CN102533758B - Small ribonucleic acid molecules for inhibiting buffalo membrane differentiation antigen 14 gene expression - Google Patents
Small ribonucleic acid molecules for inhibiting buffalo membrane differentiation antigen 14 gene expression Download PDFInfo
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Abstract
本发明的一种抑制水牛膜结合型白细胞分化抗原14基因表达的小核糖核酸分子，通过建立稳定表达增强型绿色荧光蛋白EGFP-水牛膜结合型白细胞分化抗原14融合蛋白的人胚肾293细胞系，合成不同小核糖核酸分子，并转染人胚肾293细胞系稳定细胞系；通过荧光显微镜观察增强型绿色荧光蛋白EGFP-水牛膜结合型白细胞分化抗原14融合蛋白表达的荧光强度，流式细胞术检测其蛋白表达水平，蛋白质印迹鉴定其干扰效果，筛选出能高效抑制水牛膜结合型白细胞分化抗原14基因表达的小核糖核酸分子。本发明为进一步应用核糖核酸干扰技术通过抑制水牛膜结合型白细胞分化抗原14的表达，增强水牛抵抗革兰氏阴性菌感染的能力奠定了重要基础。
A small ribonucleic acid molecule that inhibits the expression of buffalo membrane-bound leukocyte differentiation antigen-14 gene of the present invention is established by establishing a human embryonic kidney 293 cell line that stably expresses enhanced green fluorescent protein EGFP-buffalo membrane-bound leukocyte differentiation antigen-14 fusion protein , synthesized different small RNA molecules, and transfected human embryonic kidney 293 cell line stable cell line; observed the fluorescence intensity of enhanced green fluorescent protein EGFP-buffalo membrane-bound leukocyte differentiation antigen 14 fusion protein expression by fluorescence microscope, flow cytometry The protein expression level was detected by technique, the interference effect was identified by Western blot, and the small RNA molecules that could efficiently inhibit the expression of Buffalo membrane-bound leukocyte differentiation antigen 14 gene were screened out. The invention lays an important foundation for further applying ribonucleic acid interference technology to enhance the ability of buffaloes to resist Gram-negative bacteria infection by inhibiting the expression of buffalo membrane-bound leukocyte differentiation antigen 14.
Description
Technical field
The present invention relates to genetic expression, Yeast Nucleic Acid (the Ribonucleic Acid of biological technical field, RNA) interference and detection technique field, be specifically related to a kind of small ribonucleic acid molecules that suppresses the genetic expression of buffalo film mating type CD14, the invention still further relates to the biology screening method of the small ribonucleic acid molecules of this kind of inhibition buffalo film mating type CD14 genetic expression.
Background technology
CD14 (cluster of differentiation antigen, CD14) be a kind of differentiation antigen that is present in the cell surfaces such as monocyte, scavenger cell, for mediating one of important acceptor of lipopolysaccharides (lipopolysaccharide, LPS) biological effect in body.CD14 (CD14) comprises film mating type CD14 (membrane bound CD14, mCD14) and solubility CD14 (soluble CD14, sCD14) two kinds of forms: film mating type CD14 (mCD14) is that molecular weight is the glycoprotein of 55 kilodaltons (KD), its C-end invests surface of cell membrane by glycosyl phosphinositides (glucose phosphatidylinositol, GPI) structure anchor; Solubility CD14 (sCD14) molecule has 49KD and two kinds of forms of 55KD, lacks glycosyl phosphinositides (GPI) anchor.Film mating type CD14 (mCD14) is by the monocyte, the scavenger cell that contain CD14 (CD14) gene, transcribe voluntarily, translated protein polypeptide chain, in Golgi complex after saccharification, its carboxyl terminal again with phosphinositides (phosphatidylinositol, PI) combination, and be connected with cytolemma by the phospholipid moiety of phosphinositides.Under physiological condition, film mating type CD14 (membrane bound cluster of differentiation antigen, mCD14) be mainly expressed in ripe mononuclear macrophage, faint neutrophil leucocyte, mesangial cell, mammary cell and the B cell of being expressed in, the activation of mediation lipopolysaccharides (lipopolysaccharide, LPS) to these cells.
Intracellular toxin is the integral part of Gram-negative bacteria epicyte, and its main component is lipopolysaccharides (LPS), once lipopolysaccharides enters in body, by the reaction of induction body generation inflammation medium, causes the major injury of body.In the inflammatory reaction process causing gram negative bacterium, lipopolysaccharide receptor plays keying action, is the important door of lipopolysaccharide.CD14 (CD14) is considered to most important lipopolysaccharide receptor in lipopolysaccharides signal transduction, and CD14 plays a crucial role in mediation lipopolysaccharides activates target cell and inflammatory reaction; Research shows, by suppressing CD14, to destroy the target cell inflammatory reaction that lipopolysaccharides activates be a feasible methods for the treatment of.
