CN105567697A - siRNA (small interfering ribonucleic acid) capable of inhibiting chicken B lymphocyte CD86 gene expression and application of siRNA - Google Patents

siRNA (small interfering ribonucleic acid) capable of inhibiting chicken B lymphocyte CD86 gene expression and application of siRNA Download PDF

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CN105567697A
CN105567697A CN201610176019.0A CN201610176019A CN105567697A CN 105567697 A CN105567697 A CN 105567697A CN 201610176019 A CN201610176019 A CN 201610176019A CN 105567697 A CN105567697 A CN 105567697A
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sirna
chicken
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lymphocyte
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CN105567697B (en
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廖成水
王臣
张聪
张巫凡
牛明媚
杨静
李德元
李小康
张春杰
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Henan University of Science and Technology
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Abstract

The invention discloses an siRNA (small interfering ribonucleic acid) capable of inhibiting chicken B lymphocyte CD86 gene expression and application of the siRNA, belonging to the technical field of molecular biology. The siRNA (of which the sequences are disclosed as SEQ ID NO:1-2) is designed and synthesized according to the chicken B lymphocyte CD86 mRNA (messenger ribonucleic acid) sequence. The CD86 siRNA can effectively inhibit the expression of the chicken DT40 cell CD86 mRNA and CD86 protein. After interfering with the chicken CD86 gene expression, the expression level of the active peptide bursin promoted chicken DT40 cell surface membrane IgM (mIgM) antibody mRNA of the bursa of Fabricius is obviously lowered. The siRNA has favorable application prospects in the aspect of preparation of immunosuppressants for resisting avian B lymphocyte activation (or development differentiation).

