CN103088026A - Artificial miRNA for resisting hepatitis B virus and artificial miRNA combinations - Google Patents
Artificial miRNA for resisting hepatitis B virus and artificial miRNA combinations Download PDFInfo
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- CN103088026A CN103088026A CN2013100395656A CN201310039565A CN103088026A CN 103088026 A CN103088026 A CN 103088026A CN 2013100395656 A CN2013100395656 A CN 2013100395656A CN 201310039565 A CN201310039565 A CN 201310039565A CN 103088026 A CN103088026 A CN 103088026A
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Abstract
The invention relates to the technical field of molecular biology and biological medicine, and particularly relates to application of artificial miRNA (amiRNA for short) aiming at hepatitis B virus (HBV for short) and artificial miRNA combinations in treatment of HBV infectious diseases. The amiRNA and a eukaryote expression vector thereof are respectively designed and constructed specific to conserved regions of different genotypes of the HBV by means of a bioinformatics method and a molecular biology method; and 17 amiRNA and different amiRNA combinations which can obviously control duplication of the HBV are found through in-vitro and vivo screening. Therefore, the amiRNA and the amiRNA combinations are expected to become novel medicines and ways for treating diseases related to the hepatitis B virus.
Description
Technical field
The present invention relates to molecular biology and biological medicine technology field, specifically, the present invention relates to suppress hepatitis B virus (hepatitis B virus, HBV) artificial mi RNA (articifical miRNA, amiRNA) expressed sequence and be combined in the application for the treatment of HBV in catching.
Background technology
It is one of serious transmissible disease of current threat human health that HBV infects, and infects relevant liver failure, liver cirrhosis and primary hepatocellular carcinoma to HBV and approximately causes more than 100 ten thousand people dead every year.China is that HBV infects big country and belongs to high Endemic Area, HBV infects the health that not only endangers China people, cause huge economy and mental burden for patient and family thereof, and caused huge labor force and financial loss to country, and even cause the serious social concerns such as employment.Clinical effective treatment HBV drug main will comprise Interferon, rabbit and nucleoside medicine at present.The Interferon, rabbit curative ratio is limited, and side effect is more, and nucleoside medicine easily recurs after existing and easily causing HBV sudden change resistance and drug withdrawal, and therefore the research and development of novel anti-HBV medicine have important society and economic worth.
MiRNA is the approximately non-coding small molecules single stranded RNA of 22nt of a kind of size, generates after the single stranded RNA precursor that is about 70-80nt by one section length with hairpin ring structure is sheared.MiRNA sequence is near 1～7 zone that the Nucleotide seed zone is the target gene combination of 5 ' end, when miRNA and the incomplete complementation of mRNA, miRNA is attached to 3 of mRNA ' end non-translational region and suppresses its translation, when miRNA and mRNA complete complementary, can cause the said target mrna degraded.Change base under the structure that keeps hairpin miRNA stem ring precursor and the constant condition of energy feature, can produce have equal length with natural miRNA but with the fully irrelevant little RNA of natural miRNA sequence, this small RNA molecular is exactly amiRMA.In amiRNA utilizes, miRNAs precursor synthetic has the miRNA of any gene of target and lowers the expression of this specific gene with this.This specific gene efficient stable silence that realizes by the amiRNA technology is the another important breakthrough in the RNAi evolution, has become the powerful of molecular biology and medicament research and development.
Summary of the invention
The amiRNA expressed sequence and the combination thereof that the object of the present invention is to provide treatment HBV to infect, its use-pattern and carry its expression vector.Specifically, the present invention relates to have the amiRNA expressed sequence that suppresses the HBV Virus reproductivity and be combined in application in the medicine that preparation treatment HBV infects the relative disease that causes.
The amiRNA expressed sequence of target HBV provided by the invention can suppress the expression with virogene of copying of HBV.amiRNA expressed sequence provided by the invention obtains by the following method: but the RNA interfered target sequence of selection design target HBV virogene conserved regions, with this target sequence and reverse complementary sequence replace the miRNA maturation zone of mouse source pre-miRNA155, and be cloned into amiRNA expression vector pOK-basic (biotechnology communication, 2012, 23 (6): 91-95.) obtain corresponding amiRNA expression plasmid, this plasmid is carried out cotransfection experiments with the HBV infective cloned plasmids respectively, expression level by HBsAg and HBeAg in the detection supernatant obtains effective amiRNA expressed sequence.Suppress the amiRNA expressed sequence that HBV expresses, this sequence is selected from:
(1) sequence shown in any one in SEQ ID NO:1-17, perhaps
(2) with (1) in sequence (preferably at least 80%, 85%, 90%, 95%, 98% or higher) conforming sequence that has at least 70%, perhaps
(3) fragment of above-mentioned sequence or complementary sequence
The present invention also provides the combination that comprises the amiRNA expressed sequence that suppresses the HBV expression, has reached the purpose that prevents the escape that virus variation causes and improve viral inhibition.
The recombinant expression vector or the virus vector that contain amiRNA expressed sequence and combination thereof have been the invention still further relates to.
The invention still further relates in genome or carry the amiRNA expressed sequence that the present invention relates to outside genome and the cell of combination, comprise prokaryotic cell prokaryocyte and eukaryotic cell (as bacterial cell, fungal cell, vegetable cell, zooblast, preferred mammal such as people's cell, preferred liver cell and stem cell).
The invention still further relates to the purposes that amiRNA expressed sequence of the present invention and combination, carrier and cell are used for the treatment of HBV infection or HBV patient.
