CN102778570A - Method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide - Google Patents
Method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide Download PDFInfo
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Abstract
The invention discloses a method for distinguishing smooth type lipopolysaccharide and rough type lipopolysaccharide. The method comprises the following steps of: firstly synthesizing a small interfering RNA (siRNA-224.3) capable of effectively interfering mCD14 gene expression, and a negative control siRNA-NC, and preparing recombinant lentiviral vector; secondly, infecting the mice macrophage RAW264.7 with the lentiviral vector, and screening the positive clone by the puromycin, and building steady cell lines siRNA-224.3 and siRNA-NC; and finally, using an enzyme-linked immuno-sorbnent assay to detect the nitric oxide, the tumor necrosis factor alpha and the interleukin 6 in cell supernatant of the mice mononuclear macrophage steady cell line which is respectively stimulated by the smooth type lipopolysaccharide and the rough type lipopolysaccharide to express the small interfering RNA, by comparing the difference of the detection, distinguishing the smooth type lipopolysaccharide and the rough type lipopolysaccharide. In comparison with the prior art, the method provided by the invention is more convenient and more accurate.
Description
Technical field
The invention belongs to the biotechnology applied technical field, relate to a kind of method of differentiating smooth type lipopolysaccharides and rough type lipopolysaccharides.
Background technology
Leukocyte differentiation antigen 14 (cluster of differentiation antigen; CD14) be a kind of differentiation antigen that is present in cell surfaces such as monocyte, macrophage; For mediating lipopolysaccharides (lipopolysaccharide, LPS) one of important acceptor of biological effect in the body.Leukocyte differentiation antigen 14 (CD14) comprises film mating type leukocyte differentiation antigen 14 (membrane bound CD14; MCD14) and solubility leukocyte differentiation antigen 14 (soluble CD14; SCD14) two kinds of forms: film mating type leukocyte differentiation antigen 14 (mCD14) is that molecular weight is the glycoprotein of 55 kilodaltons (KD); Its C-end by the glycosyl phosphinositides (glucose phosphatidylinositol, GPI) the structure anchor invests surface of cell membrane; Solubility leukocyte differentiation antigen 14 (sCD14) molecule has 49KD and two kinds of forms of 55KD, lacks glycosyl phosphinositides (GPI) anchor.Film mating type leukocyte differentiation antigen 14 (mCD14) is by the monocyte, the macrophage that contain leukocyte differentiation antigen 14 (CD14) gene; Transcribe voluntarily, the translated protein polypeptied chain; In Golgi complex after the saccharification; (phosphatidylinositol PI) combines its c-terminus, and is connected with cell membrane by the phospholipid moiety of phosphinositides with phosphinositides again.Under the physiological condition; Film mating type leukocyte differentiation antigen 14 (mCD14) mainly is expressed in ripe mononuclear macrophage; Faint neutrophil leucocyte, mesangial cell, mammary cell and the B cell of being expressed in, mCD14 mediation lipopolysaccharides is to the activation of these cells.
Endotoxin is the ingredient of Gram-negative bacteria epicyte, and its principal ingredient is that (lipopolysaccharide, LPS), in a single day lipopolysaccharides gets in the body lipopolysaccharides, with the reaction of inducing body to produce multiple inflammatory mediator, causes the major injury of body.Lipopolysaccharides is the phosphatide of one type of specific type, and mammal is had biological action widely.According to chemical constitution, can lipopolysaccharides be divided into two big types, promptly the smooth type lipopolysaccharides (Smooth Form LPS, S-LPS) with the rough type lipopolysaccharides (Rough Form LPS, R-LPS).These two types of lipopolysaccharides have marked difference at aspects such as chemical constitution, prepared in laboratory and BAs.Set up the method for differentiating smooth type lipopolysaccharides (S-LPS) and rough type lipopolysaccharides (R-LPS), finally can be applied to the diagnosis and the treatment of clinical bacteria infectious diseases
In the inflammatory reaction process that gramnegative bacterium causes, the lipopolysaccharides acceptor plays key effect, is the important door of lipopolysaccharides effect.Leukocyte differentiation antigen 14 (CD14) is considered to most important lipopolysaccharides acceptor in the lipopolysaccharides signal transduction, and leukocyte differentiation antigen 14 activates in target cell and the inflammatory reaction at the mediation lipopolysaccharides and plays a crucial role; Research shows, is feasible methods of treatments through suppressing the leukocyte differentiation antigen 14 destruction target cell inflammatory reactions that lipopolysaccharides activated.
(RNA interference RNAi) is meant that (double stranded RNA dsRNA) causes homology messenger RNA (messenger RNA, mRNA) posttranscriptional gene of the degraded mechanism of mourning in silence by specific double stranded RNA to The RNA interference.The specific double stranded RNA of this type is cracked into little (interference) ribonucleic acid molecule (siRNAs); And the complex of mourning in silence (RNA-induced silencing complex, RISC) the effect degraded down that can cause homology messenger RNA (mRNA) to be induced at RNA..The RNA interference (RNAi, Ribonucleic acid interference) RNA simply and efficiently blocks one of most effectual way that specific gene is expressed in the mammalian cell at present, can reach the gene knockout effect, and security is good.But for The RNA interference (RNAi); Not all micro ribonucleic acid molecule (siRNAs) that synthesizes at random to target gene messenger RNA (mRNA) sequence can both cause efficient gene silencing, and the selection of interference target sequence is vital to the influence of jamming effectiveness.Therefore, be to use the important foundation that The RNA interference (RNAi) technology is carried out correlative studys such as gene function, gene therapy to the screening that can effectively suppress the micro ribonucleic acid molecule (siRNAs) that specific gene expresses.
(enzyme linked immunosorbent assay, basis ELISA) is the enzyme labeling of immobilization and the antigen or the antibody of antigen or antibody to enzyme-linked immunosorbent assay.The antigen or the antibody that are combined in surface of solid phase carriers still keep its immunologic competence, and the antigen of enzyme labeling or antibody had both kept its immunologic competence, kept the activity of enzyme again.When measuring, examined sample (measuring wherein antibody or antigen) and the antigen or the antibody of surface of solid phase carriers and reacted.With the method for washing the antigen antibody complex that forms on the solid phase carrier and other materials in the liquid are separated.Add the antigen or the antibody of enzyme labeling again, also be combined on the solid phase carrier through reaction.The amount of examined object matter is certain ratio in enzyme amount on the solid phase and the sample at this moment.After adding the substrate of enzyme reaction, substrate is become coloured product by enzymatic, and the amount of product is directly related with the amount of examined object matter in the sample, so can carry out qualitative or quantitative test according to the depth of colour generation.Because the catalytic efficiency of enzyme is very high, has amplified immunoreactive result indirectly, makes assay method reach very high susceptibility.
For the difference detection method of smooth type lipopolysaccharides and rough type lipopolysaccharides, the chemical discrimination method of prior art such as phenol-water law, phenol-chloroform-sherwood oil method all exist complex steps, the dissatisfactory deficiency of accuracy rate.
