CN1807648A - Recombinant plasmid containing effective B cell activation factor gene promotor and its preparation method and uses - Google Patents

Abstract

The invention discloses a recombination plasmid with effective BAFF gene promotor in the biomedical engineering domain, which is characterized by the following: the recombination plasmid is composed of -1349--743bp fragment of human BAFF gene upstream promotor sequence and alficetin acetylase(CAT); The usage and method are provided for preparing recombination plasmid; the recombination plasmid is exact and reliable; it's easy to activate and block-up controlling target dots of BAFF gene in transcription horizon.

Description

Contain recombinant plasmid of effective B cell activation factor gene promotor and its production and use

Technical field

The present invention relates to bio-pharmaceutical engineer technology domain, be specifically related to a kind of recombinant plasmid that contains effective B cell activation factor gene promotor and its production and use.

Background technology

(systemic lupus erythematosus is a class common disease, frequently-occurring disease SLE) to systemic lupus erythematous, mainly shows as the activation of B cell hyperproliferation, produces multiple autoantibody (the most important with anti-dsDNA antibody).This disease is mainly in 20～50 years old young and middle-aged women, about 1: 8 of M-F; If diagnoses and treatment is untimely, renal failure will appear in 60～70% patient, have a strong impact on labor force's quality.And the pathogeny of SLE is still indistinct, does not still have specific diagnostic means so far, does not more have the ideal methods of treatment.At present in the pathogenesis of numerous possible SLE, B cell transition activated theory is extensively approved, but whether it is the place of the key of SLE morbidity, is still a unknown major issue.Existing known, the B cell activation factor that the B cell-stimulating is played important regulating and controlling effect may be very relevant with the morbidity of autoimmune disease such as SLE.

B cell activation factor (B-cell activating factor, BAFF), B-cell stimulating factor (B-Lymphocyte stimulator is otherwise known as, BLyS), lymphocyte that TNF is relevant express ligand 1 (TNF and ApoL-related leukocyte-expressed ligand 1, TALL-1), cell death inducing and activation NF _-κThe TNF analogue of B (TNFhomologue that activates apoptosis, NF _-κB, and JNK THANK) etc., is a kind of multi-functional costimulatory molecules of finding in 1999.

BAFF belongs to tumour necrosis factor, and (by the coded by said gene of human chromosome 13q34, its length is 285 amino acid (aa) for tumor necrosis factor, TNF) family, is that a kind of II type film connects albumen.Wherein 47aa to have between the 73aa one the coding membrane-spanning protein the zone, length is 31 amino acid.The tenuigenin district is made up of 46 amino acid, and the cell outskirt is made up of 218 amino acid, and 2 N-glucoside positions are arranged.This cell outskirt (17kDa) can be cut off by proteolytic ferment, becomes solvable type molecule and dissociates.The main specifically expressing of BAFF is in myeloid lineage cells such as monocyte, scavenger cell, dendritic cell, and IL-10, cytokines such as IFN-γ, IL-4 can both obviously raise/reduce the myeloid lineage cell it is expressed.

Make a general survey of progress and the trend of BAFF in recent years, to be each explore the relation of BAFF and SLE immunopathogenesis since protein and mRNA level main research focus, though these researchs provide theoretical foundation and experiment basis for inquiring into the effect of BAFF in SLE, can't disclose the essence of BAFF effect at molecular level.This is because the protein expression regulation and control mainly occur in the nucleic acid level and the transcriptional level of upstream; the abnormal expression that can cause its downstream egg white level unusually that mRNA expresses; and the variation of any controlling element of transcriptional level will directly cause the unusual of mRNA expression.So research abnormal activation of BAFF gene under different condition has the meaning of particularly important to the pathogenesis of setting forth SLE.But, at present the accurate location of relevant BAFF upstream region of gene controlling element and and the protein-bonded Study of Interaction of corresponding D NA still belong to blank, more do not see the report of any changes of function about BAFF gene regulating element under the pathological conditions.Given this, we think and are necessary associating protein and the unconventionality expression of nucleic acid (mRNA) level research BAFF in SLE, the discussion B cell-stimulating that BAFF mediated is to the pathogenesis of SLE, and further carry out BAFF gene transcription regulation Study on Mechanism at molecular level, form complete from the transcriptional level to nucleic acid again to the regulation and control theory of protein level, for the pathogenic effects of opening BAFF, expand blocking-up BAFF activatory new technology/method foundation be provided, for the pharmacological agent/screening of SLE and even other autoimmune diseases provides important regulation and control target sequence.