The RNA interference (RNA interference, RNAi) refer to by specific double stranded RNA (double stranded RNA, dsRNA) cause the Post-transcriptional gene silencing mechanism of homology messenger RNA(mRNA) (messenger RNA, mRNA) degraded.The specific double stranded RNA of this class is cracked into little (interference) ribonucleic acid molecule (siRNAs), and can cause homology messenger RNA(mRNA) (mRNA) degraded under the complex body of mourning in silence (RNA-induced silencing complex, the RISC) effect of Yeast Nucleic Acid induction.The RNA interference (RNAi, Ribonucleic acid interference) RNA is one of most effectual way of simply and efficiently blocking at present neural specific gene expression in mammalian cell, can reach gene knockout effect, and security is good.But for the RNA interference (RNAi), not all small ribonucleic acid molecules (siRNAs) for target gene messenger RNA(mRNA) (mRNA) sequence random synthesis can cause efficient gene silencing, and disturbing the selection of target sequence is vital on the impact of jamming effectiveness.Therefore to effectively suppressing the screening of the small ribonucleic acid molecules (siRNAs) of neural specific gene expression, be, the important foundation that application the RNA interference (RNAi) technology is carried out the correlative studys such as gene function, gene therapy.
Summary of the invention
The object of the invention is, a kind of small ribonucleic acid molecules that suppresses the genetic expression of buffalo film mating type CD14 is provided, by suppressing the expression of buffalo film mating type CD14 (mCD14), strengthen the ability of buffalo opposing gram positive bacterial infection.
Another object of the present invention is that a kind of biology screening method that suppresses the small ribonucleic acid molecules of buffalo film mating type CD14 genetic expression is provided.
The technical solution adopted in the present invention is, a kind of small ribonucleic acid molecules that suppresses the genetic expression of buffalo film mating type CD14, and described small ribonucleic acid molecules gene order information is:
Justice, CGAGGUAAAUGACC UGACUTT (5 '-3 ');
Antisense, AGUCAGGUCAUUUACCUCGTT (5 '-3 ')).
Another technical scheme of the present invention is, a kind of biology screening method that suppresses the small ribonucleic acid molecules of buffalo film mating type CD14 genetic expression, and the method is implemented according to following steps:
The structure of step 1, recombinant expression plasmid pEGFP-mCD14, concrete steps are as follows:
PEGFP-C1 expression vector is transformed to DH5a competent cell, getting 50 microlitre bacterium liquid coats on Luria-Bertani solid medium, containing kantlex, be inverted for 37 ℃ and cultivate 24 hours, picking list colony inoculation is in 3 milliliters of Luria-Bertani nutrient solutions, containing kantlex, 37 ℃ vibrate 24 hours, centrifugal 2 minutes of 12000 revolutions per minute, collect thalline, and plasmid extraction kit extracts pEGFP-C1 plasmid in a small amount; With EcoRI and Kpn1 double digestion, reclaim polymerase chain reaction product and the pEGFP-C1 expression vector of purifying, under the effect of T4DNA ligase enzyme, object fragment after enzyme is cut is connected with carrier for expression of eukaryon pEGFP-C1, finally obtains recombinant expression plasmid pEGFP-mCD14;
The structure of the human embryo kidney 293 cells system of step 2, stably express enhanced green fluorescence protein EGFP-buffalo film mating type CD14 fusion rotein, concrete steps are as follows:
(1) in the day before yesterday of transfection, counting human embryo kidney 293 cells is also laid in 6 orifice plates, and while making transfection, cell density reaches 60-80%;
(2) on transfection same day, with the optimization of 250 microlitre serum-frees, improve she Ge Shi perfect medium I and dilute respectively 2 microgram EGFP-mCD14 plasmids and 6 microlitre transfection reagents, mix gently;
(3) hatch after 5 minutes, the plasmid of dilution and transfection reagent are mixed, after slightly mixing, in incubated at room, within 20 minutes, be beneficial to form thymus nucleic acid DNA-transfection reagent mixture;
(4) thymus nucleic acid DNA-transfection reagent mixture is directly added containing in cell and 6 orifice plates without dual anti-substratum, cruciform jog is to mix;
After transfection 24 hours, changing perfect medium cultivates 24 hours, use 0.25% trypsin digestion and cell, by 1: the dilution proportion of 10-20 goes down to posterity, after cell attachment, in the selective medium that contains aminoglycoside antibiotics 500 mcg/ml, pressurization screening, obtains the positive cell clone with antibiotics resistance; Adopt Method of Limited Dilution method that clone is diluted to 96 orifice plates and carry out mono-clonal screening, again thereby the mono-clonal amplification cultivation filtering out is obtained to the human embryo kidney 293 cells of stably express enhanced green fluorescence protein EGFP-buffalo film mating type CD14 fusion rotein, stable cell lines is carried out to fluorescent microscope detection and Western Blot evaluation