Description

A kind of siRNA and application thereof suppressing chicken B lymphocyte CD86 genetic expression
Technical field
The present invention relates to a kind of siRNA suppressing chicken B lymphocyte CD86 genetic expression, also relate to the application of this siRNA simultaneously, belong to technical field of molecular biology.
Background technology
Boissoptis in 1993 etc. have found second member CD86 (B7-2) molecule of B7 family, and the same year, Freeman etc. cloned the gene of CD86.CD86 expresses the costimulatory ligand on antigen presenting cell (antigenpresentingcell, APC) surface, is one of immune superfamily member.CD86 is by 1120bp coded by said gene, its gene order and CD80 have the homology of 26%, wherein comprise an open reading frame be made up of 981 Nucleotide, and aminoterminal has a secretion property signal peptide, infer that its whole albumen is made up of 306 amino acid, belong to I type transmembrane glycoprotein.Before modification, the molecular weight of CD86 is about 34kD, comprises an after birth outskirt be made up of 220 amino acid, a hydrophobicity cross-film district be made up of 23 amino acid and a cytoplasmic domain be made up of 60 amino acid.Wherein, after birth outskirt comprises an immunoglobulin superfamily V sample district and C sample district, connects glycosylation site containing 8 potential N; There is the potential phosphorylation site of PKC and casein kinase i I cytoplasmic domain, and prompting CD86 may have signal transduction functionality.CD86 can express with the form of monomer on various APC surface, and as the scavenger cell, B cell etc. of dendritic cell (DC), Langerhans cell, activation, it, can fast upregulation after activation generally in low-level constructive expression.
CD86 can costimulatory receptor CD28 and cytolytic T lymphocyte epitode (cytotoxicTlymphocyteassociatedantigen-4, CTLA-4) combine altogether with T cell.After CD86 and CD28/CTLA-4 combines, stimulate CD4 with CD80 is collaborative +the expression of Th1 and Th2 type cell, and Th1 type is greater than to the effect of Th2 type T cell.The I cytokines such as Th1 type cell Major Secretory IL-2, IFN-γ, TNF-α, participate in the cellular immunization that cytotoxic T cell (CTL) mediates, the II cytokines such as Th2 type cell Major Secretory IL-4, IL-5, IL-6, IL-10, IL-13, its function is to stimulate B cell proliferation and eosinophil activation, the generation of enhancing antibody (IgG, IgE), participates in humoral immunization.In a word, CD86 has vital role in adjustment immune response.
RNA disturbs (RNAinterference, RNAi) phenomenon, also claim the gene silencing mechanism depending on RNA, refer to that the specificity mediated by double-stranded RNA (double-strandedRNA, dsRNA) suppresses the phenomenon with the target gene mrna expression of its sequence homology.Calendar year 2001 Elbashir etc. in mammalian cell First Observation to RNAi phenomenon, because RNAi technology is easy to prepare specific gene disappearance phenotype individuals, the function of this gene can be studied quickly and easily, be thus widely used in gene function and gene therapy research field.Research find, siRNA (smallinterferingRNA, siRNA) can in mammalian cell inducing specific gene silencing.
SiRNA has RNA duplex structure and 3 ' end dinucleotide pendency, more difficult by nuclease degradation, has good stability and superior inhibition, be widely used in functional genomics research as gene silencing instrument.DT40 cell is by avian leukosis virus (avianleucosisvirus, ALV) differentiation-inducing, derive from a tumor cell line of chicken pre-B lymphocyte, this cell surface carries mIgM, and there is biological property identical with chicken bursa pre-B lymphocyte, therefore can carry out relevant research as good fowl bone-marrow-derived lymphocyte model.
Summary of the invention
The object of this invention is to provide a kind of siRNA suppressing chicken B lymphocyte CD86 genetic expression.
Meanwhile, the present invention also provides the application of a kind of CD86siRNA.
In order to realize above object, the technical solution adopted in the present invention is:
Suppress a siRNA for chicken B lymphocyte CD86 genetic expression, its sense strand sequence is as shown in SEQIDNO:1, and antisense strand sequence is as shown in SEQIDNO:2.
For improving the Gene interfere efficiency of siRNA, overhang by two base TT be made up of deoxynucleoside (i.e. dTdT) at 3 ' end of siRNA sequence.This overhang not with mRNA complementary, positive-sense strand 3 ' can be made to hold and more easily to unwind, thus increase its silence efficiency.
The application of siRNA, is specially the application of siRNA in the medicine of preparation suppression chicken B lymphocyte CD86 genetic expression.Described chicken B lymphocyte is chicken DT40 cell.
Further, siRNA suppresses the application in the medicine of the short chicken DT40 cell mIgM antibody mrna expression of Synthetic bursin Bursin (BS) in preparation.
Further, the application of siRNA in the immunosuppressor of anti-fowl bone-marrow-derived lymphocyte activation (or Development And Differentiation) of preparation.
Beneficial effect of the present invention:
The present invention is according to chicken B lymphocyte CD86mRNA sequences Design and synthesized micromolecule RNA interfering (sequence is as Suo Shi SEQIDNO:1 ~ 2), this CD86siRNA effectively can suppress the expression of chicken DT40 cell CD86mRNA and CD86 albumen, after interference chicken CD86 genetic expression, the expression level that Synthetic bursin Bursin urgees chicken DT40 cell surface membrane IgM (mIgM) antibody mRNA significantly reduces, and it has a good application prospect in the immunosuppressor of anti-fowl bone-marrow-derived lymphocyte activation (or Development And Differentiation) of preparation.
Accompanying drawing explanation
Fig. 1 is the expression level of fluorescence quantitative PCR detection chicken DT40 cell CD86mRNA;
Fig. 2 is the expression level that Westernblotting detects chicken DT40 cell CD86 albumen;
Fig. 3 is the impact of fluorescence quantitative PCR detection BS on chicken DT40 cell mIgM antibody mrna expression level;
Fig. 4 is that the rear BS of fluorescence quantitative PCR detection CD86siRNA interference is on the impact of chicken DT40 cell mIgM antibody mrna expression level.
Embodiment
Following embodiment is only described in further detail the present invention, but does not form any limitation of the invention.
Embodiment 1
Suppress the siRNA of chicken B lymphocyte CD86 genetic expression, its sense strand sequence is as shown in SEQIDNO:1, and antisense strand sequence, as shown in SEQIDNO:2, has overhang dTdT at 3 ' end of siRNA sequence.
Test example
1, CD86siRNA suppresses the expression of chicken B lymphocyte CD86 gene
1) Design and synthesis of CD86siRNA
According to the known mRNA sequence (NM_KU284846) of chicken B lymphocyte CD86 in GenBank gene pool, according to siRNA principle of design, the siRNA design software of Ambion company is utilized to design following sequence for coding region siRNA target position:
Positive-sense strand: 5 '-GCAGAUGAUUUGGCUAAUU-3 ',
Antisense strand: 5 '-AAUUAGCCAAAUCAUCUGC-3 '.
CD86siRNA sequence is synthesized by Shanghai JiMa pharmacy Technology Co., Ltd.For improving the Gene interfere efficiency of siRNA, overhang at 3 ' end of CD86siRNA sequence the base TT be made up of deoxynucleoside by two.
2) expression level of fluorescence quantitative PCR detection chicken DT40 cell CD86mRNA