Summary of the invention

The object of the present invention is to provide recombinant plasmid that contains effective B cell activation factor gene promotor and its production and use.

The present invention according to the people BAFF upstream region of gene 5 ' flanking region-1349 among the Genebank (AB730224)～-fragment of 329bp, behind the main nf site that this sequence of program DNAassist1.0 model analysis may exist, select-1349～-743bp is that research object is carried out clonal expression.With normal people's Whole Blood Genomic DNA is template, goes into to contain the pCAT3-Enhancer carrier of paraxin acetyltransferase (CAT) reporter gene through pcr amplification, directed cloning, constructs recombinant plasmid.Be transformed into e. coli jm109 with amplification, after enzyme is cut, is checked order and identifies, the extracting plasmid DNA is also quantitative, cultivator bone marrow leukemia cells strain: HL-60, adopt lipofectamine, this plasmid recombinant DNA is transfected in the target cell cultivated 48 hours, the pair cell lysate carries out protein quantification, measures the CAT activity of recombinant chou with CAT reporter gene ELISA detection kit.

The present invention also with constructed contain the BAFF gene promoter (1349～-743bp) recombinant plasmid is transfected in the HL-60 cell, cultivate after 24 hours, the cytokine that adds different concns: IL-10, IL-4, IFN-γ and reorganization BAFF (rBAFF) molecule are intervened, the pair cell lysate carries out protein quantification, measure the activity that its CAT expresses with CAT reporter gene ELISA detection kit, observe the variation of its transcriptional activity, inquire into the influence of different cytokines this BAFF gene promoter activity.

The result shows, through the enzyme evaluation of cutting, check order, the sequence among constructed BAFF gene recombination promoter fragment and the Genebank (1349～-743bp) consistent.IFN-γ, IL-10 and rBAFF can improve the transcriptional activity of this BAFF gene promoter recombinant chou, and IL-4 suppresses its transcriptional activity.Illustrate that cytokine IL-10, IFN-γ, IL-4, rBAFF can to a certain degree influence the synthetic and secretion of BAFF at transcriptional level.

The recombinant plasmid that the present invention is constructed contains effective BAFF gene promoter (1349～-743bp fragment) accurately, reliable, and reporter gene survey to live more active method simple, can be quantitative, it is a short-cut method at diseases related medicines of BAFF such as transcriptional level screening treatment SLE, for the regulatory mechanism of further studying people BAFF gene activation is laid a good foundation, also new approaches are provided for exploring the diseases related controls of BAFF such as SLE.

Description of drawings

Fig. 1 is a pCAT3-enhancer vector plasmid structure iron of the present invention

Fig. 2 is that recombinant plasmid enzyme of the present invention is cut the rear electrophoresis result

Fig. 3 is the sequencing result of recombinant plasmid of the present invention

Fig. 4 is the genome structure of people BAFF gene

Fig. 5 is the possible main nf binding site (DNAassist1.0) of computer simulation analysis

Fig. 6 is the influence of different cytokines to recombinant plasmid promoter activity of the present invention

Embodiment

Now reach accompanying drawing in conjunction with the embodiments, the invention will be further described.

Embodiment 1: contain the preparation of effective BAFF gene promoter recombinant plasmid

People BAFF gene promoter is positioned at the zone of the about 1020bp of this upstream region of gene 5 ' flanking region (referring to Kawasaki A, Tsuchiya N, Fukazawa T, et al.Analysis on theassociation of human BLYS (BAFF, TNFSF13B) polymorphisms withsystemic lupus erythematosus and rheumatoid arthritis[J] .Genes Immun, 2002,3 (7): 424-429), see Fig. 4.This research purpose is that the part fragment of people BAFF upstream region of gene 5 ' flanking region (1349～-743bp sequence) is started the regulation activity analysis.This sequence and reporter gene are formed the recombinant promoter plasmid,, observe the startup activity of this recombinant chou, lay a good foundation at the regulation and control target sequence of transcriptional level activation/blocking-up BAFF gene for exploring by gene transfection, reporter-gene assays activity.

