CN101781680B - Method for measuring influencing factors to BAFF-R (B cell-Activating Factor-Receptor) gene promoter activity of human - Google Patents
Method for measuring influencing factors to BAFF-R (B cell-Activating Factor-Receptor) gene promoter activity of human Download PDFInfo
- Publication number
- CN101781680B CN101781680B CN 200910213158 CN200910213158A CN101781680B CN 101781680 B CN101781680 B CN 101781680B CN 200910213158 CN200910213158 CN 200910213158 CN 200910213158 A CN200910213158 A CN 200910213158A CN 101781680 B CN101781680 B CN 101781680B
- Authority
- CN
- China
- Prior art keywords
- pgl3
- baff
- cell
- group
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for measuring influencing factors to BAFF-R (B cell-Activating Factor-Receptor) gene promoter activity of a human. The method comprises the following steps of: primer design, cloning of BAFF-R gene 5' flanking region, construction of BAFF-R gene 5' flanking region sequence loss plasmid, cell culture, interference of an IFN-gamma and NF-kappaB inhibitor to cell proliferation, transfection of a recombinant, detection of luciferase activity, interference of the IFN-gamma and NF-kappaB inhibitor to the BAFF-R promoter activity and the mRNA expression and the like. The method is reliable and easy to operate and provides an essential basis for further studying a regulatory mechanism of the BAFF-R.
Description
Technical field:
The present invention relates to the method for a kind of mensuration to the activity of human BAFF-R gene promoter influence factor.
Background technology:
BAFF-R is the specific receptors of BAFF (B cell activating factor), can combine with the BAFF specificity, and BAFF/BAFF-R forms the homeostasis that mixture is regulated the B cell, participates in the adjusting of B, the lymphocytic propagation of T and function.Research confirms, BAFF-R and part BAFF mRNA thereof and the equal abnormal expression of protein level in the cell-associated disease of B.BAFF-R is the specific receptors of BAFF, in BAFF performance biological function process, plays very vital role, regulates propagation, the survival of bone-marrow-derived lymphocyte simultaneously, keeps the stable state of bone-marrow-derived lymphocyte self environment.BAFF with can activate classical and non-classical NF-κ B signal path after BAFF-R combines, BAFF-R can promote normal B cell and NHL-B survival, propagation, this effect of BAFF-R and NF-κ B signal are by way of relevant.But what influence IFN-γ and NF-κ B have also do not see relevant report for the expression of BAFF-R.
Summary of the invention:
The object of the present invention is to provide the method for reliable, the easy-operating mensuration of a kind of method to the activity of human BAFF-R gene promoter influence factor.
Technical solution of the present invention is:
A kind of method of measuring the activity of human BAFF-R gene promoter influence factor is characterized in that: comprise the following steps:
(1) design of primers:
Press the imbrication mode at eight segmental primers of BAFF-R gene 5 ' flanking sequence design with primer-design software; Eight pairs of primers have identical downstream primer; 3 ' end of primer adds restriction enzyme HindIII site; 5 ' end adds restriction enzyme Mlu I site, adds 6 protection bases simultaneously respectively at two ends, is convenient to enzyme and cuts the MCS that is inserted into carrier; Eight segmental primer sequences and downstream primer sequence are:
Wherein 1-8 is eight segmental upstream primer sequences of different lengths, and 9 is eight fragments, 3 ' end common downstream primer sequences; Above-mentioned primer is as amplification corresponding product B 1 (549bp), B2 (691bp); The primer of B3 (878bp), B4 (973bp), B5 (1129bp), B6 (1538bp), B7 (1681bp), B8 (1823bp) (being B1, B2, B3, B4, B5, B6, B7, B8), B1～B8 are corresponding with above-mentioned primer 1～8 respectively;
(2) clone of BAFF-R gene 5 ' flanking region:
From people's whole blood, extract genomic dna as template, use LATaq enzyme pcr amplification purpose sheet degree B8 (1823bp);