By comparing, the human embryo kidney 293 cells correction enhanced green fluorescence protein EGFP-buffalo film mating type CD14 fusion rotein of stably express enhanced green fluorescence protein EGFP-buffalo film mating type CD14 fusion rotein;
According to the sequence of buffalo film mating type CD14 gene, according to the synthetic fundamental principle of small ribonucleic acid molecules siRNAs, synthetic four couples of different small ribonucleic acid molecules siRNAs for film mating type CD14;
(1) with 900 microlitre perfect mediums, 5 * 105 cells are laid in 12 orifice plates;
(2) with the Opti-MEM I substratum of 100 microlitre serum-frees, dilute small ribonucleic acid molecules siRNAs and the 9 microlitre high-efficiency transfection reagent transfection reagents that 9 microlitre concentration are 20 micro-molar concentrations respectively, both are mixed, fully mix, room temperature is placed 5-10 minute;
(3) transfection composite of formation is splashed in cell cultures hole, mix with human embryo kidney 293 cells stable cell lines, cruciform jog, to mix, is put into CO2gas incubator and is cultivated;
Cultivate after 24 hours for (4) 37 ℃, change fresh 700 microlitre Du Bicks containing 10% foetal calf serum and improve her Ge Shi perfect medium;
5.1) transfection is after 48 hours, with inverted fluorescence microscope, observe the fluorescence intensity of enhanced green fluorescence protein EGFP-buffalo film mating type CD14 expressing fusion protein, inverted fluorescence microscope, detect the protein expression situation of enhanced green fluorescence protein EGFP-buffalo film mating type CD14 fusion rotein
By comparing, siRNA4 small ribonucleic acid molecules wherein can obviously suppress the expression of enhanced green fluorescence protein EGFP-buffalo film mating type CD14 fusion rotein;
5.2) transfection is after 48 hours, the protein expression level of Flow cytometry enhanced green fluorescence protein GFP-buffalo film mating type CD14 fusion rotein, flow cytometer adopts excitation wavelength 408nm, emission wavelength 507nm, detect cell quantity and the average fluorescent strength of expressing EGFP
By comparing, siRNA4 small ribonucleic acid molecules siRNAs wherein can obviously suppress the expression of buffalo film mating type CD14;
5.3) transfection is after 48 hours, sucking-off nutrient solution, phosphate buffer wash cell layer 1-2 time, 0.125% the conventional digestion of pancreatin, collecting cell.According to the situation of cell precipitation, add cell pyrolysis liquid 50 millimolar concentration Tri(Hydroxymethyl) Amino Methane Hydrochlorides, pH is 8.0,150 nanomolar concentration sodium-chlor, the Triton X-100 of 1% millimolar concentration, 0.2% millimolar concentration sodium azide, 0.5% millimolar concentration Sodium desoxycholate, 10 mcg/ml Trypsin inhibitor,Trasylol aprotinin, 1 mcg/ml phenylmethylsulfonyl fluoride; Utilize two quinoline woods formic acid protein determination kits to measure total protein concentration, by total protein equivalent loading, under the concentration of the separation gel 12%, carry out polyacrylamide gel electrophoresis,
After electrophoresis, carry out western blotting, by comparing, siRNA4 small ribonucleic acid molecules siRNAs wherein can obviously suppress the expression of buffalo film mating type CD14,
In sum, find out that wherein significantly show can be special and efficiently suppress a small ribonucleic acid molecules siRNA of buffalo film mating type CD14 protein expression.
The invention has the beneficial effects as follows, set up the human embryo kidney 293 cells system (HEK293) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, synthetic different small ribonucleic acid molecules (the short interfering RNAs that disturbs buffalo film mating type CD14 (mCD14) genetic expression, and transfected with human embryonic kidney 293 clone stable cell lines siRNAs).By the fluorescence intensity of fluorescence microscope enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) expressing fusion protein, the protein expression level of Flow cytometry enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, western blotting (western blot) is identified its interference effect, filters out the small ribonucleic acid molecules (siRNA) that can efficiently suppress buffalo film mating type CD14 (mCD14) genetic expression.The molecular mechanism acting in immunity and inflammatory reaction for follow-up study buffalo film mating type CD14 (mCD14) and further application the RNA interference (RNAi) technology are by suppressing the expression of buffalo film mating type CD14 (mCD14), and the ability that strengthens buffalo opposing gram positive bacterial infection has been established important foundation.