Summary of the invention
The purpose of this invention is to provide a kind of method of differentiating smooth type lipopolysaccharides and rough type lipopolysaccharides, solved the chemical discrimination method complex steps of prior art, the dissatisfactory problem of accuracy rate.
The technical scheme that the present invention adopts is, a kind of method of differentiating smooth type lipopolysaccharides and rough type lipopolysaccharides, according to the following steps practical implementation:
2.1) the preparation oligonucleotides: the loop structure in the Lentivirus-shRNA template is selected TTCAAGAGA for use; 5 ' end in the positive-sense strand template adds T, cuts the cohesive end complementation that the back forms with Hpa I enzyme; 5 ' end in the antisense strand template adds AGCT, cuts the cohesive end complementation that the back forms with Xho I enzyme; The T4 ligase recombinant vector that is formed by connecting;
2.2) to the Lentivirus-shDNA template annealing, with reference to table 1, configuration annealing system:
The annealing system of table 1 Lentivirus-shDNA template
| Component | Volume (ul) |
| 10 times of bob folder DNA annealing |
5 |
| Positive-sense strand (100uM) | 5 |
| Antisense strand (100uM) | 5 |
| Water | 35 |
| |
50 |
Annealing conditions is: be 5-6min during 93-95 ° of C; Be 5-6min during 83-85 ° of C; Be 5-6min during 73-75 ° of C; Be 5-6min during 68-70 ° of C; Preserve in the environment of 4 ° of C-5 ° of C; With the annealing product that obtains dilution 50-52 doubly to final concentration be 200-220nM, be used for coupled reaction;
2.3) make up the Lentivirus-shRNA carrier, dispose linked system with reference to table 2:
The linked system of table 2Lentivirus-shRNA carrier
| Component | Volume (ul) |
| 10 times of T4DNA |
2 |
| Slow virus carrier after XhoI+Hpa I enzyme is cut | 1 |
| Template (100nM) | 1 |
| T4DNA ligase (5weissU/ul) | 1 |
| Water | 15 |
| |
20 |
After in 22 ℃ of environment, leaving standstill 1h, transform DH5 α;
2.4) transfection recombinant plasmid and prepare viral liquid
The 293T cell routine is incubated at 10% FBS+DMEM, and 37 ℃, 5% CO
2In the environmental baseline; Press 3 * 10 the previous day in transfection
6The density inoculation 293T cell of cell/ ware is in 10cm dish, and cell density can reach 90%-95% when making transfection, and every ware adds the 10%FBS+DMEM of 9ml, and 37 ℃, 5% CO
2Overnight incubation in the environment;
2.4.1) 2h changes liquid with 6 ware cells, 10%FBS+DMEM, every ware 5ml before the transfection;
2.4.2) get the EP pipe of 2 aseptic 1.5ml, add the serum-free DMEM of 500ul respectively, and be labeled as 1., 2. number pipe;
2.4.3) 1. number toward adding shuttle plasmid pGLV-U6-EGFP-shRNA-224 40ug altogether in the pipe, packaging plasmid, PG-P1-VSVG, PG-P2-REV and PG-P3-RRE three be 42ug altogether, amounts to 82ug, with both mixings; The transfection reagent RNAi-mate that adds 65 * 6=390ul in the 2. number pipe, mixing, the static 5min of room temperature;
2.4.4) behind the 5min, 2. 1. number pipe liquid join in number pipe slowly, and mixing gently, the static 20min of room temperature;
2.4.5) during 20min, the supernatant of each ware 293T cell is discarded, and wash twice with the PBS of preheating, to inhale with the tip head and remove remaining liq, every then ware cell adds the DMEM nutrient culture media of 5ml preheating;
2.4.6) behind the 20min, the mixed liquor in the 1. number pipe is on average assigned in each ware cell, put into 37 ℃ then, 5% CO
2In the incubator;
2.4.7) behind the transfection 6h, each ware cell conditioned medium liquid is sopped up, add the fresh 10%FBS+DMEM of 10ml, put into 37 ℃ then, 5% CO
2Incubator is cultivated 72h;
2.4.8) behind the transfection 72h, with the dish cell conditioned medium liquid of 6 10cm altogether 60ml all be drawn onto in two 50ml centrifuge tubes, carry out low-speed centrifugal then, centrifugal rotational speed 1800-1850rpm, centrifugation time 3-3.5min;
2.4.9) behind the low-speed centrifugal, get supernatant and filter through the filtrator of 0.45um;
2.4.10) filter after, get 15ml virus liquid and in the purifying pipe, carry out high speed centrifugation, centrifugal rotational speed 8000-8200rpm, centrifugation time 16-18min;
2.4.11) remove the waste liquid of bottom, will remain viral liquid and join and carry out the high speed centrifugation second time, centrifugal rotational speed 8000-8200rpm, centrifugation time 20-22min in the ultrafiltration pipe; Centrifugal to supernatant to 1ml, i.e. viral purification liquid 1ml, packing 5 pipes, every pipe 200ul ,-80 ℃ of conditions are preserved;
3.1) infect the previous day with complete medium with 0.5 * 10
5Individual cells/well is laid on 24 orifice plates;
3.2) remove cell culture fluid, with MOI=10, add the viral liquid 0.5ml after the dilution, at 37 ℃, 5% CO
2Spend the night under the condition;
3.3) remove cell culture fluid behind the 24h, every hole adds the complete culture solution of 0.5ml, at 37 ℃, 5% CO
2Spend the night under the condition;
3.4) behind the RAW264.7 cell infection 96h, adding final concentration is the puromycin screening of 1-1.1ug/ml, changes complete medium behind the 24h, changes liquid according to cell state, form until monoclonal, and in each positive colony amplification cultivation of microscopically picking;
The expression of step 4, employing real time fluorescent quantitative poly chain reaction technology for detection stable cell lines messenger RNA; Adopt Western blotting to detect the stable cell lines protein expression level, concrete steps are:
4.1) collecting cell is used for total RNA and extracts: the sucking-off nutrient solution, with phosphate buffer wash cell 2 times, after blowing and beating cell repeatedly, the Trizol that adds 1ml collects liquid, and the design primer adopts the corresponding composite articles of Shanghai life worker;
Extracting the kit instructions according to RNA. divides extraction total RNA.; Use the first chain kit to synthesize complementary DNA (cDNA); Adopt the ABI-7500 system, use fluorescent quantitative poly chain reaction kit, utilize and compare the expression that the CT method is measured film mating type leukocyte differentiation antigen 14;
4.2) collect albumen and carry out Western blotting and detect the stable cell lines protein expression level, concrete steps are:
Wash nutrient culture media off, with phosphate buffer wash cell 2 times, add 0.25% pancreatin-0.02%EDTA and digest 3min, the complete medium that adds 1ml again stops digestion, and the piping and druming cell makes it come off from wall; Centrifuge cell under the 1000-1050rpm condition is abandoned supernatant, the phosphate buffer re-suspended cell, centrifugal once more abandon supernatant after, add a certain amount of IP lysate, wherein contain millesimal protease inhibitors PMSF at cell lysis on ice, repeatedly piping and druming; Centrifuging and taking supernatant under the 12000-12500rpm condition; Utilize two quinoline woods formic acid protein determination kits to measure total protein concentration,, carry out polyacrylamide gel electrophoresis under the concentration of separation gel, carry out Western blotting after electrophoresis finishes 12% with appearance on the total protein equivalent;
5.1) with RAW264.7, siRNA-224.3 clone and siRNA-NC clone with unparalleled anti-nutrient culture media with 2 * 10
5Individual cells/well is laid on two 12 orifice plates, at 37 ℃, 5% CO
2Spend the night under the condition;
5.2) add the S-LPS of 800ng and the R-LPS of 800ng respectively at two 12 orifice plates, after shaking up gently, at 37 ℃, 5% CO
2Cultivated 6-6.5 hour the collecting cell culture supernatant under the condition;
5.3) according to the kit instructions, detecting cell factor nitrogen monoxide, tumor necrosis factor and interleukin-6, NO LPS group is that negative control group is: RAW264.7, siRNA-NC, siRNA-224.3; S-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with S-LPS respectively; R-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with R-LPS respectively.