Cut pcr amplification product B8 and pGL3-Basic carrier after glue reclaims purifying with restriction enzyme Mlu I, Hind III enzyme, enzyme is cut product through T
4Ligase enzyme connects, and connects mix products and transforms escherichia coli DH5 α, and enzyme is cut order-checking and identified positive colony, obtains positive recombinant plasmid, called after pGL3-B8, i.e. pGL3-B8 (1562～+ 261);
(3) structure of BAFF-R gene 5 ' flanking region sequence deletion plasmid:
With recombinant plasmid pGL3-B8 is template; Pcr amplification purpose fragment B1-B7; Method with making up recombinant plasmid pGL3-B8 is the same; Obtain 7 recombinant plasmids, respectively called after pGL3-B1, pGL3-B2, pGL3-B3, pGL3-B4, pGL3-B5, pGL3-B6, pGL3-B7 [being pGL3-B1 (288～+ 261), pGL3-B2 (430～+ 261), pGL3-B3 (617～+ 261), pGL3-B4 (712～+ 261), pGL3-B5 (868～+ 261), pGL3-B6 (1277～+ 261), pGL3-B7 (1420～+ 261)];
(4) cell cultures:
People's multiple myeloma cell line KM3 suspension growth contains 100U/ml penicillium mould, 100U/ml Streptomycin sulphate in the substratum in the RPMI1640 substratum that contains 10% foetal calf serum, place 5%CO
2, 37 ℃ of constant temperature culture, 1～2d goes down to posterity once;
(5) intervention of IFN-γ and NF-kB inhibitor on cell proliferation:
The KM3 cell adjustment concentration of cultivating going down to posterity is 2 * 10
9/ L, every hole 100 μ L are inoculated in 96 well culture plates, and after the overnight cultures, IFN-γ and the BAY11-7082 with different concns intervenes respectively, and every kind of concentration repeats 3 holes, cultivates to add WST reagent 10 μ L behind the 24h again, measures the A450 value in each hole behind the 1h;
(6) recombinant chou transfection:
The KM3 cell is divided into 11 groups: (1) pGL3-B1 group; (2) pGL3-B2 group; (3) pGL3-B3 group; (4) pGL3-B4 group; (5) pGL3-B5 group; (6) pGL3-B6 group; (7) pGL3-B7 group; (8) pGL3-B8 group; (9) pGL3-Control group; (10) pGL3-Basic group; (11) plasmid-free blank group; Each the group all with psv-β-gal plasmid as internal reference; With the eight kinds of recombinant plasmids of pGL3-B1, pGL3-B2, pGL3-B, pGL3-B4, pGL3-B, pGL3-B6, pGL3-B7, pGL3-B8 and the negative control pGL3-Basic that make up, positive control pGL3-Control, transfection KM3 cell respectively; While cotransfection psv-β-gal confidential reference items plasmid, any plasmid of not transfection of blank group;
(7) to each group in the step (6), each group is collected respectively and lysing cell behind the transfection 48h, carries out uciferase activity and detects;
(8) IFN-γ and NF-kB inhibitor intervention that BAFF-R promoter activity and mRNA are expressed: repeating step (6); And respectively organizing in the step (6) added IFN-γ and BAY11-7082 behind the recombinant plasmid transfection 24h in cell culture fluid; After continuing culturing cell 24h; Each group is collected respectively and lysing cell, detects the relative reactivity of luciferase and the expression level that the RT-PCR method detects BAFF-R mRNA.
Lysing cell in the step (7), (8), carry out the concrete grammar that uciferase activity detects and be: 1 * PBS washed cell 2 times; Add 200 μ l, 1 * lysate; Freeze thawing once back moves in the 1.5mlEppendorf pipe 12000g/min, 4 ℃ of centrifugal 2min; Get 20 μ l cracking supernatants, detect uciferase activity with Chemiluminescence Apparatus; Get 50 μ l cracking supernatants, add 50 μ l beta-galactosidase enzymes detection reagent, ELIASA is measured the activity of beta-galactosidase enzymes; The ratio of uciferase activity and betagalactosidase activity is the relative reactivity of luciferase.
Primer-design software is Primer premier5.0.
The PCR reaction conditions is 94 ℃ in the step (2), 1.5min; 94 ℃, 39S; 56 ℃, 45S, 72 ℃, 1min, 35 circulations; Last 72 ℃ are extended 5min; The PCR product is through 1% agarose gel electrophoresis, and ethidium bromide staining is also taken a picture and identified.
Ratio in the step (6) between lipofectamine and the plasmid is 3 μ l: 4 μ g, the ratio of plasmid to be measured and internal reference plasmid is 4: 1.