Accompanying drawing explanation
Fig. 1 is the evaluation figure of the recombinant expression plasmid pEGFP-mCD14 in the inventive method, in figure, from left to right indicates respectively: M refers to (λ HindIII); Sequence number 1 and sequence number 2 refer to former plasmid pEGFP-CD14; Sequence number 3 refers to that pEGFP-CD14 is through EcoR I and Kpn1 double digestion; Sequence number 4 refers to that pEGFP-CD14 is through Kpn1 single endonuclease digestion; Sequence number 5 refers to that pEGFP-CD14 is through EcoR I single endonuclease digestion; Sequence number 6 refers to that pEGFP-CD14 is through BamH1 single endonuclease digestion 2h; Sequence number 7 refers to that pEGFP-CD14 is through BamH1 single endonuclease digestion 2.5h;
Fig. 2 is the structure schematic diagram of the human embryo kidney 293 cells system of the stably express buffalo film mating type CD14 in the inventive method, and wherein scheming a is the fluorescence microscope photo of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP); Figure b is the fluorescence microscope photo of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein; Figure c analyzes the Western blot of the HEK293 clone of set up stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Fig. 3 is the fluorescence intensity figure of enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) expressing fusion protein after the fluorescence microscope transfection small ribonucleic acid molecules (siRNAs) in the inventive method
Wherein,
Figure a is the fluorescence microscope photo of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure b is the fluorescence microscope photo after the transfected invalid siRNA of HEK293 clone (scramble siRNA) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure c is the fluorescence microscope photo after the siRNA (siRNA EGFP) that expresses of the transfected inhibition enhanced green fluorescence protein of HEK293 clone (EGFP) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure d is the fluorescence microscope photo after the transfected siRNA195 of HEK293 clone (siRNA1) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure e is the fluorescence intensity analysis after the transfected siRNA 664 of HEK293 clone (siRNA2) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure f is the fluorescence intensity analysis after the transfected siRNA902 of HEK293 clone (siRNA3) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure g is the fluorescence intensity analysis after the transfected siRNA949 of HEK293 clone (siRNA4) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
The protein expression level figure of enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein after Fig. 4 Flow cytometry transfection small ribonucleic acid molecules (siRNAs), wherein,
Figure a is the fluorescence intensity analysis of HEK293 clone;
Figure b is the fluorescence intensity analysis of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure c be after the transfected invalid siRNA of HEK293 clone (Scramble siRNA) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein fluorescence intensity analysis;
Figure d is the fluorescence intensity analysis after the siRNA (siRNA EGFP) that expresses of the transfected inhibition enhanced green fluorescence protein of HEK293 clone (EGFP) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure e is the fluorescence intensity analysis after the transfected siRNA195 of HEK293 clone (siRNA1) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure f is the fluorescence intensity analysis after the transfected siRNA664 of HEK293 clone (siRNA2) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure g is the fluorescence intensity analysis after the transfected siRNA902 of HEK293 clone (siRNA3) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure h is the fluorescence intensity analysis after the transfected siRNA944 of HEK293 clone (siRNA4) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Fig. 5 is the result of the expression level of western blotting (western blot) enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein of analyzing transfection small ribonucleic acid molecules (siRNAs) stable cell lines.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.
The present invention is according to a kind of buffalo film mating type CD14 (mCD14) complementary DNA (cDNA) molecule (cDNA) sequence, and the gene order of this complementary RNA molecule (shorthand) is:
1 atggtgtgcg tgccctacct gttgctgctg ctgctgccgc cactgctgcg tgtgtctgcg
61 gacacaacag aaccctgcga gctggacgac gacgatttcc gttgtgtctg caacttcacg
121 gatccgaagc ctgactggtc tagcgccgtt cagtgtatgg ttgccgtaga ggtggagatc