Chicken DT40 cell, purchased from Ji Niou bio tech ltd, Guangzhou, is inoculated in 1640 substratum containing 10%FBS by chicken DT40 cell, is placed in 37 DEG C, 5%CO 2middle cultivation, changes liquid 1 time in every 2 days.Treat chicken DT40 cell cultures to 3 × 10 6/ mL, is sub-packed in 24 porocyte culture plates, every hole 400 μ L, changes fresh culture before transfection.Experiment is divided into CD86siRNA group, random controls group (SCR) and blank group (control), random controls group adopts the RNA double-strand (scramble of stochastic sequence, SCR), purchased from Shanghai JiMa pharmacy Technology Co., Ltd, catalog number (Cat.No.): A06001.Use Lipofectamine tM2000 transfection reagent boxes (Invitrogen company) are according to test kit specification sheets transfection CD86siRNA and random controls, the nucleic acid transfection final concentration of CD86siRNA group, random controls group is 100nmol/L, blank group adds serum-free 1640 substratum with medicine equal-volume (100 μ L), transfection final volume 500 μ L.Chicken DT40 cell after transfection continues to cultivate 48h, and extract each group of cell total rna with total RNA extraction reagent box (Qiagen company), the total serum IgE UV detector of extraction surveys OD 260nm/ OD 280nm, detect its purity and concentration.
Utilize OneStep rT-PCRKit (Invitrogen company) is that internal reference detects chicken DT40 cell CD86mRNA expression level with GAPDH: total serum IgE reverse transcription is become cDNA, carries out fluorescence quantitative PCR detection by RocheLightCycler480.Carry out the analysis of Ct value with Rotor-Gene software, adopt 2 -Δ Δ Ctmethod analyzes the expression level of CD86mRNA.Wherein RT-PCR the primer designed, designed (as Suo Shi SEQIDNO:3 ~ 6), is synthesized by Beijing Liuhe Huada Genomics Technology Co., Ltd.
CD86RT-PCR primer is as follows:
Upstream primer: 5 '-CATAATATTACTCCCGG-3 ',
Downstream primer: 5 '-CTTCGTGTACCACTTCA-3 '.
GAPDHRT-PCR primer is as follows:
Upstream primer: 5 '-GTTTACATGTTCAAATATGA-3 ',
Downstream primer: 5 '-GGTGAAGACACCAGTGGACT-3 '.
Fluorescence quantitative PCR detection result as shown in Figure 1, wherein the CD86mRNA expression level of CD86siRNA group compares remarkable decline (P < 0.05) with random controls group with blank group, shows that CD86siRNA effectively can suppress the expression of chicken DT40 cell CD86mRNA.
3) Westernblotting detects the expression level of chicken DT40 cell CD86 albumen
By chicken DT40 cell cultures to 3 × 10 6/ mL, is sub-packed in 24 porocyte culture plates, every hole 400 μ L, changes fresh culture before transfection.Experiment is divided into CD86siRNA group, random controls group (SCR) and blank group (control), random controls group adopts the RNA double-strand (scramble of stochastic sequence, SCR), purchased from Shanghai JiMa pharmacy Technology Co., Ltd, catalog number (Cat.No.): A06001.Use Lipofectamine tM2000 transfection reagent boxes are according to test kit specification sheets transfection CD86siRNA and random controls, the nucleic acid transfection final concentration of CD86siRNA group, random controls group is 100nmol/L, blank group adds serum-free 1640 substratum with medicine equal-volume (100 μ L), transfection final volume 500 μ L.Chicken DT40 cell after transfection continues to cultivate 48h, collecting cell, cell protein is extracted according to cell pyrolysis liquid (RIPA) specification sheets, and utilize Bradford determination of protein concentration kit measurement extract the concentration of albumen, get the solution containing 50 μ g albumen, add albumen sample-loading buffer, be placed in boiling water and heat 5min, the albumen sample handled well is carried out SDS-PAGE, through transferring film, close, add primary antibodie, 4 DEG C of night incubation, TBST washes film 3 times, each 5min, add two anti-(HRP anti-chicken CD86 monoclonal antibodies, abcam company of the U.S.), 37 DEG C of shaking tables hatch 1h, TBST washes film 3 times, each 5min, then with ECL luminescence colour developing (PIERCE).The gray scale of CD86 and β-actin is analyzed, with the relative expression levels of β-actin (the anti-chicken β of HRP-actin monoclonal antibody, abcam company of the U.S.) for internal reference calculating CD86 albumen by ImageJ image analysis software.
Transfection final concentration is that the CD86siRNA of 100nmol/L is after chicken DT40 cell 48h, result as shown in Figure 2, transfection CD86siRNA group, expression level remarkable decline compared with random controls group (P < 0.05) of CD86 albumen, and the expression level of the CD86 albumen of transfection random controls group compared with blank group without considerable change, show that in the present invention, CD86siRNA effectively can suppress the expression of chicken DT40 cell CD86 albumen.
2, CD86siRNA is suppressing the application in chicken DT40 cell CD86 genetic expression
1) Synthetic bursin Bursin (BS) is on the impact of chicken DT40 cell mIgM antibody mrna expression level
By chicken DT40 cell cultures to 3 × 10 6/ mL, adding 2 μ g/mLBS stimulates chicken DT40 cell 4h, and with PBS group for blank group, collecting cell also extracts total serum IgE, according to OneStep total serum IgE reverse transcription is become cDNA by RT-PCRKit specification sheets, carries out fluorescence quantitative PCR detection by RocheLightCycler480.Carry out the analysis of Ct value with Rotor-Gene software, adopt 2 -Δ Δ Ctmethod analyzes BS to the impact of chicken DT40 cell mIgM antibody mrna expression level.
As shown in Figure 3, chicken DT40 cell mIgM antibody mrna expression level remarkable rising compared with PBS control group (P < 0.05) of BS treatment group, shows that BS can promote the expression of chicken DT40 cell mIgM antibody mRNA to result.
2) CD86siRNA suppresses BS to urge the impact of chicken DT40 cell mIgM antibody mrna expression level
By chicken DT40 cell cultures to 3 × 10 6/ mL, add 2 μ g/mLBS and stimulate chicken DT40 cell 4h, experiment is divided into CD86siRNA group, random controls group (SCR) and blank group (control), random controls group adopts the RNA double-strand (scramble of stochastic sequence, SCR), purchased from Shanghai JiMa pharmacy Technology Co., Ltd, catalog number (Cat.No.): A06001.Use Lipofectamine tM2000 transfection reagent boxes are according to test kit specification sheets transfection CD86siRNA, and the nucleic acid transfection final concentration of CD86siRNA group is 100nmol/L, and blank group adds serum-free 1640 substratum of equal-volume (100 μ L), transfection final volume 500 μ L.Chicken DT40 cell after transfection continues to cultivate 48h, extracts each group of cell total rna, utilize OneStep with total RNA extraction reagent box rT-PCRKit take GAPDH as the expression level that internal reference detects chicken DT40 cell mIgMmRNA: total serum IgE reverse transcription is become cDNA, carries out fluorescence quantitative PCR detection by RocheLightCycler480, RT-PCR primer sequence is as shown in SEQIDNO:3 ~ 6.Carry out the analysis of Ct value with Rotor-Gene software, adopt 2 -Δ Δ Ctafter method analyzes CD86siRNA interference, BS is on the impact of chicken DT40 cell mIgM antibody mrna expression level.
Result as shown in Figure 4, (P < 0.05=shows that CD86siRNA can suppress the expression of chicken DT40 cell mIgM antibody mRNA in expression level remarkable reduction compared with control group of the rear BS treatment group chicken DT40 cell mIgM antibody mRNA of CD86siRNA interference.