The method of the expression level of RT-PCR method detection BAFF-R mRNA is in the step (8): collect the KM3 cell that receives IFN-γ and BAY11-7082 effect front and back respectively; Trizol extracts total RNA; And carry out quantitatively with ultraviolet spectrophotometer; Obtain cDNA with RNA rt test kit rt, pcr amplification purpose band is with β
2M is as internal reference; The used upstream primer of amplification BAFF-R: 5 '-GGAAGACCCAGGAACCAC-3 ', downstream primer: 5 '-TTTGGTGTGCTTGCCTT-3 ' is (297bp); Amplification β
2The upstream primer that M is used: 5 '-CTATCCAGCGTACTCCAA-3 ', downstream primer: 5 '-GCAGGCATACTCATCTTTT-3 ' (242bp).
The inventive method is reliable, easy to operate.Fragment with the human BAFF-R gene upper reaches 5 ' flanking region-1562～+ 261 bp different lengthss is connected 8 recombinant chous of structure as promotor with luciferase reporter gene carrier pGL3-Basic; With recombinant chou transfection people multiple myeloma cell line KM3; Through detecting the relative reactivity of luciferase, confirm the zone that promoter activity is the strongest.After containing the active recombinant chou transfection of strong promoter KM3, use IFN-γ (10 μ g/L), BAY11-7082 (1 μ mol/L) to intervene respectively; Through detecting the relative reactivity and the BAFF-R mRNA change of Expression of luciferase, inquire into IFN-γ and BAY11-7082 influence to the BAFF-R genetic transcription.The result shows that promoter activity zone is-1420～+ 261, explains in this zone to have cis-acting elements, makes the activity of promotor apparently higher than other regional promotors.And possibly also have relevant negative regulatory sequence in other regional promoter sequences, make the active relatively low of promotor.IFN-γ has promoter action to the BAFF-R gene promoter activity and can raise the expression of BAFF-R mRNA, the expression that NF-kB inhibitor (BAY11-7082) has restraining effect and can reduce BAFF-R mRNA the BAFF-R gene promoter activity.Show that IFN-γ and NF-κ B signal path participated in BAFF-R gene transcription regulation and control, these results provide basic foundation for the further regulation mechanism of research BAFF-R.BAFF-R with can activate NF-κ B signal path after BAFF combines, the NF-kB inhibitor can influence the expression of promoter activity and the mRNA thereof of BAFF-R again.NF-κ B can regulate the expression of BAFF; And a certain amount of BAFF also can regulate the activity of NF-κ B signal path; This explains to interknit between each factor in organism and conditions each other, has constituted complicated endless chain between them, thereby kept the stable state of organismic internal environment jointly.
Description of drawings:
Below in conjunction with accompanying drawing and embodiment the present invention is described further.
Fig. 1 is that the enzyme of recombinant plasmid pGL3-B1～B8 is cut the qualification result diagrammatic sketch.
Fig. 2 is different concns IFN-γ and the BAY11-7082 diagrammatic sketch that influences to KM3 cell proliferation.
Fig. 3 is the relative reactivity diagrammatic sketch of luciferase behind the different recombinant plasmid transfection KM3 cells.
Fig. 4 is IFN-γ and the BAY11-7082 diagrammatic sketch that influences to the BAFF-R gene promoter activity.
Fig. 5 is IFN-γ and the BAY11-7082 diagrammatic sketch that influences to the BAFF-R gene mRNA expression.
A:pGL3-B1 among Fig. 3 (288～+ 261); B:pGL3-B2 (430～+ 261); C:pGL3-B3 (617～+ 261); D:pGL3-B4 (712～+ 261); E:pGL3-B5 (868～+ 261); F:pGL3-B6 (1277～+ 261); G:pGL3-B7 (1420～+ 261); H:pGL3-B8 (1562～+ 261); I:pGL3-Basic (
aP＜0.05).
A:pGL3-Basic among Fig. 4; B: negative control; C:IFN-γ; D:BAY11-7082.
