggctct ccacgctttg cctgaccctg cttgctcaac tctacgtctt tgtttcgttt 5100

tctgttctgc gccgttacag atccaagctg tgaccggcgc ctacgctaga tgaccgagta 5160

caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc 5220

cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga 5280

gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt 5340

gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc 5400

gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc 5460

cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt 5520

cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt 5580

gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc 5640

gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt 5700

gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgag tcgacaatca 5760

acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg ttgctccttt 5820

tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt cccgtatggc 5880

tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg agttgtggcc 5940

cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg 6000

gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc tccctattgc 6060

cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg 6120

cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg 6180

tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc 6240

agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct 6300

tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggta cctttaagac 6360

caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg 6420

aagggctaat tcactcccaa cgaaaataag atctgctttt tgcttgtact gggtctctct 6480

ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc 6540

ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg 6600

gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtagtagtt 6660

catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga 6720

ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 6780

caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 6840

cttatcatgt ctggctctag ctatcccgcc cctaactccg cccagttccg cccattctcc 6900

gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga 6960

gctattccag aagtagtgag gaggcttttt tggaggccta gacttttgca gagacggccc 7020

aaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc 7080

acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta 7140

actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca 7200

gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc 7260

cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 7320

tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 7380

gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 7440

ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 7500

aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 7560

tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 7620

ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 7680

gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 7740

tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 7800

caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 7860

ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 7920

cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 7980

ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 8040

cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 8100

gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 8160

aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 8220

acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 8280

gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 8340

cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 8400

cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 8460

tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 8520

cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 8580

gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 8640

cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 8700

ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 8760

gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 8820

taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 8880

gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 8940

acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 9000

aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 9060

cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 9120

atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt 9180

gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 9240

cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca 9300

gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca 9360

gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca 9420

gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 9480

ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt 9540

gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag 9600

ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgccaagct 9660

g 9661

Claims (8)

1. a kind of preparation method of the monoclonal cell system of detectable pyrogen, which is characterized in that including：

(1) NF- κ B or IFN-β gene are assembled into the Lentiviral that can express puromycin, structure contains NF- The recombinant plasmid of κ 1 B genes；

(2) recombinant plasmid that step (1) obtains is stably transfected into epithelial cell line；

(3) epithelial cell line that step (2) obtains is screened using puromycin, airflow classification monoclonal cell is used in combination；

(4) monoclonal cell for obtaining step (3) using high dose LPS stimulate, wherein to LPS have reaction then be NF- κ B or IFN-β positive colony；

(5) positive colony for obtaining step (4) is stimulated using the LPS of lower dosage, obtains extremely sensitive positive colony cell.

2. according to the method described in claim 1, it is characterized in that, the NF- κ B or IFN-β gene is located at pNF κ On B-luc pGL3-Control plasmids.

3. according to the method described in claim 1, it is characterized in that, the Lentiviral is pCDH-CMV-MCS- EF1 serial carriers.

4. according to the method described in claim 1, it is characterized in that, the epithelial cell line behaviour alveolar epithelial cells A549。

5. according to the method described in claim 1, it is characterized in that, the LPS of the high dose, a concentration of 500ng/ml.

6. according to the method described in claim 1, it is characterized in that, described to have reaction to LPS be to pass through luciferase reporting Measuring.

7. according to the method described in claim 1, it is characterized in that, the lower dosage LPS, concentration are respectively 1,10, 50,100ng/ml or 10,25,50,100ng/ml.

8. according to the method described in claim 1, it is characterized in that, the LPS stimulations, time 6h.