The invention has the beneficial effects as follows that according to the sequence of film mating type leukocyte differentiation antigen 14 genes, design has specific siRNA-224.3; And packing slow virus carrier; Efficient infecting mouse macrophage, the poor efficiency when having overcome with transfection reagent, thus build stable cell lines fast.Then; Use enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay; ELISA) detect respectively with the nitrogen monoxide (nitrogenmonoxide in mouse monokaryon macrophage (RAW264.7) the stable cell lines cell conditioned medium of expressing this small interference ribonucleic acid molecule under smooth type lipopolysaccharides and the rough type lipopolysaccharides incentive condition; NO), tumor necrosis factor (Tumor necrosis factor-α, TNF-α) and interleukin-6 (interleukin-6, IL-6); Through comparing its difference, differentiate smooth type lipopolysaccharides and rough type lipopolysaccharides.More simple accurately than existing chemical discrimination method (phenol-water law, phenol-chloroform-sherwood oil method), thus be that smooth type lipopolysaccharides and the difference of rough type lipopolysaccharides are studied and the functional analysis of lipopolysaccharides provides foundation.
Description of drawings
Fig. 1 is the slow virus carrier flow package figure in the inventive method;
Fig. 2 is after the inventive method stable cell lines is set up; Behind real time fluorescent quantitative poly chain reaction (real time PCR) detection transfection small interference ribonucleic acid molecule (siRNAs), the expression figure of film mating type leukocyte differentiation antigen 14 messenger RNA (mRNA);
Fig. 3 is that the Western-blot in the inventive method detects its protein level figure;
Fig. 4 is that RAW264.7, siRNA-NC stable cell lines, the siRNA-224.3 stable cell lines in the inventive method stimulates back collecting cell culture supernatant with S-LPS and R-LPS respectively, and Elisa detects the expression figure of cell factor NO;
Fig. 5 is that RAW264.7, siRNA-NC stable cell lines, the siRNA-224.3 stable cell lines in the inventive method stimulates back collecting cell culture supernatant with S-LPS and R-LPS respectively, and Elisa detects the expression figure of cytokine TNF-α;
Fig. 6 is that RAW264.7, siRNA-NC stable cell lines, the siRNA-224.3 stable cell lines in the inventive method stimulates back collecting cell culture supernatant with S-LPS and R-LPS respectively, and Elisa detects the expression figure of cell factor IL-6.
Embodiment
The present invention is a kind of method that can differentiate smooth type lipopolysaccharides and rough type lipopolysaccharides fast, according to the following steps practical implementation:
2.1) Oligo Design (preparation oligonucleotides): the loop structure in the Lentivirus-shRNA template has been selected TTCAAGAGA for use, to avoid forming termination signal; 5 ' end in the positive-sense strand template has added T, cuts the cohesive end complementation that the back forms with Hpa I enzyme; 5 ' end in the antisense strand template has added AGCT, cuts the cohesive end complementation that the back forms with Xho I enzyme; The T4 ligase recombinant vector that is formed by connecting;
Like Fig. 1, be slow virus carrier flow package figure, after soon siRNA-224.3 was converted into shRNA, the loop structure had been selected TTCAAGAGA for use, held in 5 ' of positive-sense strand template and had added T, cut the cohesive end complementation that the back forms with the HpaI enzyme; 5 ' end in the antisense strand template has added AGCT, cuts the cohesive end complementation that the back forms with Xho I enzyme.
2.2) to the Lentivirus-shDNA template annealing, with reference to table 1, configuration annealing system:
The annealing system of table 1Lentivirus-shDNA template
| Component | Volume (ul) |
| 10 times of bob folder |
5 |
| Positive-sense strand (100uM) | 5 |
| Antisense strand (100uM) | 5 |
| Water | 35 |
| |
50 |
Condition is: be 5min during 95 ° of C; Be 5min during 85 ° of C; Be 5min during 75 ° of C; Be 5min during 70 ° of C; Preserve in the environment of 5 ° of C; With 50 times of the annealing product that obtains dilutions to final concentration is 200nM, is used for coupled reaction;
2.3) make up the Lentivirus-shRNA carrier, dispose linked system with reference to table 2:
The linked system of table 2Lentivirus-shRNA carrier
| Component | Volume (ul) |
| 10 times of T4DNA |
2 |
| Slow virus carrier after XhoI+Hpa I enzyme is cut | 1 |
| Template (100nM) | 1 |
| T4DNA ligase (5weissU/ul) | 1 |
| Water | 15 |
| |
20 |
After in 22 ℃ of environment, leaving standstill 1h, transform DH5 α;
2.4) transfection recombinant plasmid and prepare viral liquid
The 293T cell routine is incubated at 10% FBS+DMEM, and 37 ℃, 5% CO
2In the environmental baseline; Press 3 * 10 the previous day in transfection
6The density inoculation 293T cell of cell/ ware is in 10cm dish, and cell density can reach 95% when making transfection, and every ware adds the 10%FBS+DMEM of 9ml, and 37 ℃, 5% CO
2Overnight incubation in the environment;
2.4.1) 2h changes liquid with 6 ware cells, 10%FBS+DMEM, every ware 5ml before the transfection;
2.4.2) get the EP pipe of 2 aseptic 1.5ml, add the serum-free DMEM of 500ul respectively, and be labeled as 1., 2. number pipe;
2.4.3) toward 1. adding shuttle plasmid pGLV-U6-EGFP-shRNA-224 40ug altogether in number pipe, packaging plasmid (PG-P1-VSVG, PG-P2-REV and PG-P3-RRE) is 42ug altogether, amounts to 82ug, with both mixings; The transfection reagent RNAi-mate that adds 65 * 6=390ul in the 2. number pipe, mixing, the static 5min of room temperature;
2.4.4) behind the 5min, 2. 1. number pipe liquid join in number pipe slowly, and mixing gently, the static 20min of room temperature;
2.4.5) during 20min, the supernatant of each ware 293T cell is discarded, and wash twice with the PBS of preheating, to inhale with the tip head and remove remaining liq, every then ware cell adds the DMEM nutrient culture media of 5ml preheating;
2.4.6) behind the 20min, the mixed liquor in the 1. number pipe is on average assigned to (attention will dropwise join in the cell) in each ware cell, put into 37 ℃ then, 5% CO
2In the incubator;
2.4.7) behind the transfection 6h, each ware cell conditioned medium liquid is sopped up, add the fresh 10%FBS+DMEM of 10ml, put into 37 ℃ then, 5% CO
2Incubator is cultivated 72h;