Embodiment:
A kind of method of measuring the activity of human BAFF-R gene promoter influence factor comprises the following steps: (1) design of primers:
Press the imbrication mode at eight segmental primers of BAFF-R gene 5 ' flanking sequence design with primer-design software; Eight pairs of primers have identical downstream primer; 3 ' end of primer adds restriction enzyme HindIII site; 5 ' end adds restriction enzyme Mlu I site, adds 6 protection bases simultaneously respectively at two ends, is convenient to enzyme and cuts the MCS that is inserted into carrier; Eight segmental primer sequences and downstream primer sequence are:
Wherein 1-8 is eight segmental upstream primer sequences of different lengths, and 9 is eight fragments, 3 ' end common downstream primer sequences; Above-mentioned primer is as amplification corresponding product B 1 (549bp), B2 (691bp); The primer of B3 (878bp), B4 (973bp), B5 (1129bp), B6 (1538bp), B7 (1681bp), B8 (1823bp) (being B 1, B2, B3, B4, B5, B6, B7, B8), B1～B8 are corresponding with above-mentioned primer 1～8 respectively;
(2) clone of BAFF-R gene 5 ' flanking region:
From people's whole blood, extract genomic dna as template, use LATaq enzyme pcr amplification purpose sheet degree B8 (1823bp);
Cut pcr amplification product B8 and pGL3-Basic carrier after glue reclaims purifying with restriction enzyme Mlu I, Hind III enzyme, enzyme is cut product through T
4Ligase enzyme connects, and connects mix products and transforms escherichia coli DH5 α, and enzyme is cut order-checking and identified positive colony, obtains positive recombinant plasmid, called after pGL3-B8, i.e. pGL3-B8 (1562～+ 261);
(3) structure of BAFF-R gene 5 ' flanking region sequence deletion plasmid:
With recombinant plasmid pGL3-B8 is template; Pcr amplification purpose fragment B1-B7; Method with making up recombinant plasmid pGL3-B8 is the same; Obtain 7 recombinant plasmids, respectively called after pGL3-B1, pGL3-B2, pGL3-B3, pGL3-B4, pGL3-B5, pGL3-B6, pGL3-B7 [being pGL3-B1 (288～+ 261), pGL3-B2 (430～+ 261), pGL3-B3 (617～+ 261), pGL3-B4 (712～+ 261), pGL3-B5 (868～+ 261), pGL3-B6 (1277～+ 261), pGL3-B7 (1420～+ 261)];
(4) cell cultures:
People's multiple myeloma cell line KM3 suspension growth contains 100U/ml penicillium mould, 100U/ml Streptomycin sulphate in the substratum in the RPMI1640 substratum that contains 10% foetal calf serum, place 5%CO
2, 37 ℃ of constant temperature culture, 1～2d goes down to posterity once;
(5) intervention of IFN-γ and NF-kB inhibitor on cell proliferation:
The KM3 cell adjustment concentration of cultivating going down to posterity is 2 * 10
9/ L; Every hole 100 μ L are inoculated in 96 well culture plates, and after the overnight cultures, IFN-γ and the BAY11-7082 with different concns intervenes respectively; Promptly use concentration (A, B, C, D, E) to be respectively: 0, the IFN-γ of 5,10,15,20 μ g/L and concentration (A, B, C, D, E) are: 0, the BAY11-7082 of 0.5,1,1.5,2 μ mol/L intervenes; Every kind of concentration repeats 3 holes, cultivates to add WST reagent 10 μ L behind the 24h again, measures the A450 value in each hole behind the 1h;
(6) recombinant chou transfection:
The KM3 cell is divided into 11 groups: (1) pGL3-B1 group; (2) pGL3-B2 group; (3) pGL3-B3 group; (4) pGL3-B4 group; (5) pGL3-B5 group; (6) pGL3-B6 group; (7) pGL3-B7 group; (8) pGL3-B8 group; (9) pGL3-Control group; (10) pGL3-Basic group; (11) plasmid-free blank group; Each the group all with psv-β-gal plasmid as internal reference; With the eight kinds of recombinant plasmids of pGL3-B1, pGL3-B2, pGL3-B, pGL3-B4, pGL3-B, pGL3-B6, pGL3-B7, pGL3-B8 and the negative control pGL3-Basic that make up, positive control pGL3-Control, transfection KM3 cell respectively; While cotransfection psv-β-gal confidential reference items plasmid, any plasmid of not transfection of blank group;
(7) to each group in the step (6), each group is collected respectively and lysing cell behind the transfection 48h, carries out uciferase activity and detects;
(8) IFN-γ and NF-kB inhibitor intervention that BAFF-R promoter activity and mRNA are expressed: repeating step (6); And respectively organizing in the step (6) added IFN-γ (10 μ g/L) and BAY11-7082 (1 μ mol/L) behind the recombinant plasmid transfection 24h in cell culture fluid; After continuing culturing cell 24h; Each group is collected respectively and lysing cell, detects the relative reactivity of luciferase and the expression level that the RT-PCR method detects BAFF-R mRNA.