181 agtggcggcg gccgcagcct ggaacagttt ctcaagggag ccgacaccaa cccgaagcag
241 tatgctgaca caatcaaggc tctgcgcgtt cggcgactca agctgggcgc tgcacaggtt
301 cctgctcagc ttctggtcgc cgttctgcgc gcgctcgggt actctcgtct caaggaacta
361 acgcttgagg acctggaagt aaccggccca atgcccccga agcctctgga agccactggg
421 cctgcgttca ccaccctcag tctccgtaac gtatcgtggg caacaggagg tgcctggctc
481 ggcgaactgc agcagtggct caagcctggg ctcagggtgc tgaacattgc ccaagcacac
541 tcgcttgccc ttccgtgcgc agggctctcc accttcgagg cgctcaccac cctagacctg
601 tctgacaatc ccagtctcgg cgacagcggg ctgatggcag ctctctgtcc gaacaagttc
661 ccggccctcc aatatctagc gctacgcaac gccgggatgg agacgctgag cggagtgtgc
721 gcggcgctgg cggcagcgag ggtgcagccc caaagcctgg acctcagcca caactcgctg
781 cgcgtcaccg ccctgggcgc tacccgatgt gtctggccca gtgcactaag ctctctgaat
841 ttgtcgttcg ctgggctgga gcaagtgcct aagggactgc cccccaagct cagcgtgctt
901 gatctcagct gcaacaagct aagcagggag ccgcggcgag acgagctgcc cgaggtaaat
961 gacctgactc tggatggaaa tccctttctg gaccctggag ccctccagca ccaaaatgac
1021 ccgatgatct ccggcgtggt cccagcctgt gcgcgttctg ccttgaccat gggggtgtca
1081 ggagccctgg cgctgcttca aggagcccga ggcttcgcgt aa
The above-mentioned small ribonucleic acid molecules (siRNAs) that can suppress the genetic expression of buffalo film mating type CD14 of the present invention, its biology screening principle is first to set up the human embryo kidney 293 cells system (HEK293) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein; Then according to the sequence of buffalo film mating type CD14 (mCD14) gene, synthetic four pairs of different small ribonucleic acid molecules (siRNAs) for film mating type CD14 (mCD14), application high-efficiency transfection reagent (HiPerFect Transfection Reagent) are by this this stable cell lines of four pairs of small ribonucleic acid molecules (siRNAs) transfection; Again, by the expression of fluorescence microscope enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, the protein expression level of Flow cytometry enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, western blotting (western blot) is identified its interference effect, filters out the small ribonucleic acid molecules (siRNA) that can efficiently suppress buffalo film mating type CD14 (mCD14) genetic expression; Finally, comprehensive these data, relatively find that wherein one (the 4th No. 949) small ribonucleic acid molecules (siRNAs) can obviously suppress the expression of buffalo film mating type CD14 (mCD14).
The structure of step 1, recombinant expression plasmid pEGFP-mCD14, concrete steps are as follows:
PEGFP-C1 expression vector is transformed to DH5a competent cell, get 50 microlitres (μ l) bacterium liquid and coat (containing kantlex) on Luria-Bertani solid medium, be inverted for 37 ℃ and cultivate 24 hours, picking list colony inoculation is (containing kantlex) in 3 milliliters of (ml) Luria-Bertani nutrient solutions, 37 ℃ vibrate 24 hours, centrifugal 2 minutes of 12000 revolutions per minute (rpm), collect thalline, and plasmid extraction kit extracts pEGFP-C1 plasmid in a small amount; With EcoRI and Kpn1 double digestion, reclaim polymerase chain reaction (PCR) product and the pEGFP-C1 expression vector of purifying, under the effect of T4DNA ligase enzyme, object fragment after enzyme is cut (mCD14 Insert Fragment) is connected with carrier for expression of eukaryon pEGFP-C1, the final recombinant expression plasmid pEGFP-mCD14 that obtains, enzyme is cut evaluation and is seen Fig. 1, analysis situation in Fig. 1 shows, pEGFP-mCD14 builds correct.
The structure of the human embryo kidney 293 cells system (HEK293) of step 2, stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, concrete steps are as follows:
(1) in the day before yesterday of transfection, counting human embryo kidney 293 cells (HEK293) is also laid in 6 orifice plates, and while making transfection, cell density reaches 60-80%;
(2) on transfection same day, with the optimization of 250 microlitres (μ l) serum-free, improveing she Ge Shi perfect medium I (Opti-MEM I) dilutes respectively 2 micrograms (μ g) EGFP-mCD14 plasmid and 6 microlitres (μ l) transfection reagent (LipofectamineTM 2000, Invitrogen, USA), mix gently;
(3) hatch after 5 minutes, by plasmid and the transfection reagent (LipofectamineTM2000 of dilution, Invitrogen, USA) mix, (LipofectamineTM 2000 after slightly mixing, in incubated at room, within 20 minutes, to be beneficial to form thymus nucleic acid (DNA)-transfection reagent, Invitrogen, USA) mixture;
(4) thymus nucleic acid (DNA)-transfection reagent (LipofectamineTM2000, Invitrogen, USA) mixture is directly added containing in cell and 6 orifice plates without dual anti-substratum, cruciform jog is to mix.