2.4.8) behind the transfection 72h, the dish cell conditioned medium liquid (60ml altogether) of 6 10cm all is drawn onto in two 50ml centrifuge tubes, carry out low-speed centrifugal then, centrifugal rotational speed 1800rpm, centrifugation time 3min;
2.4.9) behind the low-speed centrifugal, get supernatant and filter through the filtrator of 0.45um;
2.4.10) filter after, get 15ml virus liquid and in the purifying pipe, carry out high speed centrifugation, centrifugal rotational speed 8000rpm, centrifugation time 18min;
2.4.11) remove the waste liquid of bottom, will remain viral liquid and join and carry out the high speed centrifugation second time, centrifugal rotational speed 8000rpm, centrifugation time 20min in the ultrafiltration pipe; Centrifugal to supernatant to 1ml, i.e. viral purification liquid 1ml, packing 5 pipes, every pipe 200ul ,-80 ℃ of conditions are preserved;
3.1) infect the previous day with complete medium with 0.5 * 10
5Individual cells/well is laid on 24 orifice plates;
3.2) remove cell culture fluid, with MOI=10 (infected at an MOI of 10), add the viral liquid 0.5ml after the dilution, at 37 ℃, 5% CO
2Spend the night under the condition;
3.3) remove cell culture fluid behind the 24h, every hole adds the complete culture solution of 0.5ml, at 37 ℃, 5% CO
2Spend the night under the condition;
3.4) behind the RAW264.7 cell infection 96h, adding final concentration is puromycin (puro) screening of 1ug/ml, changes complete medium behind the 24h, changes liquid according to cell state, form until monoclonal, and in each positive colony amplification cultivation of microscopically picking;
The expression of step 4, employing real time fluorescent quantitative poly chain reaction (real time PCR) technology for detection stable cell lines messenger RNA (mRNA); Adopt Western blotting (western blot) to detect the stable cell lines protein expression level, concrete steps are:
4.1) collecting cell is used for total RNA and extracts: the sucking-off nutrient solution, with phosphate buffer (PBS) washed cell 2 times, after blowing and beating cell repeatedly, the Trizol that adds 1ml collects liquid, and the design primer adopts the corresponding composite articles of Shanghai life worker;
Film mating type leukocyte differentiation antigen 14 (mCD14):
Upstream primer is: 5 '-AAC TCG CTC AAT CTG TCT TTCAC-3 ',
Downstream primer is: 5 '-GCT CAT CTG GGC TAG GGT TC-3 ';
Glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH):
Upstream primer is: 5 '-TGT GTC CGT CGT GGA TCT GA-3 ',
Downstream primer is: 5 '-CCT GCT TCA CCA CCT TCT TGA-3 ',
Extract kit (PureLink TM RNA Mini Kit) instructions according to RNA. and divide extraction total RNA. (RNA); Use the first chain kit (M-MLV first strand Kit) to synthesize complementary DNA (cDNA) (DNA); Adopt the ABI-7500 system; Use fluorescent quantitative poly chain reaction kit (
Green qPCR SuperMix-UDG Kit); Utilize and compare (mCD14) expression that the CT method is measured film mating type leukocyte differentiation antigen 14; The result sees shown in Figure 2; Show that the siRNA-224.3 stable cell lines compares with negative cells RAW264.7, siRNA-NC, mouse film mating type leukocyte differentiation antigen 14mRNA level is suppressed 95% in the siRNA-224.3 stable cell lines;
4.2) collect albumen and carry out Western blotting (western blot) and detect the stable cell lines protein expression level, concrete steps are:
Wash nutrient culture media off, with phosphate buffer (PBS) washed cell 2 times, add 0.25% pancreatin-0.02%EDTA and digest 3min, the complete medium that adds 1ml again stops digestion, and the piping and druming cell makes it come off from wall; Centrifuge cell under the 1000rpm condition is abandoned supernatant, phosphate buffer (PBS) re-suspended cell, centrifugal once more abandon supernatant after, add a certain amount of IP lysate (containing millesimal protease inhibitors PMSF) at cell lysis on ice, repeatedly piping and druming; Centrifuging and taking supernatant under the 12000rpm condition; Utilize two quinoline woods formic acid (bicinchoninic acid; BCA) protein determination kit is measured total protein concentration, with appearance on the total protein equivalent, carries out polyacrylamide gel electrophoresis (SDS-PAGE) under the concentration of the separation gel 12%; After finishing, electrophoresis carries out Western blotting (western blot)
One anti-is: the anti-film mating type of rabbit leukocyte differentiation antigen 14 (mCD14) polyclonal antibody (rabbit polyclonal anti-mCD14; 1:300 doubly dilutes); The anti-glyceraldehyde-3-phosphate dehydrogenase of rabbit (GAPDH) monoclonal antibody (GAPDH rabbit mAB, 1:1000 doubly dilutes)
Two anti-are: horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (HRP labeled goat anti-rabbit IgG, 1:3000 doubly dilutes).
The result sees shown in Figure 3, shows that the siRNA-224.3 stable cell lines compares with negative cells RAW264.7, siRNA-NC, and mouse film mating type leukocyte differentiation antigen 14 protein levels are suppressed 90% in the siRNA-224.3 stable cell lines;
5.1) with RAW264.7, siRNA-224.3 clone and siRNA-NC clone with unparalleled anti-nutrient culture media with 2 * 10
5Individual cells/well is laid on two 12 orifice plates, at 37 ℃, 5% CO
2Spend the night under the condition;
5.2) add the S-LPS of 800ng and the R-LPS of 800ng respectively at two 12 orifice plates, after shaking up gently, at 37 ℃, 5% CO
2Cultivated 6 hours the collecting cell culture supernatant under the condition;
5.3) according to the kit instructions; Detect cell factor nitrogen monoxide (NO), tumor necrosis factor (TNF-α) and interleukin-6 (IL-6); The result sees Fig. 4, Fig. 5, Fig. 6; Horizontal ordinate is RAW264.7, siRNA-NC, siRNA-224.3NO LPS group, S-LPS stimulating group, R-LPS stimulating group; Ordinate is the content of nitrogen monoxide in the cells and supernatant (NO), tumor necrosis factor (TNF-α) and interleukin-6 (IL-6).NO LPS group is that negative control group is: RAW264.7, siRNA-NC, siRNA-224.3; S-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with S-LPS respectively; R-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with R-LPS respectively.Wherein, three kinds of cell factors all raise during the S-LPS group was organized with R-LPS, but the cell factor of siRNA-224.3 significantly reduces in the S-LPS group, and the siRNA-224.3 cell factor changes indifference in the R-LPS group.Explanation thus, CD14 combines with S-LPS, thereby obtains conclusion, and the inventive method utilizes siRNA-224.3 clone can identify S-LPS and R-LPS convenient, exactly.