Lysing cell in the step (7), (8), carry out the concrete grammar that uciferase activity detects and be: 1 * PBS washed cell 2 times; Add 200 μ l, 1 * lysate; Freeze thawing once back moves in the 1.5mlEppendorf pipe 12000g/min, 4 ℃ of centrifugal 2min; Get 20 μ l cracking supernatants, detect uciferase activity with Chemiluminescence Apparatus; Get 50 μ l cracking supernatants, add 50 μ l beta-galactosidase enzymes detection reagent, ELIASA is measured the activity of beta-galactosidase enzymes; The ratio of uciferase activity and betagalactosidase activity is the relative reactivity of luciferase.
Primer-design software is Primer premier5.0.
The PCR reaction conditions is 94 ℃ in the step (2), 1.5min; 94 ℃, 39S; 56 ℃, 45S, 72 ℃, 1min, 35 circulations; Last 72 ℃ are extended 5min; The PCR product is through 1% agarose gel electrophoresis, and ethidium bromide staining is also taken a picture and identified.
Ratio in the step (6) between lipofectamine and the plasmid is 3 μ l: 4 μ g, the ratio of plasmid to be measured and internal reference plasmid is 4: 1.
The method of the expression level of RT-PCR method detection BAFF-R mRNA is in the step (8): collect the KM3 cell that receives IFN-γ and BAY11-7082 effect front and back respectively; Trizol extracts total RNA; And carry out quantitatively with ultraviolet spectrophotometer; Obtain cDNA with RNA rt test kit (TaKaRa) rt, pcr amplification purpose band is with β
2M is as internal reference; The used upstream primer of amplification BAFF-R: 5 '-GGAAGACCCAGGAACCAC-3 ', downstream primer: 5 '-TTTGGTGTGCTTGCCTT-3 ' is (297bp); Amplification β
2The upstream primer that M is used: 5 '-CTATCCAGCGTACTCCAA-3 ', downstream primer: 5 '-GCAGGCATACTCATCTTTT-3 ' (242bp).
Then the uciferase activity that obtains is detected data and use statistical procedures, the luciferase relative reactivity representes that with X ± s group difference adopts the t check to compare.
Discuss:
BAFF belongs to the member of tnf family cytokines, through (TACI, BCMA BAFF-R) combine, and participate in the adjusting of B, T lymphopoiesis and function with its 3 acceptors; The BAFF over-expression can impel bone-marrow-derived lymphocyte constantly to breed, secrete autoantibody, thereby causes the morbidity of a series of autoimmune diseases; Because the BAFF over-expression makes the generation of the lymphopoiesis B of causing cell malignancies out of control.BAFF-R is its specific receptors in 3 acceptors of BAFF, can combine with BAFF is specific, and BAFF-R is necessary for the B cell survival of BAFF mediation with maturation.Only limit at present the dependency aspect of itself and disease for the research of BAFF-R; Relevant report is not seen in research to BAFF-R gene transcription regulation mechanism aspect as yet; Thereby on its transcriptional control mechanism, study for the BAFF-R gene; Illustrate what the signal transduction path of BAFF-R mediation was correlated with, can be for new treatment target spot being provided with the B cell related diseases.
Among the present invention with the human BAFF-R gene upper reaches 5 ' flanking region-1562～+ fragment of 261bp different lengths is connected with luciferase reporter gene carrier pGL3-Basic as promotor and makes up 8 recombinant chous; With recombinant chou transfection people multiple myeloma cell line KM3; Through detecting the relative reactivity of luciferase, confirm the zone that promoter activity is the strongest.After containing the active recombinant chou transfection of strong promoter KM3, use IFN-γ (10 μ g/L), BAY11-7082 (1 μ mol/L) to intervene respectively; Through detecting the relative reactivity and the BAFF-R mRNA change of Expression of luciferase, inquire into IFN-γ and BAY11-7082 influence to the BAFF-R genetic transcription.The result shows that promoter activity zone is-1420～+ 261, explains in this zone to have cis-acting elements, makes the activity of promotor apparently higher than other regional promotors.And possibly also have relevant negative regulatory sequence in other regional promoter sequences, make the active relatively low of promotor.IFN-γ has promoter action to the BAFF-R gene promoter activity and can raise the expression of BAFF-RmRNA, the expression that NF-kB inhibitor (BAY11-7082) has restraining effect and can reduce BAFF-R mRNA the BAFF-R gene promoter activity.Show that IFN-γ and NF-κ B signal path participated in BAFF-R gene transcription regulation and control, these results provide basic foundation for the further regulation mechanism of research BAFF-R.BAFF-R with can activate NF-κ B signal path after BAFF combines, our result of study finds that the NF-kB inhibitor can influence the expression of promoter activity and the mRNA thereof of BAFF-R again., BAFF also finds that NF-κ B can regulate the expression of BAFF when being studied; And a certain amount of BAFF also can regulate the activity of NF-κ B signal path; Interknit between each factor in this explanation organism and condition each other; Constitute complicated endless chain between them, thereby kept the stable state of organismic internal environment jointly.