After transfection 24 hours, changing perfect medium cultivates 24 hours, use 0.25% trypsin digestion and cell, by 1: the dilution proportion of 10-20 goes down to posterity, after cell attachment, in the selective medium that contains aminoglycoside antibiotics (G418,500 mcg/ml), pressurization screening, obtains the positive cell clone with antibiotics resistance, adopt Method of Limited Dilution method that clone is diluted to 96 orifice plates and carry out mono-clonal screening, again thereby the mono-clonal amplification cultivation filtering out is obtained to the human embryo kidney 293 cells (HEK293) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, stable cell lines is carried out to fluorescent microscope detection and Western Blot evaluation, the results are shown in Figure 2 each components, analysis situation in Fig. 2 shows, human embryo kidney 293 cells (HEK293) correction enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, contrast no, illustrate that stable cell lines builds correct.
In Fig. 2, figure a is the fluorescence microscope photo of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP); Figure b is the fluorescence microscope photo of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein; Figure c analyzes the Western blot of the HEK293 clone of set up stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein.
According to the sequence of buffalo film mating type CD14 (mCD14) gene, according to following fundamental principle, synthesize small ribonucleic acid molecules (siRNAs):
1) GC content 30%-52%;
2) in the 15-19 base of sense chain, at least contain 3 A/U;
3) do not contain inverted repeats;
4) the 19th base of sense chain is A;
5) the 3rd base of sense chain is A;
6) the 10th base of sense chain is U;
7) the 19th base of sense chain is not G/C;
8) the 13rd base of sense chain is not G,
According to mentioned above principle, in the embodiment of the present invention, four pairs of different small ribonucleic acid molecules (siRNAs) for film mating type CD14 (mCD14) have been synthesized, by Shanghai JiMa pharmacy Technology Co., Ltd, synthesized, the storage conditions of this small ribonucleic acid molecules (siRNAs) is, be placed in diethylpyrocarbonate (DEPC, diethypyrocarbonate) in DEPC water,-20 ℃ of preservations, table 1 is for synthetic small ribonucleic acid molecules (siRNAs) sequence of buffalo film mating type CD14 (mCD14)
Four pairs of small ribonucleic acid molecules (siRNAs) sequence that table 1 embodiment of the present invention is synthetic
(1) with 900 microlitres (μ l) perfect medium, 5 * 105 cells are laid in 12 orifice plates;
(2) use respectively the Opti-MEM I substratum of 100 microlitres (μ l) serum-free to dilute small ribonucleic acid molecules (siRNAs) and 9 microlitres (μ l) high-efficiency transfection reagent (HiPerfect) transfection reagent that 9 microlitres (μ l) concentration is 20 micro-molar concentrations (μ M), both are mixed, fully mix, room temperature is placed 5-10 minute;
(3) transfection composite of formation is splashed in cell cultures hole, mix with human embryo kidney 293 cells (HEK293) stable cell lines, cruciform jog, to mix, is put into CO2gas incubator and is cultivated;
Cultivate after 24 hours for (4) 37 ℃, change fresh Du of 700 microlitres (μ l) containing 10% foetal calf serum Bick and improve her Ge Shi perfect medium (Dulbecco ' s Modified Eagle ' s Medium, DMEM);
5.1) transfection is after 48 hours, with inverted fluorescence microscope, observe the fluorescence intensity of enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) expressing fusion protein, inverted fluorescence microscope, model is: Olympus X71, Japan, detect the protein expression situation of enhanced green fluorescence protein (EGFP) buffalo film mating type CD14 (mCD14) fusion rotein, the results are shown in Figure 3 each components, fluorescence microscope shows, the fluorescence intensity of enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein of the 4th (No. 949) small ribonucleic acid molecules of transfection (siRNAs) stable cell lines is suppressed.
The demonstration of each component in Fig. 3 shows:
Figure a is the fluorescence microscope photo of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure b is the fluorescence microscope photo after the transfected invalid siRNA of HEK293 clone (scramble siRNA) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure c is the fluorescence microscope photo after the siRNA (siRNA EGFP) that expresses of the transfected inhibition enhanced green fluorescence protein of HEK293 clone (EGFP) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure d is the fluorescence microscope photo after the transfected siRNA195 of HEK293 clone (siRNA1) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure e is the fluorescence intensity analysis after the transfected siRNA 664 of HEK293 clone (siRNA2) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure f is the fluorescence intensity analysis after the transfected siRNA902 of HEK293 clone (siRNA3) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure g is the fluorescence intensity analysis after the transfected siRNA949 of HEK293 clone (siRNA4) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein.
Above data declaration, siRNA949 (siRNA4) small ribonucleic acid molecules (siRNAs) can obviously suppress the expression of enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein.
