CN101333528B - SiRNA sequence for HCMV UL122 gene and applications - Google Patents
SiRNA sequence for HCMV UL122 gene and applications Download PDFInfo
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- CN101333528B CN101333528B CN2008100633117A CN200810063311A CN101333528B CN 101333528 B CN101333528 B CN 101333528B CN 2008100633117 A CN2008100633117 A CN 2008100633117A CN 200810063311 A CN200810063311 A CN 200810063311A CN 101333528 B CN101333528 B CN 101333528B
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Abstract
The invention provides a cDNA sequence of siRNA aiming at an HCMV UL122 gene: 5'-GGAAGAACAGGGTGAAGAAGT-3'. According to the design principle of the siRNA, the cDNA sequence of the selected siRNA is 5'-GGAAGAACAGGGTGAAGAAGT-3', and the GC content is 47.6 percent, and the selected siRNA is corresponding to 618-638 nucleotide sites in UL122 mRNA, siRNA eukaryotic expression vectors aiming at the HCMV UL122 gene are constructed, and plasmids are extracted after transforming Escherichia coli for transfecting AD293 cells, thereby effectively inhibiting the UL122 gene mRNA and the protein expression level, and the sequence can be applied to the preparation of drugs for curing cytomegalovirus infection of human.
Description
Technical field
The invention belongs to biotechnology, relate to molecular biology and gene engineering technology field, be specifically related at design, the screening of the siRNA sequence of Human cytomegalic inclusion disease virus (HCMV) UL122 gene and suppress the application of HCMV in vivo and in vitro.
Background technology
1.HCMV the treatment present situation
HCMV belongs to Betaherperesvirinae, infects extensively in the crowd, but mostly is asymptomatic subclinical infection greatly.HCMV infects then can cause the serious disease of multisystem in the crowd (as AIDS, organ transplantation patient etc.) of immunologic hypofunction, and may be relevant with the generation of multiple malignant tumour.Simultaneously, HCMV also is the topmost infectious factors that causes in congenital malformation and newborn infant's inborn defect.Therefore, control HCMV infection is one of focus of domestic and international medical attention always.At present, the medicine of HCMV mainly comprise ganciclovir (ganciclovir, GCV), Trisodium phosphonoformate hexahydrate (fascarnet, FOS) and cidofovir (cidofovir, CDV), but because the appearance of poisonous side effect of medicine and HCMV persister, urgent need is explored new treatment approach control HCMV and is infected.
2.UL122 the present Research of gene and expressing protein thereof
The genomic expression of HCMV has certain sequential, albumen mutually in the time of can being divided at once three kinds of early stage (IE), early stage (E) and late periods (L).Main IE gene UL122 produces the mRNA that length is respectively 2.25kb (exons 1,2,3,5) and 1.70kb (exons 1,2,3,5 ') through alternative splicing, and IE encodes respectively
286 (86kDa, 580 aa) and IE
2Two kinds of nuclear phosphoproteins of 55 (55kDa, 425 aa).Studies show that the UL122 gene is the propagation indispensable gene of HCMV, the HCMV mutant strain of disappearance UL122 can not produce proliferative and infect.The IE of UL122 genes encoding
286 albumen are in the performance keying action of aspects such as virus replication and adjusting host cell cycle.IE
286 not only can the multiple virus of trans-activation and the promotor of cell, and can be as suppressing the proteic expression of the son main IE of downward modulation.In addition, IE
286 suppress the apoptosis of infected host cell by number of ways, comprising: combine with apoptosis inducing factor p53 and make its inactivation; Suppress its activation by the modification of disturbing p53; Prevention does not rely on the apoptosis pathway of p53 such as the death receptor signal pathway that TNF regulates; Induce NF-κ B to transcribe, NF-κ B stops apoptosis by inducing cIAPs under proper condition.
3.RNA interferential present Research
(RNA interference is that sequence-specific double-stranded RNA (dsRNA) makes degradation of homologous mRNA in the cell RNAi), thereby produces the process of expression of specific gene silence in the RNA interference.It is the sequence-specific immunoprotection mechanism of a kind of microRNA mediation of body, can resist the genic effect of invasive.Since 1998 were in the news first, the RNAi technology was applied to rapidly in the research of antiviral and related fields with its distinctive superiority, makes important progress in the antiviral research of HIV, HBV, HCV, influenza virus etc. at present.