According to the step of embodiment 1, implement according to following concrete parameter:
2.1) the preparation oligonucleotides: the loop structure in the Lentivirus-shRNA template is selected TTCAAGAGA for use; 5 ' end in the positive-sense strand template adds T, cuts the cohesive end complementation that the back forms with Hpa I enzyme; 5 ' end in the antisense strand template adds AGCT, cuts the cohesive end complementation that the back forms with Xho I enzyme; The T4 ligase recombinant vector that is formed by connecting;
2.2) to the Lentivirus-shDNA template annealing, with reference to table 1 configuration annealing system,
Annealing conditions is: be 5min during 94 ° of C; Be 6min during 84 ° of C; Be 6min during 74 ° of C; Be 5min during 69 ° of C; Preserve in the environment of 4 ° of C; With 51 times of the annealing product that obtains dilutions to final concentration is 210nM, is used for coupled reaction;
2.3) make up the Lentivirus-shRNA carrier, with reference to table 2 configuration linked system,
After in 22 ℃ of environment, leaving standstill 1h, transform DH5 α;
2.4) transfection recombinant plasmid and prepare viral liquid
The 293T cell routine is incubated at 10% FBS+DMEM, and 37 ℃, 5% CO
2In the environmental baseline; Press 3 * 10 the previous day in transfection
6The density inoculation 293T cell of cell/ ware is in 10cm dish, and cell density can reach 93% when making transfection, and every ware adds the 10%FBS+DMEM of 9ml, and 37 ℃, 5% CO
2Overnight incubation in the environment;
2.4.1) 2h changes liquid with 6 ware cells, 10%FBS+DMEM, every ware 5ml before the transfection;
2.4.2) get the EP pipe of 2 aseptic 1.5ml, add the serum-free DMEM of 500ul respectively, and be labeled as 1., 2. number pipe;
2.4.3) 1. number toward adding shuttle plasmid pGLV-U6-EGFP-shRNA-224 40ug altogether in the pipe, packaging plasmid, PG-P1-VSVG, PG-P2-REV and PG-P3-RRE three be 42ug altogether, amounts to 82ug, with both mixings; The transfection reagent RNAi-mate that adds 65 * 6=390ul in the 2. number pipe, mixing, the static 5min of room temperature;
2.4.4) behind the 5min, 2. 1. number pipe liquid join in number pipe slowly, and mixing gently, the static 20min of room temperature;
2.4.5) during 20min, the supernatant of each ware 293T cell is discarded, and wash twice with the PBS of preheating, to inhale with the tip head and remove remaining liq, every then ware cell adds the DMEM nutrient culture media of 5ml preheating;
2.4.6) behind the 20min, the mixed liquor in the 1. number pipe is on average assigned in each ware cell, put into 37 ℃ then, 5% CO
2In the incubator;
2.4.7) behind the transfection 6h, each ware cell conditioned medium liquid is sopped up, add the fresh 10%FBS+DMEM of 10ml, put into 37 ℃ then, 5% CO
2Incubator is cultivated 72h;
2.4.8) behind the transfection 72h, with the dish cell conditioned medium liquid of 6 10cm altogether 60ml all be drawn onto in two 50ml centrifuge tubes, carry out low-speed centrifugal then, centrifugal rotational speed 1850rpm, time 3min;
2.4.9) behind the low-speed centrifugal, get supernatant and filter through the filtrator of 0.45um;
2.4.10) filter after, get 15ml virus liquid and in the purifying pipe, carry out high speed centrifugation, centrifugal rotational speed 8200rpm, centrifugation time 18min;
2.4.11) remove the waste liquid of bottom, will remain viral liquid and join and carry out the high speed centrifugation second time, centrifugal rotational speed 8200rpm, centrifugation time 20min in the ultrafiltration pipe; Centrifugal to supernatant to 1ml, i.e. viral purification liquid 1ml, packing 5 pipes, every pipe 200ul ,-80 ℃ of conditions are preserved;
3.1) infect the previous day with complete medium with 0.5 * 10
5Individual cells/well is laid on 24 orifice plates;
3.2) remove cell culture fluid, with MOI=10, add the viral liquid 0.5ml after the dilution, at 37 ℃, 5% CO
2Spend the night under the condition;
3.3) remove cell culture fluid behind the 24h, every hole adds the complete culture solution of 0.5ml, at 37 ℃, 5% CO
2Spend the night under the condition;
3.4) behind the RAW264.7 cell infection 96h, adding final concentration is the puromycin screening of 1.05ug/ml, changes complete medium behind the 24h, changes liquid according to cell state, form until monoclonal, and in each positive colony amplification cultivation of microscopically picking;
The expression of step 4, employing real time fluorescent quantitative poly chain reaction technology for detection stable cell lines messenger RNA; Adopt Western blotting to detect the stable cell lines protein expression level, concrete steps are:
4.1) collecting cell is used for total RNA and extracts: the sucking-off nutrient solution, with phosphate buffer wash cell 2 times, after blowing and beating cell repeatedly, the Trizol that adds 1ml collects liquid, and the design primer adopts the corresponding composite articles of Shanghai life worker;
Extracting the kit instructions according to RNA. divides extraction total RNA.; Use the first chain kit to synthesize complementary DNA (cDNA); Adopt the ABI-7500 system, use fluorescent quantitative poly chain reaction kit, utilize and compare the expression that the CT method is measured film mating type leukocyte differentiation antigen 14;
4.2) collect albumen and carry out Western blotting and detect the stable cell lines protein expression level, concrete steps are:
Wash nutrient culture media off, with phosphate buffer wash cell 2 times, add 0.25% pancreatin-0.02%EDTA and digest 3min, the complete medium that adds 1ml again stops digestion, and the piping and druming cell makes it come off from wall; Centrifuge cell under the 1050rpm condition is abandoned supernatant, the phosphate buffer re-suspended cell, centrifugal once more abandon supernatant after, add a certain amount of IP lysate, wherein contain millesimal protease inhibitors PMSF at cell lysis on ice, repeatedly piping and druming; Centrifuging and taking supernatant under the 12500rpm condition; Utilize two quinoline woods formic acid protein determination kits to measure total protein concentration,, carry out polyacrylamide gel electrophoresis under the concentration of separation gel, carry out Western blotting after electrophoresis finishes 12% with appearance on the total protein equivalent;
5.1) with RAW264.7, siRNA-224.3 clone and siRNA-NC clone with unparalleled anti-nutrient culture media with 2 * 10
5Individual cells/well is laid on two 12 orifice plates, at 37 ℃, 5% CO
2Spend the night under the condition;
5.2) add the S-LPS of 800ng and the R-LPS of 800ng respectively at two 12 orifice plates, after shaking up gently, at 37 ℃, 5% CO
2Cultivated 6.5 hours the collecting cell culture supernatant under the condition;
5.3) according to the kit instructions, detecting cell factor nitrogen monoxide, tumor necrosis factor and interleukin-6, NO LPS group is that negative control group is: RAW264.7, siRNA-NC, siRNA-224.3; S-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with S-LPS respectively; R-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with R-LPS respectively.