Materials and methods

1 material

1.1 main agents

Whole Blood Genomic DNA extraction test kit, ProofSart high-fidelity PCR test kit, plasmid extraction test kit are U.S. QIAGEN company product; Restriction enzyme Mlu I, Xhol, T ₄Dna ligase, DNA marker are all available from Dalian TakaRa bio-engineering corporation; Glue reclaims test kit available from MBI company in a small amount; Fugene 6 lipofectamine, CAT ELISA detection kit are U.S. Roche company product; Calf serum, GI1640 substratum are available from U.S. Gibco company; 50ml Tissue Culture Flask, six, 12 porocyte culture plates are U.S. Corning company product; Primer is synthetic, the dna sequencing analysis is finished by Shanghai associating gene biological technology company.

1.2 carrier, bacterial strain, cell strain

Expression vector pCAT3-enhancer Vector, CAT express positive plasmid, β-gal plasmid, e. coli jm109: all available from U.S. Promega company;

People's bone marrow leukemia cells strain HL-60: hematology of hospital is so kind as to give by the Changhai.

1.3 key instrument

The PE-9600PCR instrument (U.S. PE Biosystems company) that increases, ultraviolet spectrophotometer Du-600 (U.S. Backman company), horizontal strip electrophoresis instrument (Sweden Bio-Rad company), KUBOTA 5420 table model high speed centrifuges (Japanese Kubota company), 550 type enzyme-linked immunosorbent assay instruments (Model 550 Microplate Reader, Bio-Rad company produces).

2 experimental techniques

2.1 the structure of recombinant chou

PCAT3-enhancer Vector vector plasmid does not contain promotor, but contains SV enhanser, chloramphenicol acetyltransferase (CAT) reporter gene (see figure 1).The purpose fragment of being inserted in the recombinant chou is the promoter fragment of the BAFF that will study.

2.1.1 design of primers

According to the promoter sequence (Accession:AB730224) of the people BAFF that Genebank reported, the main nf site (see figure 5) that this sequence of application program DNA assist1.0 model analysis may exist, Primer Premier 5.0 primer-design softwares design primer.The restriction enzyme site that adds restriction enzyme Mlu I, Xhol at the two ends of primer respectively, the fragment length that intend to insert is respectively 606bp, make up according to this contain-1349 of people BAFF gene promoter sequence～-recombinant chou phBAFF3 that the 743bp fragment is connected with pCAT3-enhancer.The segmental primer of this recombinant promoter that increases is as follows:

Forward primer is: 5 '-AGCCTA CTCGAGAGCCTGGGTCTGGAGTTC-3 '

Reverse primer is: 5 '-ATGTCT ACGCGTGGTCGCAGGGTGTTGAGT-3 '

5 ' end of forward and reverse primer contains 6 protection bases and restriction enzyme MluI or Xhol recognition site (underscore part) respectively.Primer is synthetic by associating genome company.

2.1.2 the extraction of genomic dna

Get normal people's whole blood 200 μ l, operate to extract genomic dna according to " QIAamp DNA bloodmini kit " test kit specification sheets of QINGEN company, on ultraviolet spectrophotometer the genomic dna that extracts is carried out quantitatively, packing-80 ℃ preservation is standby.

2.1.3 the segmental acquisition of purpose

With the genomic dna is template, with " the ProofStart high-fidelity PCR test kit " of QINGEN company this recombinant chou purpose fragment that increases, by preliminary experiment reaction conditions is adjusted, specific as follows:

10×PCR buffer 5μl

2.5mM dNTP 6μl

10 μ M forward primers, 5 μ l

10 μ M reverse primers, 5 μ l

ProofStart archaeal dna polymerase 1 μ l

Human gene group DNA (100ng/ μ l) 3 μ l

Amount to 50 μ l

Reaction parameter:

95℃ 2min

2.1.4PCR the purifying of product

The purifying of PCR product is pressed MBI company " glue reclaims test kit in a small amount " specification sheets operation, presses the label prompting adds 19 times of volumes in Washing Buffer liquid dehydrated alcohol in advance.