Claims (6)
1. a mensuration is characterized in that: comprise the following steps: the method for activity of human BAFF-R gene promoter influence factor
(1) design of primers:
Press the imbrication mode at eight segmental primers of BAFF-R gene 5 ' flanking sequence design with primer-design software; Eight pairs of primers have identical downstream primer; 3 ' end of primer adds restriction enzyme HindIII site; 5 ' end adds restriction enzyme Mlu I site, adds 6 protection bases simultaneously respectively at two ends, is convenient to enzyme and cuts the MCS that is inserted into carrier; Eight segmental primer sequences and downstream primer sequence are:
Wherein 1-8 is eight segmental upstream primer sequences of different lengths, and 9 is eight fragments, 3 ' end common downstream primer sequences; Above-mentioned primer is as the primer of the corresponding product B of amplification 1, B2, B3, B4, B5, B6, B7, B8, and B1～B8 is corresponding with above-mentioned primer 1～8 respectively;
(2) clone of BAFF-R gene 5 ' flanking region:
From people's whole blood, extract genomic dna as template, use LATaq enzyme pcr amplification purpose fragment B8;
Cut pcr amplification product B8 and pGL3-Basic carrier after glue reclaims purifying with restriction enzyme Mlu I, Hind III enzyme; Enzyme is cut product and is connected through the T4 ligase enzyme; Connect mix products and transform escherichia coli DH5 α; Enzyme is cut order-checking and is identified positive colony, obtains positive recombinant plasmid, called after pGL3-B8;
(3) structure of BAFF-R gene 5 ' flanking region sequence deletion plasmid:
With recombinant plasmid pGL3-B8 is template; Pcr amplification purpose fragment B1-B7; Method with making up recombinant plasmid pGL3-B8 is the same, obtains 7 recombinant chous, respectively called after pGL3-B1, pGL3-B2, pGL3-B3, pGL3-B4, pGL3-B5, pGL3-B6, pGL3-B7;
(4) cell cultures:
People's multiple myeloma cell line KM3 suspension growth contains 100U/ml penicillium mould, 100U/ml Streptomycin sulphate in the substratum in the RPMI1640 substratum that contains 10% foetal calf serum, place 5%CO
2, 37 ℃ of constant temperature culture, 1～2d goes down to posterity once;
(5) intervention of IFN-γ and NF-kB inhibitor on cell proliferation:
The KM3 cell adjustment concentration of cultivating going down to posterity is 2 * 10
9/ L, every hole 100 μ L are inoculated in 96 well culture plates, and after the overnight cultures, IFN-γ and the BAY11-7082 with different concns intervenes respectively, and every kind of concentration repeats 3 holes, cultivates to add WST reagent 10 μ L behind the 24h again, measures the A450 value in each hole behind the 1h;
(6) recombinant chou transfection:
The KM3 cell is divided into 11 groups: (1) pGL3-B1 group; (2) pGL3-B2 group; (3) pGL3-B3 group; (4) pGL3-B4 group; (5) pGL3-B5 group; (6) pGL3-B6 group; (7) pGL3-B7 group; (8) pGL3-B8 group; (9) pGL3-Control group; (10) pGL3-Basic group; (11) plasmid-free blank group; Each the group all with psv-β-gal plasmid as internal reference; With the eight kinds of recombinant plasmids of pGL3-B1, pGL3-B2, pGL3-B3, pGL3-B4, pGL3-B5, pGL3-B6, pGL3-B7, pGL3-B8 and the negative control pGL3-Basic that make up, positive control pGL3-Control, transfection KM3 cell respectively; While cotransfection psv-β-gal confidential reference items plasmid, any plasmid of not transfection of blank group;
(7) to each group in the step (6), each group is collected respectively and lysing cell behind the transfection 48h, carries out uciferase activity and detects;
(8) IFN-γ and NF-kB inhibitor intervention that BAFF-R promoter activity and mRNA are expressed: repeating step (6); And respectively organizing in the step (6) added IFN-γ and BAY11-7082 behind the recombinant plasmid transfection 24h in cell culture fluid; After continuing culturing cell 24h; Each group is collected respectively and lysing cell, detects the relative reactivity of luciferase and the expression level that the RT-PCR method detects BAFF-R mRNA.