Wherein, three kinds of cell factors all raise during the S-LPS group was organized with R-LPS, but the cell factor of siRNA-224.3 significantly reduces in the S-LPS group, and the siRNA-224.3 cell factor changes indifference in the R-LPS group.Explanation thus, CD14 combines with S-LPS, thereby obtains conclusion, and the inventive method utilizes siRNA-224.3 clone can identify S-LPS and R-LPS convenient, exactly.
According to the step of embodiment 1, concrete parameter below adopting is implemented:
2.1) the preparation oligonucleotides: the loop structure in the Lentivirus-shRNA template is selected TTCAAGAGA for use; 5 ' end in the positive-sense strand template adds T, cuts the cohesive end complementation that the back forms with Hpa I enzyme; 5 ' end in the antisense strand template adds AGCT, cuts the cohesive end complementation that the back forms with Xho I enzyme; The T4 ligase recombinant vector that is formed by connecting;
2.2) to the Lentivirus-shDNA template annealing, with reference to table 1 configuration annealing system,
Annealing conditions is: be 5-6min during 93-95 ° of C; Be 5-6min during 83-85 ° of C; Be 5-6min during 73-75 ° of C; Be 5-6min during 68-70 ° of C; Preserve in the environment of 4 ° of C-5 ° of C; With the annealing product that obtains dilution 50-52 doubly to final concentration be 200-220nM, be used for coupled reaction;
2.3) make up the Lentivirus-shRNA carrier, with reference to table 2 configuration linked system,
After in 22 ℃ of environment, leaving standstill 1h, transform DH5 α;
2.4) transfection recombinant plasmid and prepare viral liquid
The 293T cell routine is incubated at 10% FBS+DMEM, and 37 ℃, 5% CO
2In the environmental baseline; Press 3 * 10 the previous day in transfection
6The density inoculation 293T cell of cell/ ware is in 10cm dish, and cell density can reach 90% when making transfection, and every ware adds the 10%FBS+DMEM of 9ml, and 37 ℃, 5% CO
2Overnight incubation in the environment;
2.4.1) 2h changes liquid with 6 ware cells, 10%FBS+DMEM, every ware 5ml before the transfection;
2.4.2) get the EP pipe of 2 aseptic 1.5ml, add the serum-free DMEM of 500ul respectively, and be labeled as 1., 2. number pipe;
2.4.3) 1. number toward adding shuttle plasmid pGLV-U6-EGFP-shRNA-224 40ug altogether in the pipe, packaging plasmid, PG-P1-VSVG, PG-P2-REV and PG-P3-RRE three be 42ug altogether, amounts to 82ug, with both mixings; The transfection reagent RNAi-mate that adds 65 * 6=390ul in the 2. number pipe, mixing, the static 5min of room temperature;
2.4.4) behind the 5min, 2. 1. number pipe liquid join in number pipe slowly, and mixing gently, the static 20min of room temperature;
2.4.5) during 20min, the supernatant of each ware 293T cell is discarded, and wash twice with the PBS of preheating, to inhale with the tip head and remove remaining liq, every then ware cell adds the DMEM nutrient culture media of 5ml preheating;
2.4.6) behind the 20min, the mixed liquor in the 1. number pipe is on average assigned in each ware cell, put into 37 ℃ then, 5% CO
2In the incubator;
2.4.7) behind the transfection 6h, each ware cell conditioned medium liquid is sopped up, add the fresh 10%FBS+DMEM of 10ml, put into 37 ℃ then, 5% CO
2Incubator is cultivated 72h;
2.4.8) behind the transfection 72h, with the dish cell conditioned medium liquid of 6 10cm altogether 60ml all be drawn onto in two 50ml centrifuge tubes, carry out low-speed centrifugal then, centrifugal rotational speed 1840rpm, centrifugation time 3min;
2.4.9) behind the low-speed centrifugal, get supernatant and filter through the filtrator of 0.45um;
2.4.10) filter after, get 15ml virus liquid and in the purifying pipe, carry out high speed centrifugation, centrifugal rotational speed 8100rpm, centrifugation time 17min;
2.4.11) remove the waste liquid of bottom, will remain viral liquid and join and carry out the high speed centrifugation second time, centrifugal rotational speed 8100rpm, centrifugation time 21min in the ultrafiltration pipe; Centrifugal to supernatant to 1ml, i.e. viral purification liquid 1ml, packing 5 pipes, every pipe 200ul ,-80 ℃ of conditions are preserved;
3.1) infect the previous day with complete medium with 0.5 * 10
5Individual cells/well is laid on 24 orifice plates;
3.2) remove cell culture fluid, with MOI=10, add the viral liquid 0.5ml after the dilution, at 37 ℃, 5% CO
2Spend the night under the condition;
3.3) remove cell culture fluid behind the 24h, every hole adds the complete culture solution of 0.5ml, at 37 ℃, 5% CO
2Spend the night under the condition;
3.4) behind the RAW264.7 cell infection 96h, adding final concentration is the puromycin screening of 1.1ug/ml, changes complete medium behind the 24h, changes liquid according to cell state, form until monoclonal, and in each positive colony amplification cultivation of microscopically picking;
The expression of step 4, employing real time fluorescent quantitative poly chain reaction technology for detection stable cell lines messenger RNA; Adopt Western blotting to detect the stable cell lines protein expression level, concrete steps are:
4.1) collecting cell is used for total RNA and extracts: the sucking-off nutrient solution, with phosphate buffer wash cell 2 times, after blowing and beating cell repeatedly, the Trizol that adds 1ml collects liquid, and the design primer adopts the corresponding composite articles of Shanghai life worker;
Extracting the kit instructions according to RNA. divides extraction total RNA.; Use the first chain kit to synthesize complementary DNA (cDNA); Adopt the ABI-7500 system, use fluorescent quantitative poly chain reaction kit, utilize and compare the expression that the CT method is measured film mating type leukocyte differentiation antigen 14;
4.2) collect albumen and carry out Western blotting and detect the stable cell lines protein expression level, concrete steps are:
Wash nutrient culture media off, with phosphate buffer wash cell 2 times, add 0.25% pancreatin-0.02%EDTA and digest 3min, the complete medium that adds 1ml again stops digestion, and the piping and druming cell makes it come off from wall; Centrifuge cell under the 1030rpm condition is abandoned supernatant, the phosphate buffer re-suspended cell, centrifugal once more abandon supernatant after, add a certain amount of IP lysate, wherein contain millesimal protease inhibitors PMSF at cell lysis on ice, repeatedly piping and druming; Centrifuging and taking supernatant under the 12200rpm condition; Utilize two quinoline woods formic acid protein determination kits to measure total protein concentration,, carry out polyacrylamide gel electrophoresis under the concentration of separation gel, carry out Western blotting after electrophoresis finishes 12% with appearance on the total protein equivalent;
5.1) with RAW264.7, siRNA-224.3 clone and siRNA-NC clone with unparalleled anti-nutrient culture media with 2 * 10
5Individual cells/well is laid on two 12 orifice plates, at 37 ℃, 5% CO
2Spend the night under the condition;
5.2) add the S-LPS of 800ng and the R-LPS of 800ng respectively at two 12 orifice plates, after shaking up gently, at 37 ℃, 5% CO
2Cultivated 6.25 hours the collecting cell culture supernatant under the condition;
5.3) according to the kit instructions, detecting cell factor nitrogen monoxide, tumor necrosis factor and interleukin-6, NO LPS group is that negative control group is: RAW264.7, siRNA-NC, siRNA-224.3; S-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with S-LPS respectively; R-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with R-LPS respectively.