Claims (6)

1. suppress a siRNA for chicken B lymphocyte CD86 genetic expression, it is characterized in that: the sense strand sequence of described siRNA is as shown in SEQIDNO:1, and antisense strand sequence is as shown in SEQIDNO:2.
2. siRNA according to claim 1, is characterized in that: overhang at 3 ' end of siRNA sequence the base TT be made up of deoxynucleoside by two.
3. siRNA suppresses the application in the medicine of chicken B lymphocyte CD86 genetic expression in preparation as claimed in claim 1 or 2.
4. application according to claim 3, is characterized in that: described chicken B lymphocyte is chicken DT40 cell.
5. siRNA urgees the application in the medicine of chicken DT40 cell mIgM antibody mrna expression at preparation suppression Synthetic bursin Bursin as claimed in claim 1 or 2.
6. the application of siRNA in the immunosuppressor of the anti-fowl bone-marrow-derived lymphocyte Development And Differentiation of preparation as claimed in claim 1 or 2.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106362164A (en) * 2016-09-22 2017-02-01 河南科技大学 SiRNA inhibiting expression of CD86 genes in mouse B lymphocytes and application thereof
CN116426525A (en) * 2023-03-24 2023-07-14 江苏省家禽科学研究所 ASO for inhibiting chicken Wnt3a gene expression and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LEE ET AL: "Role of chicken melanoma differentiation-associated gene 5", 《ARCH VIROL》 *
YIN ET AL: "Bursopentin (BP5) from chicken bursa of fabricius attenuates the immune function of dendritic cells", 《AMINO ACIDS》 *
王臣等: "囊素三肽鸡B淋巴细胞相互作用蛋白的筛选及功能分析", 《科技成果》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106362164A (en) * 2016-09-22 2017-02-01 河南科技大学 SiRNA inhibiting expression of CD86 genes in mouse B lymphocytes and application thereof
CN116426525A (en) * 2023-03-24 2023-07-14 江苏省家禽科学研究所 ASO for inhibiting chicken Wnt3a gene expression and application thereof
CN116426525B (en) * 2023-03-24 2024-03-22 江苏省家禽科学研究所 ASO for inhibiting chicken Wnt3a gene expression and application thereof

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