5.2) transfection is after 48 hours, the protein expression level of Flow cytometry enhanced green fluorescence protein (GFP)-buffalo film mating type CD14 (mCD14) fusion rotein.Flow cytometer adopts excitation wavelength 408nm, and emission wavelength 507nm detects cell quantity and the average fluorescent strength of expressing EGFP.The results are shown in Figure 4 each components, the Flow cytometry in Fig. 4 shows, cell quantity and the average fluorescent strength of the expression EGFP of the 4th (No. 949) small ribonucleic acid molecules of transfection (siRNAs) stable cell lines are suppressed.The demonstration of each component in Fig. 4 is respectively:
Figure a is the fluorescence intensity analysis of HEK293 clone;
Figure b is the fluorescence intensity analysis of the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure c be after the transfected invalid siRNA of HEK293 clone (Scramble siRNA) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein fluorescence intensity analysis;
Figure d is the fluorescence intensity analysis after the siRNA (siRNA EGFP) that expresses of the transfected inhibition enhanced green fluorescence protein of HEK293 clone (EGFP) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure e is the fluorescence intensity analysis after the transfected siRNA195 of HEK293 clone (siRNA1) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure f is the fluorescence intensity analysis after the transfected siRNA664 of HEK293 clone (siRNA2) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure g is the fluorescence intensity analysis after the transfected siRNA902 of HEK293 clone (siRNA3) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
Figure h is the fluorescence intensity analysis after the transfected siRNA944 of HEK293 clone (siRNA4) of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein.
Above data declaration, siRNA949 (siRNA4) small ribonucleic acid molecules (siRNAs) can obviously suppress the expression of buffalo film mating type CD14 (mCD14).
In each figure in Fig. 4: the fluorescence intensity of X-axis for measuring, Y-axis is cell number,
FL1, the green intensity within the scope of 515-545nm;
Counts, cell count.
5.3) transfection is after 48 hours, sucking-off nutrient solution, phosphate buffered saline buffer (PBS) washed cell layer 1-2 time, 0.125% the conventional digestion of pancreatin, collecting cell.According to the situation of cell precipitation, add cell pyrolysis liquid [50 millimolar concentrations (mM) Tri(Hydroxymethyl) Amino Methane Hydrochlorides (Tris-HCL) (pH is 8.0), 150 nanomolar concentrations (nM) sodium-chlor (NaCl), 1% millimolar concentration Triton X-100 (TritonX-100), 0.2% millimolar concentration sodium azide (Sodium azide), 0.5% millimolar concentration Sodium desoxycholate (Sodium deoxycholate sodium deoxycholate), 10 mcg/ml (μ g/ml) Trypsin inhibitor,Trasylol aprotinin, 1 mcg/ml (μ g/ml) phenylmethylsulfonyl fluoride (phenylmethanesulfonyl fluoride).Utilize two quinoline woods formic acid (bicinchoninic acid, BCA) protein determination kits to measure total protein concentration, by total protein equivalent loading, under the concentration of the separation gel 12%, carry out polyacrylamide gel electrophoresis (SDS-PAGE),
After electrophoresis, carry out western blotting (western blot), main antibody, primary antibodie is: the anti-film mating type of rabbit CD14 (mCD14) polyclonal antibody (rabbit polyclonal anti-mCD14,1: 1000 times of dilution), the anti-glyceraldehyde-3-phosphate dehydrogenase of rabbit (GAPDH) monoclonal antibody (GAPDH rabbit mAB, 1: 1000 times of dilution); Two resist and are: horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (HR labeled goat anti-rabbit IgG, 1: 6000 times of dilution).The results are shown in Figure 5, western blotting in Fig. 5 (western blot) result shows, enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 of the 4th (No. 949) small ribonucleic acid molecules of transfection (siRNAs) stable cell lines (membrane bound cluster of differentiation antigen, mCD14) expression level of fusion rotein is suppressed, description of contents in Fig. 5, siRNA949 (siRNA4) small ribonucleic acid molecules (siRNAs) can obviously suppress the expression of buffalo film mating type CD14 (mCD14).
Sign explanation in Fig. 5:
PEGFP-CD14: the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, untransfected,
Scramble siRNA: the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, the invalid siRNA of transfection,
EGFP-siRNA: the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, transfection suppresses the siRNA (siRNA EGFP) that enhanced green fluorescence protein (EGFP) is expressed
CD14-siRNA1: the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, transfection siRNA195 (siRNA1),
CD14-siRNA2: the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, transfection siRNA664 (siRNA2),
CD14-siRNA3: the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, transfection siRNA902 (siRNA3),
CD14-siRNA4: the HEK293 clone of stably express enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein, transfection siRNA944 (siRNA4),
HEK293:HEK293 clone, untransfected.
EGFP-CD14: enhanced green fluorescence protein (EGFP)-buffalo film mating type CD14 (mCD14) fusion rotein;
GAPDH: the expression of the anti-glyceraldehyde-3-phosphate dehydrogenase of rabbit.