1) dna fragmentation separates with 1% agarose gel electrophoresis;

2) corresponding molecular weight Marker reclaims segmental gel strips with desire and downcuts, and puts in the 1.5ml centrifuge tube, adds the Bind Solution of 3 times of volumes;

3) 55 ℃ of water-baths are 5 minutes, and per 2 minutes mixings once;

4) by 2 μ l suspensions/μ g DNA, add Silica Powder Suspension suspension, 55 ℃ of water-baths 5 minutes, the whirlpool was outstanding once in per 2 minutes;

5) high speed centrifugation formed flaky precipitate after 1 minute, removed supernatant;

6) add the Washing Buffer of 500 μ l ice precooling, behind the mixing centrifugal 30 seconds, remove supernatant, triplicate;

7) add 50 μ l TE liquid, gentle resuspended post precipitation, 55 ℃ of water-baths 5 minutes;

8) centrifugal 30 seconds, collect supernatant ,-20 ℃ of preservations are standby;

9) get 10 μ l DNA and carry out the evaluation of 1% agarose gel electrophoresis; Get 7 μ l DNA with 10 times of pure water dilutions, be used for uv-spectrophotometric instrument detection by quantitative.

2.1.5 purpose fragment and vector plasmid are cut, connected to enzyme

With restriction enzyme Mlu I, this section of Xhol double digestion purpose fragment and carrier pCAT3-enhancer Vector, endonuclease reaction system and parameter are as follows:

Dna fragmentation 30 μ l

Mlu I(10U) 2μl

Xhol(10U) 2μl

10×Buffer 4μl

Deionization H ₂O 2 μ l

Amount to 40 μ l

Vector plasmid 1 μ l

Mlu I(10U) 2μl

Xhol(10U) 2μl

10×Buffer 4μl

Deionization H ₂O 31 μ l

Amount to 40 μ l

37 ℃ act on 2 hours, after agarose electrophoresis identifies that enzyme is cut efficient, cut plasmid and purpose fragment after glue recovery enzyme is cut, quantitatively.Purpose fragment after enzyme is cut has identical sticking end with linear pCAT3-enhancerVector, connects through the T4 ligase enzyme.The ligation system:

T4DNA ligase enzyme 1 μ l

Plasmid 300ng

Purpose fragment 150ng

10 * ligation damping fluid, 2.5 μ l

Supply reaction volume (25 μ l), 16 ℃ of connections are spent the night.

2.1.6 preparation of competence bacterium and conversion

1) e. coli jm109 is inoculated in the LB substratum, shaking culture is spent the night;

2) bacterial cultures that spends the night in the inoculation of 1: 10 ratio is in 2ml LB substratum, shaking culture 3 hours;

Abandon supernatant after 8000g is centrifugal, add 400 μ l 0.1mol/L CaCl ₂(initial incubation thing amount 1/5), mixing is put ice bath 10min on ice;

3) the centrifugal 10min of 4000g, careful supernatant discarded reclaims bacterium.Add 1/25 long-pending 0.1mol/L CaCl of initial incubation object ₂, packing 50 μ l/ pipe;

4) add above-mentioned connection product 5 μ l, mixing, ice bath 30 minutes;

5) 42 ℃ were impacted 90 seconds, and ice bath 5 minutes adds 800 μ l LB substratum, cultivated shaking culture 3 hours 40 minutes for 37 ℃;

6) centrifugal, abandon the part supernatant, the remainder mixing is inoculated in and contains on the antibiotic LB plate culture medium, cultivates 12～16h for 37 ℃;

7) do the contrast that vector plasmid, plasmid-free connect product, digested plasmid simultaneously.

2.2 a large amount of amplifications, the extraction of recombinant plasmid, CAT positive expression plasmid and vector plasmid

2.2.1 a large amount of amplifications of recombinant plasmid, CAT positive expression plasmid and vector plasmid

Through the amicillin resistance screening, after enzyme is cut preliminary evaluation, order-checking correctly in a small amount; The e. coli jm109 a large amount of amplification positive colonies in containing the 100ml LB substratum of penbritin that conversion had recombinant plasmid, CAT positive expression plasmid, vector plasmid.

2.2.2 a large amount of extractions of recombinant plasmid, CAT positive expression plasmid and vector plasmid

Because plasmid is used for following cell transfecting, therefore, the strict aseptic technique of the process need of extraction." QIAfilter Plasmid Midi Kits " test kit with QIAGEN company extracts each recombinant plasmid in a large number, and operation is all undertaken by explanation.In advance RNaseA is added in the P1 liquid to final concentration be 100 μ g/ml, with P3 liquid 4 ℃ of precoolings.