2. mensuration according to claim 1 is to the method for activity of human BAFF-R gene promoter influence factor; It is characterized in that: lysing cell in the step (7), (8), carry out the concrete grammar that uciferase activity detects and be: 1 * PBS washed cell 2 times, add 200 μ l, 1 * lysate, freeze thawing once back moves in the 1.5mlEppendorf pipe; 12000g/min; 4 ℃ of centrifugal 2min get 20 μ l cracking supernatants, detect uciferase activity with Chemiluminescence Apparatus; Get 50 μ l cracking supernatants, add 50 μ l beta-galactosidase enzymes detection reagent, ELIASA is measured the activity of beta-galactosidase enzymes; The ratio of uciferase activity and betagalactosidase activity is the relative reactivity of luciferase.
3. mensuration according to claim 1 is characterized in that the method for activity of human BAFF-R gene promoter influence factor: primer-design software is Primer premier5.0.
4. mensuration according to claim 1 and 2 is characterized in that the method for activity of human BAFF-R gene promoter influence factor: the PCR reaction conditions is 94 ℃ in the step (2), 1.5min; 94 ℃, 39S; 56 ℃, 45S, 72 ℃, 1min, 35 circulations; Last 72 ℃ are extended 5min; The PCR product is through 1% agarose gel electrophoresis, and ethidium bromide staining is also taken a picture and identified.
5. mensuration according to claim 1 and 2 is to the method for activity of human BAFF-R gene promoter influence factor; It is characterized in that: the ratio in the step (6) between lipofectamine and the plasmid is 3 μ l: 4 μ g, the ratio of plasmid to be measured and internal reference plasmid is 4: 1.
6. mensuration according to claim 1 and 2 is to the method for activity of human BAFF-R gene promoter influence factor; It is characterized in that: the method for the expression level of RT-PCR method detection BAFF-RmRNA is in the step (8): collect the KM3 cell that receives IFN-γ and BAY11-7082 effect front and back respectively; Trizol extracts total RNA, and carries out quantitatively with ultraviolet spectrophotometer, obtains cDNA with RNA rt test kit rt; Pcr amplification purpose band, with β 2M as internal reference; The used upstream primer of amplification BAFF-R: 5 '-GGAAGACCCAGGAACCAC-3 ', downstream primer: 5 '-TTTGGTGTGCTTGCCTT-3 ' is (297bp); The used upstream primer of amplification β 2M: 5 '-CTATCCAGCGTACTCCAA-3 ', downstream primer: 5 '-GCAGGCATACTCATCTTTT-3 ' (242bp).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910213158 CN101781680B (en) | 2009-10-19 | 2009-10-19 | Method for measuring influencing factors to BAFF-R (B cell-Activating Factor-Receptor) gene promoter activity of human |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910213158 CN101781680B (en) | 2009-10-19 | 2009-10-19 | Method for measuring influencing factors to BAFF-R (B cell-Activating Factor-Receptor) gene promoter activity of human |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101781680A CN101781680A (en) | 2010-07-21 |
| CN101781680B true CN101781680B (en) | 2012-05-09 |
Family
ID=42521833
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910213158 Expired - Fee Related CN101781680B (en) | 2009-10-19 | 2009-10-19 | Method for measuring influencing factors to BAFF-R (B cell-Activating Factor-Receptor) gene promoter activity of human |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101781680B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101781679B (en) * | 2009-10-19 | 2012-05-23 | 南通大学附属医院 | Method for detecting activity of human BAFF-R gene promoter |
| CN106399461A (en) * | 2016-09-14 | 2017-02-15 | 妙顺（上海）生物科技有限公司 | Method of detection of transcription factor expression activity by luciferase reporter gene system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001012812A2 (en) * | 1999-08-17 | 2001-02-22 | Biogen, Inc. | Baff receptor (bcma), an immunoregulatory agent |
| CN1844400A (en) * | 2006-04-14 | 2006-10-11 | 中国人民解放军第二军医大学 | Recombinant plasmids containing B cell activation factor gene promoters with different lengths and their preparation method and application |