In sum, the inventive method has been set up the human embryo kidney 293 cells system (HEK293) of stably express buffalo film mating type CD14 (mCD14); Small ribonucleic acid molecules (siRNAs) by synthetic four couple for buffalo film mating type CD14 (mCD14) gene, this stable cell lines of transfection, therefrom filter out remarkable energy special and efficiently suppress buffalo film mating type CD14 (mCD14) protein expression the 4th to (949#) small ribonucleic acid molecules (siRNAs), its gene order information is:
Justice, CGAGGUAAAUGACC UGACUTT (5 '-3 ');
Antisense, AGUCAGGUCAUUUACCUCGTT (5 '-3 ')).
Adopt the inventive method to screen small ribonucleic acid molecules (siRNAs), the molecular mechanism acting in immunity and inflammatory reaction for follow-up study mating type CD14 (mCD14), by suppressing the expression of buffalo film mating type CD14 (mCD14), the ability that strengthens buffalo opposing gram positive bacterial infection has been established important foundation with further application the RNA interference (RNAi) technology.
Appendix 1:
Buffalo film mating type CD14 (mCD14) complementary DNA (cDNA) molecule (cDNA) the sequence information computer-readable version of institute of the present invention foundation is:
<110> king Fengyang
<120> buffalo film mating type CD14 gene C DS
<160>1
<210>1
<211>1122
<212>DNA
<213> buffalo
<400>1
atggtgtgcg tgccctacct gttgctgctg ctgctgccgc cactgctgcg tgtgtctgcg 60
gacacaacag aaccctgcga gctggacgac gacgatttcc gttgtgtctg caacttcacg 120
gatccgaagc ctgactggtc tagcgccgtt cagtgtatgg ttgccgtaga ggtggagatc 180
agtggcggcg gccgcagcct ggaacagttt ctcaagggag ccgacaccaa cccgaagcag 240
tatgctgaca caatcaaggc tctgcgcgtt cggcgactca agctgggcgc tgcacaggtt 300
cctgctcagc ttctggtcgc cgttctgcgc gcgctcgggt actctcgtct caaggaacta 360
acgcttgagg acctggaagt aaccggccca atgcccccga agcctctgga agccactggg 420
cctgcgttca ccaccctcag tctccgtaac gtatcgtggg caacaggagg tgcctggctc 480
ggcgaactgc agcagtggct caagcctggg ctcagggtgc tgaacattgc ccaagcacac 540
tcgcttgccc ttccgtgcgc agggctctcc accttcgagg cgctcaccac cctagacctg 600
tctgacaatc ccagtctcgg cgacagcggg ctgatggcag ctctctgtcc gaacaagttc 660
ccggccctcc aatatctagc gctacgcaac gccgggatgg agacgctgag cggagtgtgc 720
gcggcgctgg cggcagcgag ggtgcagccc caaagcctgg acctcagcca caactcgctg 780
cgcgtcaccg ccctgggcgc tacccgatgt gtctggccca gtgcactaag ctctctgaat 840
ttgtcgttcg ctgggctgga gcaagtgcct aagggactgc cccccaagct cagcgtgctt 900
gatctcagct gcaacaagct aagcagggag ccgcggcgag acgagctgcc cgaggtaaat 960
gacctgactc tggatggaaa tccctttctg gaccctggag ccctccagca ccaaaatgac 1020
ccgatgatct ccggcgtggt cccagcctgt gcgcgttctg ccttgaccat gggggtgtca 1080
ggagccctgg cgctgcttca aggagcccga ggcttcgcgt aa 1122
Appendix 2:
The small ribonucleic acid molecules sequence information computer-readable version of inhibition buffalo film mating type CD14 of the present invention genetic expression is:
<110> king Fengyang
<120> suppresses the small ribonucleic acid molecules of buffalo film mating type CD14 genetic expression
<160>1
<210>1
<211>21
<212>RNA
<213> buffalo
<400>1
cgagguaaau gaccugacut t 21
Claims (2)
1. a small ribonucleic acid molecules that suppresses the genetic expression of buffalo film mating type CD14, is characterized in that, described small ribonucleic acid molecules gene order information is:
Positive-sense strand: 5 '-CGAGGUAAAUGACCUGACUTT-3 ';
Antisense strand: 5 '-AGUCAGGUCAUUUACCUCGTT-3 '.
2. the purposes of small ribonucleic acid molecules claimed in claim 1 in suppressing the genetic expression of buffalo film mating type CD14, it is characterized in that, this small ribonucleic acid molecules is applied to the RNA interference technology, energy is special and efficiently suppress buffalo film mating type CD14 protein expression, strengthens the ability of buffalo opposing gram positive bacterial infection.
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| 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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