1) collect LB bacterium liquid with the aseptic centrifuge tube of 50ml, 4500rpm, 4 ℃ of centrifugal 25min do although fall interior precipitation and button;

2) add P1 liquid 4ml in each pipe respectively, blow and beat mixing repeatedly;

3) add P2 liquid 4ml in each pipe respectively, gentleness is put upside down mixing 4～6 times, and room temperature leaves standstill 5min;

4) mouth of pipe with the QIAGEN collection tube clogs with lid, and puts it in the suitable centrifuge tube, adds P3 liquid 4ml in containing each pipe of bacterial lysate respectively, and gentleness is put upside down mixing 4～6 times;

5) immediately above-mentioned lysate is poured in the ready QIAGEN collection tube, room temperature leaves standstill 10min;

6) each uses QBT liquid 4ml balance QIAGEN-tip100 post;

When 7) treating that this post drains fully, take off the lid of collection tube, collection tube is connected on the QIAGEN-tip100 post;

8) touch collection tube with the piston pressurization, the supernatant part of lysate is flowed in the tip100 post fully;

9) treat that lysate leaches fully from the tip100 post, twice on the QC liquid difference wash-out tip post of each usefulness 10ml;

10) use the aseptic centrifuge tube of 15ml instead and make collection tube, with the QF liquid wash-out tip post of 5ml;

11) respectively add the isopropanol precipitating DNA of 0.7 times of volume in the aseptic centrifuge tube of above-mentioned each 15ml, centrifugal 1 hour of 4100rpm behind the mixing, 4 ℃ absorb supernatant;

12) use the 70% ethanol concentration of DNA of 2ml more respectively, centrifugal 1 hour of 4100rpm, 4 ℃ carefully absorb supernatant, prevent to destroy the DNA precipitation;

13) after 10 minutes, respectively use TE liquid (pH8.0) dissolving DNA of the sterilization of 2ml at air drying;

14) it is quantitative that ultraviolet spectrophotometer carries out DNA.

2.3HL-60 the cultivation of cell

People's bone marrow leukemia cells strain HL-60 1640 substratum that contain 10% calf serum, at 37 ℃, 5%CO ₂Cultivate, go down to posterity under the condition, select the 3rd～8 subtituted culturing cell to experimentize.

2.4 recombinant DNA transfection

The liposome transfection method is adopted in the DNA transfection, and operation is undertaken by FuGENE6 liposome reagent operation instruction.Transfection the day before yesterday, the HL-60 cell is with 3 * 10 ⁵Cells/well is inoculated in 6 well culture plates.When cell grows to 60%～80% fusion, renew bright substratum.With the recombinant plasmid of the above-mentioned BAFF gene promoter of 1 μ g, β-gal positive expression plasmid pSV β-gal that 1 μ g makes internal reference and FuGENE6 transfection reagent with the composite form transfection to the HL-60 cell.The CAT positive expression plasmid that transfection 1 μ g is set simultaneously is as positive control; The pCAT3-enhancer vector plasmid of 1 μ g is as negative control.After the transfection 48 hours, cells transfected cracking, CAT reporter gene activity to be measured.

2.5 protein quantification

48h behind the HL-60 cell transfecting recombinant chou stops cultured cells, uses the PBS damping fluid (pH7.4) of precooling to wash 3 times, and with lysis buffer (Roche) lysing cell, cell pyrolysis liquid carries out protein quantification with ultraviolet spectrophotometer.

2.6 β-gal determination of activity

Add (Promega) mixing of 100 μ l " β-gal assay 2 * buffer " in the 100 μ l cell pyrolysis liquids, put 37 ℃ 12 hours, add 1M yellow soda ash mixing again, microplate reader 420nm measures optical density(OD).

2.7CAT determination of activity

With the activity of ELISA test kit (Roche) detection CAT reporter gene, operation is undertaken by operation instruction.200 μ l cell pyrolysis liquids are added 96 orifice plates of coated antibody, and reaction finishes the back and adds the substrate colour developing, and microplate reader 405nm and 492nm measure optical density value.