The effect of RNAi is mainly by the dsRNA mediation that is about 21～23nt, and wherein a class is called siRNA (small interfering RNA, siRNA), it and purpose mRNA sequence have strict pairing, can cause the corresponding mRNA degraded.Because the specificity degraded that RNAi is siRNA targeting mRNA to be caused, the key that therefore will produce effective RNAi is to select suitable siRNA action target spot.At present the selection principle of RNAi target spot may be summarized to be following some: 1. select the mRNA zone of GC content about 50%; 2. avoid selecting the initial position of promotor downstream 50～100nt with the zone in interior or the terminator upstream 50～100nt; 3. avoid surpassing the overlapping of three G or three C the reticent mechanism of RNA interfering i because poly G or poly C can form the class polymers; 4. select the sequence that begins with two AA, this will make synthetic siRNA be more prone to; 5. guarantee that target sequence and other genes do not have homology.At present, the method for preparing siRNA comprises that mainly chemical synthesis, in-vitro transcription method, long segment dsRNA transcribe 5 kinds of the siRNA expression cassette methods of method and PCR preparation etc. through RNase III edman degradation Edman, siRNA expression vector.
Summary of the invention
The purpose of this invention is to provide the cDNA sequence of a kind of siRNA at human cytomegalovirus UL 122 gene, its nucleotide sequence is: 5 '-GGAAGAACAGGGTGAAGAAGT-3 '.
Another object of the present invention provides the application of this cDNA sequence in preparation treatment human cytomegalovirus infection's medicine.
The present invention is according to the siRNA principle of design, and the GC content of its cDNA sequence of the siRNA of selection is 47.6%, 618-638 nucleotide site among the corresponding UL122mRNA; Structure extracts plasmid at the siRNA carrier for expression of eukaryon of HCMV UL122 gene behind the transformed into escherichia coli, transfection AD293 cell can effectively suppress UL122 gene mRNA and protein expression level, therefore can be applicable to the preparation of HCMV medicine.
Usefulness of the present invention is: the siRNA sequence that provides still is difficult the report of this gene as the RNAi target spot both at home and abroad at present at the propagation indispensable gene UL122 of HCMV.Carrier for expression of eukaryon based on this sequence can effectively suppress UL122 gene mRNA level and protein expression level in cell, therefore can be used as a novel targets of HCMV medicine exploitation.
Embodiment
The present invention is further described in conjunction with the embodiments.
Embodiment one siRNA carrier for expression of eukaryon vitro inhibition UL122-EGFP Expression of Fusion Protein
1.siRNA design: according to the siRNA principle of design, from UL122mRNA initiator codon AUG downstream 100nt search AA sequence, the adjacent 21nt sequence of its 3 ' end is as candidate's target spot, therefrom select GC content in 40～55% siRNA sequence, and compare by the Blast function and the human genomic sequence of GenBank database, guaranteeing does not have homology.Final its cDNA sequence of siRNA of selecting is 5 '-GGAAGAACAGGGTGAAGAAGT-3 ', and GC content is 47.6%, 618-638 nucleotide site among the corresponding UL122mRNA.
2. express the synthetic and preparation of the required shDNA of siRNA: the positive-sense strand sequence of expressing the required shDNA of siRNA is
5 '-GATCC
GGAAGAACAGGGTGAAGAAGTTTCG
ACTTCTTCACCCTGTTCT TCCTTTTTA-3 ', the antisense strand sequence is
5 '-AGCTTAAAAA
GGAAGAACAGGGTGAAGAAGTCGAA
ACTTCTTCACCC TGTTCTTCCG-3 ' after trust biotech firm is synthetic, forms positive and negative adopted chain annealing double-stranded.
3.siRNA Construction of eukaryotic: eukaryon expression plasmid pSilencer2.0-U6 (U.S. Ambion company) BamH I and Hind III double digestion with U6 promotor, be connected with the shDNA after the annealing is double-stranded, transformed into escherichia coli competent cell DH5 α, 37 ℃ of overnight incubation, picking clone extracting plasmid is served Hai Yingjun company and is carried out the dna sequencing evaluation, chooses the correct plasmid amplification of sequencing result, preservation.
4.siRNA the experiment of expression plasmid vitro inhibition UL 122-EGFP expressing fusion protein
(1) reporter plasmid pUL122-EGFP: be the plasmid of expressing green fluorescent protein (EGFP) and UL122 fusion rotein, the Hind III and the EcoR I site structure that are inserted pEGFP-N1 (U.S. Clontech company) by the UL122 gene cDNA form, because EGFP is positioned at the downstream of UL122, and a shared promotor, so the expression of EGFP can reflect the expression of UL122 indirectly.