Wherein, three kinds of cell factors all raise during the S-LPS group was organized with R-LPS, but the cell factor of siRNA-224.3 significantly reduces in the S-LPS group, and the siRNA-224.3 cell factor changes indifference in the R-LPS group.Explanation thus, CD14 combines with S-LPS, thereby obtains conclusion, and the inventive method utilizes siRNA-224.3 clone can identify S-LPS and R-LPS convenient, exactly.
In sum; The present invention differentiates the method for smooth type lipopolysaccharides and rough type lipopolysaccharides; At first, the synthetic small interference ribonucleic acid molecule (siRNA-224.3) and the negative control siRNA-NC that can effectively disturb mouse mCD14 gene expression, and be prepared into recombinant slow virus; Secondly,, and filter out positive colony, make up stable cell lines siRNA-224.3, siRNA-NC with puromycin with slow-virus infection mouse macrophage RAW264.7; At last; Use enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay; ELISA) detect use respectively smooth type lipopolysaccharides and rough type lipopolysaccharides (lipopolysaccharide, LPS) incentive condition down the nitrogen monoxide in mouse monokaryon macrophage (RAW264.7) the stable cell lines cell conditioned medium of this small interference ribonucleic acid molecule of expression (nitrogenmonoxide, NO), tumor necrosis factor (Tumor necrosis factor-α; TNF-α) and interleukin-6 (interleukin-6; IL-6), through comparing its difference, realize differentiating smooth type lipopolysaccharides and rough type lipopolysaccharides; Compare existing method, convenient, accurate.
Appendix
Computer-readable gene order table 1
< 110>Wang Fengyang
< 120>the micro ribonucleic acid molecule of inhibition mouse film mating type leukocyte differentiation antigen 14 gene expressions
<160>1
<210>1
<211>21
<212>RNA
< 213>mouse
<400>1
gggcaguuca?cugauauuatt?21
Claims (3)
1. a method of differentiating smooth type lipopolysaccharides and rough type lipopolysaccharides is characterized in that, this method is according to the following steps practical implementation:
Step 1, according to the sequence of film mating type leukocyte differentiation antigen 14 genes, according to the cardinal rule of small interference ribonucleic acid molecule design, synthetic a pair of small interference ribonucleic acid molecule siRNA-224.3 to film mating type leukocyte differentiation antigen 14;
Step 2, will synthesize good siRNA-224.3 packing slow virus carrier
2.1) the preparation oligonucleotides: the loop structure in the Lentivirus-shRNA template is selected TTCAAGAGA for use; 5 ' end in the positive-sense strand template adds T, cuts the cohesive end complementation that the back forms with Hpa I enzyme; 5 ' end in the antisense strand template adds AGCT, cuts the cohesive end complementation that the back forms with Xho I enzyme; The T4 ligase recombinant vector that is formed by connecting;
2.2) to the Lentivirus-shDNA template annealing, dispose the annealing system with reference to table 1:
The annealing system of table 1Lentivirus-shDNA template
Annealing conditions is: be 5-6min during 93-95 ° of C; Be 5-6min during 83-85 ° of C; Be 5-6min during 73-75 ° of C; Be 5-6min during 68-70 ° of C; Preserve in the environment of 4 ° of C-5 ° of C; With the annealing product that obtains dilution 50-52 doubly to final concentration be 200-220nM, be used for coupled reaction;
2.3) make up the Lentivirus-shRNA carrier, dispose linked system with reference to table 2:
The linked system of table 2Lentivirus-shRNA carrier
After in 22 ℃ of environment, leaving standstill 1h, transform DH5 α;
2.4) transfection recombinant plasmid and prepare viral liquid
The 293T cell routine is incubated at 10% FBS+DMEM, and 37 ℃, 5% CO
2In the environmental baseline; Press 3 * 10 the previous day in transfection
6The density inoculation 293T cell of cell/ ware is in 10cm dish, and cell density can reach 90%-95% when making transfection, and every ware adds the 10%FBS+DMEM of 9ml, and 37 ℃, 5% CO
2Overnight incubation in the environment;
2.4.1) 2h changes liquid with 6 ware cells, 10%FBS+DMEM, every ware 5ml before the transfection;
2.4.2) get the EP pipe of 2 aseptic 1.5ml, add the serum-free DMEM of 500ul respectively, and be labeled as 1., 2. number pipe;
2.4.3) 1. number toward adding shuttle plasmid pGLV-U6-EGFP-shRNA-224 40ug altogether in the pipe, packaging plasmid, PG-P1-VSVG, PG-P2-REV and PG-P3-RRE three be 42ug altogether, amounts to 82ug, with both mixings; The transfection reagent RNAi-mate that adds 65 * 6=390ul in the 2. number pipe, mixing, the static 5min of room temperature;
2.4.4) behind the 5min, 2. 1. number pipe liquid join in number pipe slowly, and mixing gently, the static 20min of room temperature;
2.4.5) during 20min, the supernatant of each ware 293T cell is discarded, and wash twice with the PBS of preheating, to inhale with the tip head and remove remaining liq, every then ware cell adds the DMEM nutrient culture media of 5ml preheating;
2.4.6) behind the 20min, the mixed liquor in the 1. number pipe is on average assigned in each ware cell, put into 37 ℃ then, 5% CO
2In the incubator;
2.4.7) behind the transfection 6h, each ware cell conditioned medium liquid is sopped up, add the fresh 10%FBS+DMEM of 10ml, put into 37 ℃ then, 5% CO
2Incubator is cultivated 72h;
2.4.8) behind the transfection 72h, with the dish cell conditioned medium liquid of 6 10cm altogether 60ml all be drawn onto in two 50ml centrifuge tubes, carry out low-speed centrifugal then, centrifugal rotational speed 1800-1850rpm, centrifugation time 3-3.5min;
2.4.9) behind the low-speed centrifugal, get supernatant and filter through the filtrator of 0.45um;
2.4.10) filter after, get 15ml virus liquid and in the purifying pipe, carry out high speed centrifugation, centrifugal rotational speed 8000-8200rpm, centrifugation time 16-18min;
2.4.11) remove the waste liquid of bottom, will remain viral liquid and join and carry out the high speed centrifugation second time, centrifugal rotational speed 8000-8200rpm, centrifugation time 20-22min in the ultrafiltration pipe; Centrifugal to supernatant to 1ml, i.e. viral purification liquid 1ml, packing 5 pipes, every pipe 200ul ,-80 ℃ of conditions are preserved;