2.8 data processing

All CAT measurement results are all proofreaied and correct with mole number, protein concentration and the β-gal activity of corresponding transfection plasmid, and experiment repeats 4 times, adopt the relative reactivity of each recombinant chou of mean value computation of 4 experiments.SAS 6.12 softwares are all adopted in the statistical treatment of experimental data, and measurement data is checked and variance analysis with t, and P＜0.05 thinks that difference has statistical significance.

3 results

3.1 contain the evaluation of effective BAFF gene promoter recombinant plasmid

With people BAFF upstream region of gene 5 ' flanking region-1349～-the 329bp fragment is a target sequence, template adopts the normal healthy people Whole Blood Genomic DNA, it is that the fragment of 606bp is as promotor that pcr amplification obtains length, form recombinant chou phBAFF3 with the carrier pCAT3-enhancer that does not contain promotor, this recombinant chou is identified correct (see figure 2) through restriction enzyme Mlu I and Xhol double digestion.Be that carrier is the pCAT3-enhancer of 4.3kb, the length of inserting promotor is 606bp, corresponding to-1349 of people BAFF upstream region of gene promoter sequence～-the 743bp fragment.Through order-checking, insert sequence (AB730224) (see figure 3) in full accord of fragment and GenBank report.

Relative molecular weight indication Marker is wide range DNA marker (TakaRa) among Fig. 2, and the purpose band is the band of phBAFF3 (606bp) positive colony behind Mlu I, Xhol double digestion.

It among Fig. 3 the forward sequencing result of recombinant chou phBAFF3.

3.2 the cell CAT expression level of transfection recombinant chou is measured

People's bone marrow leukemia cells HL-60 behind the above-mentioned recombinant chou that contains effective BAFF gene promoter of transfection 48 hours, the ELISA method is measured cell CAT expression amount.Measure the protein concentration and the β-gal activity of cell pyrolysis liquid simultaneously, β-gal activity is observed the CAT expression amount as internal reference down to keep transfection efficiency the same terms.After the CAT measurement result was proofreaied and correct with mole number, protein concentration and the β-gal of corresponding transfection plasmid is active, the relative reactivity that calculates this recombinant chou was: (0.17 ± 0.03).

Embodiment 2: the parts of fine intracellular cytokine is to the influence of BAFF transcriptional control effect

Studies show that in a large number the immunopathogenesis of various autoimmune diseases such as the unconventionality expression of BAFF and SLE is closely related.The foreign literature report, the BAFF molecule is subjected to the influence of IL-10, the various kinds of cell factors such as IFN-γ, IL-4, these cytokines can obviously raise/reduce the proteic synthetic and secretion of BAFF in the expression of myeloid lineage cells such as monocyte, scavenger cell, dendritic cell.But how these cytokines regulate the expression of BAFF molecule at the myeloid lineage cell, whether influence synthesizing of BAFF and secretion by transcriptional level, still do not have so far clearly and explain.This research with constructed in " embodiment 1 ", contain-1349 of strong transcriptional activity～-the recombinant chou phBAFF3 of 743bp sequence is an experiment porch, with its transient transfection people bone marrow leukemia cells HL-60, adding cytokine IL-10, IFN-γ, IL-4, recombinant human B AFF standard substance (rBAFF) are intervened, and inquire into the influence of these cytokines to the BAFF gene promoter activity.

Materials and methods

1 material

1.1 plasmid, cell strain

The recombinant plasmid phBAFF3 that makes up in " embodiment 1 ";

CAT expresses positive plasmid, β-gal plasmid: U.S. Promega company;

People's bone marrow leukemia cells HL-60: hematology of hospital is so kind as to give by the Changhai

1.2 main agents

Calf serum, GI1640 substratum (U.S. Gibco company); The 50ml Tissue Culture Flask, six, 12 porocyte culture plates (U.S. Corning company); The Fugene transfection reagent, CAT ELISA detection kit (Roche company); Recombinanthumanifn-(rh IFN-γ), recombinant human IL-10 (rh IL-10), recombinant human IL-4 (rh IL-4) is all available from U.S. R﹠amp; D company; Recombinant human B AFF standard substance (rBAFF) are U.S. PeproTech company product.