(2) siRNA expression plasmid and reporter plasmid cotransfection AD293 cell: after HEKC AD293 inoculates 12 porocyte culture plates, treat that cell reaches 80%～90% fusion rate, with the Lipofectamine of Invitrogen company 2000 reagent with siRNA expression plasmid and reporter plasmid pUL122-EGFP cotransfection cell, working method reference reagent specification sheets, establish the negative control group of transfection empty plasmid pSilencer 2.0-U6 and the blank group of not transfection plasmid simultaneously, change nutrient solution (DMEM that contains 10% new-born calf serum) after 5 hours and continue to cultivate.
(3) fluorescence quantitative RT-RCR detects the expression of UL122mRNA in the AD293 cell: 48h behind the plasmid transfection, collect the AD293 cell, Trizol method extracting cell total rna, the SYBR PrimeScript RT-RCP Kit of employing TAKARA company detects the mRNA of viral UL122, experimental technique reference reagent box specification sheets.UL122 gene PCR primer sequence is: upstream 5 '-CGGCTGTATCGTGATCTCTG-3 ', downstream 5 '-CAGGAGGAGGAAGACGAAGA-3 '.Internal reference adopts GAPDH, and the PCR primer sequence is: upstream 5 '-GAAGGTGAAGGTCGGAGTC-3 ', downstream 5 '-GAAGATGGTGATGGGATTTC-3 '.The result shows, compares with negative control group, and the siRNA expression plasmid is 59.4% to the inhibiting rate that UL122mRNA expresses.
(4) flow cytometer detects UL122 protein expression situation in the AD293 cell: 72h behind the plasmid transfection, collect every hole inner cell, and be resuspended among the PBS after washing 2 times with the PBS damping fluid.Under the 488nm excitation wavelength, detect every porocyte average fluorescent strength (mean fluorescence intensity with flow cytometer, MFI) and fluorescencepositive cell ratio α, calculate every porocyte total fluorescence intensity (total fluorescenceintensity, TFI)=MFI * α.The result shows that compare with negative control group, the siRNA expression plasmid is 82.0% to the inhibiting rate of UL122 protein expression.
The present invention binds and closes most preferred embodiment and be described, and after having read foregoing of the present invention, those skilled in the art can do various modifications to the present invention, and these equivalent form of values fall within the appended claims of the present invention institute restricted portion equally.
The sequence that the present invention relates to
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the cDNA sequence of the siRNA of HCMV UL122 gene design
<400>1
GGAAGAACAG?GGTGAAGAAG?T?21
<210>2
<211>57
<212>DNA
＜213〉artificial sequence
<220>
＜223〉sequence of the required shDNA positive-sense strand of preparation UL122 gene siRNA
<400>2
GATCCGGAAG?AACAGGGTGA?AGAAGTTTCG?ACTTCTTCAC?CCTGTTCTTC?CTTTTTA?57
<210>3
<211>57
<212>DNA
＜213〉artificial sequence
<220>
＜223〉sequence of the required shDNA antisense strand of preparation UL122 gene siRNA
<400>3
AGCTTAAAAA?GGAAGAACAG?GGTGAAGAAG?TCGAAACTTC?TTCACCCTGT?TCTTCCG?57
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉UL122 gene PCR upstream primer sequence
<400>4
CGGCTGTATC?GTGATCTCTG?20
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉UL122 gene PCR downstream primer sequence
<400>5
CAGGAGGAGG?AAGACGAAGA?20
<210>6
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAPDH gene PCR upstream primer sequence
<400>6
GAAGGTGAAG?GTCGGAGTC?19
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉GAPDH gene PCR downstream primer sequence
<400>7
GAAGATGGTG?ATGGGATTTC?20
Claims (1)
1. the application of cDNA in preparation treatment human cytomegalovirus infection's medicine at the siRNA of human cytomegalovirus UL 122 gene, the nucleotide sequence of described cDNA is: 5 '-GGAAGAACAGGGTGAAGAAGT-3 '.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2008100633117A CN101333528B (en) | 2008-08-01 | 2008-08-01 | SiRNA sequence for HCMV UL122 gene and applications |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100633117A CN101333528B (en) | 2008-08-01 | 2008-08-01 | SiRNA sequence for HCMV UL122 gene and applications |
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| CN101333528B true CN101333528B (en) | 2011-05-04 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112175948A (en) * | 2020-09-17 | 2021-01-05 | 暨南大学 | Small nucleic acid for inhibiting human cytomegalovirus infection and preparation and application thereof |
| CN114990116B (en) * | 2022-05-24 | 2024-04-26 | 青岛大学 | SiRNA for inhibiting human cytomegalovirus immediate early gene and application thereof |
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