Step 3, slow-virus infection mouse macrophage RAW264.7 cell make up stable cell lines, and concrete steps are:
3.1) infect the previous day with complete medium with 0.5 * 10
5Individual cells/well is laid on 24 orifice plates;
3.2) remove cell culture fluid, with MOI=10, add the viral liquid 0.5ml after the dilution, at 37 ℃, 5% CO
2Spend the night under the condition;
3.3) remove cell culture fluid behind the 24h, every hole adds the complete culture solution of 0.5ml, at 37 ℃, 5% CO
2Spend the night under the condition;
3.4) behind the RAW264.7 cell infection 96h, adding final concentration is the puromycin screening of 1-1.1ug/ml, changes complete medium behind the 24h, changes liquid according to cell state, form until monoclonal, and in each positive colony amplification cultivation of microscopically picking;
The expression of step 4, employing real time fluorescent quantitative poly chain reaction technology for detection stable cell lines messenger RNA; Adopt Western blotting to detect the stable cell lines protein expression level, concrete steps are:
4.1) collecting cell is used for total RNA and extracts: the sucking-off nutrient solution, with phosphate buffer wash cell 2 times, after blowing and beating cell repeatedly, the Trizol that adds 1ml collects liquid, and the design primer adopts the corresponding composite articles of Shanghai life worker;
Extracting the kit instructions according to RNA. divides extraction total RNA.; Use the first chain kit to synthesize complementary DNA (cDNA); Adopt the ABI-7500 system, use fluorescent quantitative poly chain reaction kit, utilize and compare the expression that the CT method is measured film mating type leukocyte differentiation antigen 14;
4.2) collect albumen and carry out Western blotting and detect the stable cell lines protein expression level; Concrete steps comprise: wash nutrient culture media off; With phosphate buffer wash cell 2 times; Add 0.25% pancreatin-0.02%EDTA and digest 3min, the complete medium that adds 1ml again stops digestion, and the piping and druming cell makes it come off from wall; Centrifuge cell under the 1000-1050rpm condition is abandoned supernatant, the phosphate buffer re-suspended cell, centrifugal once more abandon supernatant after, add a certain amount of IP lysate, wherein contain millesimal protease inhibitors PMSF at cell lysis on ice, repeatedly piping and druming; Centrifuging and taking supernatant under the 12000-12500rpm condition; Utilize two quinoline woods formic acid protein determination kits to measure total protein concentration,, carry out polyacrylamide gel electrophoresis under the concentration of separation gel, carry out Western blotting after electrophoresis finishes 12% with appearance on the total protein equivalent;
Step 5, respectively with S-LPS and R-LPS800ng irritation cell system, the collecting cell culture supernatant, Elisa detects cell factor NO, TNF-α and IL-6, concrete steps are following:
5.1) with RAW264.7, siRNA-224.3 clone and siRNA-NC clone with unparalleled anti-nutrient culture media with 2 * 10
5Individual cells/well is laid on two 12 orifice plates, at 37 ℃, 5% CO
2Condition is spent the night;
5.2) add the S-LPS of 800ng and the R-LPS of 800ng respectively at two 12 orifice plates, after shaking up gently, at 37 ℃, 5% CO
2Cultivated 6-6.5 hour the collecting cell culture supernatant under the condition;
5.3) according to the kit instructions, detecting cell factor nitrogen monoxide, tumor necrosis factor and interleukin-6, NO LPS group is that negative control group is: RAW264.7, siRNA-NC, siRNA-224.3; S-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with S-LPS respectively; R-LPS stimulating group: RAW264.7, siRNA-NC, siRNA-224.3 stimulate with R-LPS respectively.
2. the method for discriminating smooth type lipopolysaccharides according to claim 1 and rough type lipopolysaccharides; It is characterized in that: the design primer described step 4.1) adopts film mating type leukocyte differentiation antigen 14: upstream primer is: 5 '-AAC TCG CTC AAT CTG TCT TTC AC-3 '
Downstream primer is: 5 '-GCT CAT CTG GGC TAG GGT TC-3 ';
Glyceraldehyde-3-phosphate dehydrogenase:
Upstream primer is: 5 '-TGT GTC CGT CGT GGA TCT GA-3 ',
Downstream primer is: 5 '-CCT GCT TCA CCA CCT TCT TGA-3 '.
3. the method for discriminating smooth type lipopolysaccharides according to claim 1 and rough type lipopolysaccharides is characterized in that: after finishing, the electrophoresis described step 4.1) carries out Western blotting, wherein
One anti-is: the anti-film mating type of rabbit leukocyte differentiation antigen 14 polyclonal antibodies, and 1:300 doubly dilutes, the anti-glyceraldehyde-3-phosphate dehydrogenase monoclonal antibody of rabbit, 1:1000 doubly dilutes;
Two anti-are: horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G, 1:3000 doubly dilutes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5705398A (en) * | 1994-03-02 | 1998-01-06 | The Scripps Research Institute | Methods for identifying inhibitors of LPS-mediated LBP binding |
| WO1998020347A1 (en) * | 1996-11-01 | 1998-05-14 | Eisai Co., Ltd. | Assay for lipopolysaccharide antagonists |
| CN101227922A (en) * | 2005-05-06 | 2008-07-23 | 斯克里普斯研究学院 | Compositions and methods for modulating cells via CD14 and toII-like receptor 4 signaling pathway |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5705398A (en) * | 1994-03-02 | 1998-01-06 | The Scripps Research Institute | Methods for identifying inhibitors of LPS-mediated LBP binding |
| WO1998020347A1 (en) * | 1996-11-01 | 1998-05-14 | Eisai Co., Ltd. | Assay for lipopolysaccharide antagonists |
| CN101227922A (en) * | 2005-05-06 | 2008-07-23 | 斯克里普斯研究学院 | Compositions and methods for modulating cells via CD14 and toII-like receptor 4 signaling pathway |
Non-Patent Citations (2)
| Title |
|---|
| MING LEI ET AL.: "siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 张葵: "CD14的研究进展及临床意义", 《临床检验杂志》 * |
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|---|---|---|---|---|
| CN107488635A (en) * | 2017-09-22 | 2017-12-19 | 中国人民解放军第三军医大学第三附属医院 | Monoclonal cell and its production and use |
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