1.3 key instrument

Ultraviolet spectrophotometer Du-600 (U.S. Backman company), horizontal strip electrophoresis instrument (Sweden Bio-Rad company), KUBOTA 5420 table model high speed centrifuges (Japanese Kubota company), 550 type enzyme-linked immunosorbent assay instruments (Model 550 Microplate Reader, Bio-Rad company produces).

2 experimental techniques

2.1HL-60 the cultivation of cell

People's bone marrow leukemia cells strain HL-60 1640 substratum that contain 10% calf serum, at 37 ℃, 5%CO ₂Cultivate, go down to posterity under the condition, select the 3rd～8 subtituted culturing cell to experimentize.

2.2 recombinant DNA transfection and cytokine intervention

2.2.1 recombinant DNA transfection

FuGENE6 (Roche) lipofectamine method is adopted in the DNA transfection, and operation is undertaken by the reagent operation instruction.Transfection the day before yesterday, the HL-60 cell is with 3 * 10 ⁵Cells/well is inoculated in 6 well culture plates.When cell grows to 60%～80% fusion, renew bright substratum.With 1 μ gphBAFF3 recombinant plasmid, 1 μ g do the β-gal positive expression plasmid pSV β-gal of internal reference and FuGENE6 transfection reagent with the composite form transfection to the HL-60 cell.The CAT positive expression plasmid that transfection 1 μ g is set simultaneously is as positive control; The pCAT3-enhancer vector plasmid of 1 μ g is as negative control.After the transfection 24 hours, change contain 2% calf serum fresh 1640 substratum with tranquillization.

2.2.2 cytokine intervention

Behind the HL-60 cell transfecting recombinant chou 24 hours, change fresh 1640 substratum that contain 2% calf serum, according to preliminary experiment and document design, selection cytokine intervention dosage be: IFN-γ (5ng/ml), IL-10 (100ng/mL), IL-4 (100ng/mL), rhBAFF (200ng/mL), adding above-mentioned cytokine intervenes, cultivate after 48 hours, measure the CAT reporter gene activity.Every kind of cytokine repeats 2 holes, and experiment repeats 5 times.

2.3 protein quantification

Stop cultured cells and use the PBS damping fluid (pH7.4) of precooling to wash 3 times, with lysis buffer (Roche) lysing cell, cell pyrolysis liquid carries out protein quantification with ultraviolet spectrophotometer.

2.4 β-gal determination of activity

Add (Promega) mixing of 100 μ l " β-gal assay 2 * buffer " in the 100 μ l cell pyrolysis liquids, put 37 ℃ 12 hours, add 1M yellow soda ash mixing again, microplate reader 420nm measures optical density(OD).

2.5CAT determination of activity

With the activity of ELISA test kit (Roche) detection CAT reporter gene, operation is undertaken by operation instruction.200 μ l cell pyrolysis liquids are added 96 orifice plates of coated antibody, and reaction finishes the back and adds the substrate colour developing, and microplate reader 405nm and 492nm measure optical density value.

2.6 data processing

All CAT measurement results are all proofreaied and correct with mole number, protein concentration and the β-gal activity of corresponding transfection plasmid, and experiment repeats 5 times, adopt the relative reactivity of each recombinant chou of mean value computation of 5 experiments.SAS 6.12 softwares are all adopted in the statistical treatment of experimental data, and measurement data is checked and variance analysis with t, and P＜0.05 thinks that difference has statistical significance.

3 results

3.1 the evaluation of recombinant chou

Recombinant chou phBAFF3 transformed into escherichia coli JM109 increases large quantity extracting plasmid behind the bacterium, and the recombinant chou that takes a morsel is cut through restriction enzyme Mlu I and Xhol enzyme and identified correct (Fig. 2).Be the pCAT3-enhancer that carrier is 4.3kb, the length of inserting promotor is: 606bp, corresponding to-1349 of people BAFF upstream region of gene promoter sequence～-the 743bp fragment.

Relative molecular weight indication Marker is wide range DNA marker (TakaRa) among Fig. 2, the specific amplified band at the 750bp place of contiguous Marker is target DNA band (606bp), and the amplified band at the 5000bp place of contiguous Marker is the DNA band of pCAT3-enhancer carrier.