| CN101271113A (en) * | 2008-04-25 | 2008-09-24 | 南京师范大学 | ELISA reagent kit for detecting human B lymphocyte stimulus factor and its production method |
-
2009
- 2009-10-19 CN CN 200910213158 patent/CN101781680B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001012812A2 (en) * | 1999-08-17 | 2001-02-22 | Biogen, Inc. | Baff receptor (bcma), an immunoregulatory agent |
| CN1844400A (en) * | 2006-04-14 | 2006-10-11 | 中国人民解放军第二军医大学 | Recombinant plasmids containing B cell activation factor gene promoters with different lengths and their preparation method and application |
| CN101271113A (en) * | 2008-04-25 | 2008-09-24 | 南京师范大学 | ELISA reagent kit for detecting human B lymphocyte stimulus factor and its production method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101781680A (en) | 2010-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lu et al. | MicroRNA-21 limits in vivo immune response-mediated activation of the IL-12/IFN-γ pathway, Th1 polarization, and the severity of delayed-type hypersensitivity | |
| Mahesh et al. | MicroRNA-155: a master regulator of inflammation | |
| Kim et al. | Human cytomegalovirus microRNA miR-US4-1 inhibits CD8+ T cell responses by targeting the aminopeptidase ERAP1 | |
| Chan et al. | Cutting edge: a natural antisense transcript, AS-IL1α, controls inducible transcription of the proinflammatory cytokine IL-1α | |
| Toth et al. | Interferon action and the double‐stranded RNA‐dependent enzymes ADAR1 adenosine deaminase and PKR protein kinase | |
| Mayoral et al. | MicroRNA-221–222 regulate the cell cycle in mast cells | |
| Lu et al. | Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses | |
| Philippe et al. | MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes | |
| Zhang et al. | Interferon-β-induced miR-155 inhibits osteoclast differentiation by targeting SOCS1 and MITF | |
| Xu et al. | MicroRNA-216a inhibits NF-κB-mediated inflammatory cytokine production in teleost fish by modulating p65 | |
| Ji et al. | The miR-17-92 microRNA cluster is regulated by multiple mechanisms in B-cell malignancies | |
| Sullivan et al. | MicroRNA-deficient NK cells exhibit decreased survival but enhanced function | |
| CN101781679B (en) | Method for detecting activity of human BAFF-R gene promoter | |
| Jin et al. | MicroRNA-181 expression regulates specific post-transcriptional level of SAMHD1 expression in vitro | |
| Quynh et al. | The cytosolic sensor, DDX41, activates antiviral and inflammatory immunity in response to stimulation with double-stranded DNA adherent cells of the olive flounder, Paralichthys olivaceus | |
| Li et al. | Molecular characterisation of porcine miR-155 and its regulatory roles in the TLR3/TLR4 pathways | |
| CN101781680B (en) | Method for measuring influencing factors to BAFF-R (B cell-Activating Factor-Receptor) gene promoter activity of human | |
| Maharjan et al. | Human aldosterone synthase gene polymorphism promotes miRNA binding and regulates gene expression | |
| Chumakov et al. | Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector | |
| CN105132424A (en) | MicroRNA inhibitor, microRNA inhibitor expression vector, building method of microRNA inhibitor expression vector and application of microRNA inhibitor expression vector | |
| Periyasamy et al. | Isolation, characterization and evaluation of the Pichia pastoris sorbitol dehydrogenase promoter for expression of heterologous proteins | |
| CN112760320B (en) | Exogenous artificial miRNA for effectively inhibiting replication of porcine epidemic diarrhea virus and application thereof | |
| Hao et al. | Regulation of NKCC2B by TNF-α in response to salt restriction | |
| US10787664B2 (en) | Compounds of chemically modified oligonucleotides and methods of use thereof | |
| Zhang et al. | MicroRNA sponge knockdowns miR-483-5p and upregulates serum ALT/AST in transgenic mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120509 Termination date: 20131019 |