3.2 cytokine is to the influence of BAFF gene promoter activity

The HL-60 cell is after transfection recombinant promoter phBAFF3 cultivates 24h, change the fresh 1640 substratum tranquillization 24h of 2% calf serum, add cytokine IFN-γ (5ng/ml), IL-10 (100ng/mL), IL-4 (100ng/mL), rhBAFF (200ng/mL) intervention respectively, collecting cell behind the 48h, ELISA method are measured cell CAT expression amount.Measure the protein concentration and the β-gal activity of cell pyrolysis liquid simultaneously, β-gal activity compares the CAT expression amount as internal reference down to keep transfection efficiency the same terms.Cell CAT expression amount with the transfection phBAFF3 that do not add the cytokine intervention is 1, the CAT relative expression quantities of all the other each groups the results are shown in Table 1.

Table 1. different cytokines is to the influence of BAFF gene promoter activity (n=5, X ± s)

Cytokines	Relative value of reporter gene activity	P Value(compared to control)
			Control(no cytokine) IFN-γ(5ng/ml) IL-10(100ng/ml) IL-4(100ng/ml) rBAFF(200ng/ml)	1.000±0.001 3.527±0.542 2.306±0.350 0.717±0.212 1.443±0.402	＜0.05 ＜0.05 ＜0.05 ＝0.05

The result shows, cell with the transfection of phBAFF3 recombinant chou after IFN-γ, IL-10, rBAFF handle, the CAT expression amount all obviously rises (P≤0.05), and cell with the transfection of phBAFF3 recombinant chou after IL-4 handles CAT expression amount then significantly descend (P＜0.05).Wherein, IFN-γ (5ng/ml) can make 3.5 times of the active risings of the CAT of recombinant chou phBAFF3, IL-10 (100ng/ml) can make 2.3 times of the active risings of the CAT of recombinant chou, rBAFF (200ng/ml) can make 1.4 times of the active risings of the CAT of recombinant chou, and IL-4 (100ng/ml) makes the CAT activity of recombinant chou be reduced to 72% (Fig. 6) of control group.The Control group is not for adding the recombinant chou phBAFF3 that cytokine is intervened among the figure.

Claims (6)

1, a kind of recombinant plasmid that contains effective B cell activation factor BAFF gene promoter, it is characterized in that it by-1349 of people BAFF upstream region of gene promoter sequence～-743bp fragment and chloramphenicol acetyltransferase CAT reporter gene form.

2, the described a kind of preparation method who contains the recombinant plasmid of effective B cell activation factor BAFF gene promoter of claim 1 comprises the steps:

I.pCAT3-enhancer Vector vector plasmid does not contain promotor, but contains SV enhanser, chloramphenicol acetyltransferase CAT reporter gene;

II.-1349 of people BAFF upstream region of gene promoter sequence～-the segmental design of primers of 743bp:

Forward primer: 5 '-AGCCTA CTCGAGAGCCTGGGTCTGGAGTTC-3 '

Reverse primer: 5 '-ATGTCT ACGCGTGGTCGCAGGGTGTTGAGT-3 '

5 ' end of primer contains 6 protection bases and restriction enzyme Mlu I or Xhol recognition site respectively;

III. be template with normal people's Whole Blood Genomic DNA, to-1349 of people BAFF upstream region of gene promoter sequence～-743bp segmentally carries out pcr amplification, enzyme is cut and purifying, the above-mentioned purpose fragment is inserted pCAT3-enhancer Vector vector plasmid, make up the recombinant promoter plasmid;

IV. by the cytogene transfection, CAT reporter gene ELISA detection reagent is measured the CAT activity of recombinant chou, observes the startup activity of recombinant plasmid.

3, the described a kind of recombinant plasmid that contains effective B cell activation factor BAFF gene promoter of claim 1 is characterized in that cytokine IFN-γ, IL-10 and rBAFF improve its startup activity, and cytokine IL-4 suppresses its startup activity.

4, the described a kind of recombinant plasmid that contains effective B cell activation factor BAFF gene promoter of claim 1 has the effect that activates or block the target for modulation of BAFF gene at transcriptional level.

5, the application of the described a kind of recombinant plasmid that contains effective B cell activation factor BAFF gene promoter of claim 1 aspect the diseases related cytokine class medicine of screening treatment BAFF.

6, the described a kind of application of recombinant plasmid aspect the cytokine class medicine of screening therapy system lupus erythematosus that contains effective B cell activation factor BAFF gene promoter of claim 5.

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