Enforcement of the present invention has important Social benefit and economic benefit to the hepatitis B of serious harm human health and the treatment of relative disease thereof.
Description of drawings
Essence for a better understanding of the present invention, the present invention will be described in more detail below in conjunction with accompanying drawing.
Fig. 1 is pOK-basic carrier collection of illustrative plates, is used for structure and the expression of amiRNA.
Fig. 2 shown 17 amiRNA expressed sequence plasmids of target with HBV infective cloned plasmids cotransfection HepG2 cell after the ability expressed of the inhibition HBV that demonstrates, can suppress the expression of HBsAg and HBeAg in the HepG2 cell.
Fig. 3 shown the plasmid that contains series connection amiRNA expressed sequence (combination) with HBV infective cloned plasmids cotransfection HepG2 cell after the ability of the inhibition HBV expression that demonstrates, can suppress the expression of HBsAg and HBeAg in the HepG2 cell.
After Fig. 4 has shown the plasmid and HBV infective cloned plasmids water power injection mouse tail vein that contains series connection amiRNA expressed sequence (combination), respectively at 2,5,9 and 14 days tail venous blood samplings, the HBsAg content that detects after the proper ratio dilution.Series connection amiRNA expressed sequence (combination) demonstrates the ability of stronger inhibition HBV replication in vivo, can suppress the expression of HBV infective cloned plasmids, reduces the level of HBsAg in serum.
After Fig. 5 has shown the plasmid and HBV infective cloned plasmids water power injection mouse tail vein that contains series connection amiRNA expressed sequence (combination), respectively at 2,5,9 and 14 days tail venous blood samplings, the HBeAg content that detects after the proper ratio dilution.Series connection amiRNA expressed sequence (combination) demonstrates the ability of stronger inhibition HBV replication in vivo, can suppress the expression of HBV infective cloned plasmids, reduces the level of HBeAg in serum.
Embodiment
The invention provides a kind of novel method of carrying out anti-HBV treatment by amiRNA expressed sequence and combination thereof, below the present invention is described in further detail by example.Should be appreciated that specific examples described herein only is used for explaining the present invention, unless stated otherwise, term of the present invention has the normally used implication in this area.
But the invention provides the amiRNA expressed sequence of target HBV, comprise following sequence or have 70% with it at least any one or several sequence in (preferably at least 80%, 85%, 90%, 95%, 98% or higher) conforming sequence: SEQ ID NO:1-17.
Consistence (identity) can be calculated according to method well known in the art.A preferred example that is suitable for the algorithm of definite sequence identity and sequence similarity percentage ratio is BLAST and BLAST2.0 algorithm.Carrying out software that BLAST analyzes can be obtained by the public by state-run biotechnology information center.
Refer to the sequence of expressing or design the target HBV that the mode such as synthetic obtains by design according to amiRNA expressed sequence of the present invention.The promotor of using when it is expressed by the plasmid form can be any promotor that is suitable at the cells goal gene.Can be composing type, can be also epigamic.
The design ﹠ formulation of the amiRNA expression vector plasmid of embodiment one target HBV
The design of the amiRNA expressed sequence of target HBV: take the HBV reference sequences as target sequence, but select the RNA interfered target sequence of design target HBV virogene conserved regions, with this target sequence and reverse complementary sequence replace the miRNA maturation zone of mouse source pre-miRNA155, and be cloned into amiRNA expression vector pOK-basic (legend 1) and obtain corresponding amiRNA expression plasmid.
Embodiment two cotransfection experiments screenings can suppress the amiRNA expressed sequence of HBV
Materials and methods
1. cell cultures
Cell used is for being liver cancer cell HepG2, with cultivating under 37 ℃ of the DMEM cell culture fluids that contains 5% foetal calf serum (FBS, Hyclone), 5%CO2 condition.
2.pOK-basic-amiRNA plasmid is by the invention provides; Hbv replication type plasmid pHBVl.31 contains 1.31 copy HBV DNA and the complete copy unit is arranged, it can complete copying of virus in transfection HepG 2 cell, the secretion of HBsAg (HBsAg) and e antigen (HBeAg) can be detected in the cell culture fluid supernatant, reach cell conditioned medium in cell and the virus replication intermediate can be detected.
3. cell transfecting
The HepG2 cell is cultivated in 37 ℃, 5%CO2 incubator in containing the DMEM nutrient solution of 5%FBS.The observation of cell growth conditions is good, is cultured to the logarithmic growth after date, with HepG2 cell inoculation 24 orifice plates, 8 * 10
4Cells/well, 37 ℃, 5%CO2 was hatched 24-36 hour, after growing to the 70%-90% cell and converging, adopt the GenJet transfection reagent also with reference to specification sheets operation transfection, establish simultaneously the clone have target not anyone and mouse the amiRNA stochastic sequence carrier in contrast.
4.HBsAg, HBeAg detects
48h collecting cell nutrient solution after transfection, good cell culture fluid will be collected, according to HBsAg, HBeAg quantitative ELISA detection kit (AUTOBIO DIAGNOSTICS CO., LTD) specification sheets operation steps detects, and obtaining transfection has HBsAg and the HBeAg concentration that contains in the supernatant of different amiRNA.
Result
With cotransfection the HBsAg in the culture supernatant of HepG2 cell of contrast amiRNA expression plasmid and HBV infective cloned plasmids, HBeAg albumen in contrast, calculate the relative level of HBsAg, HBeAg albumen in each transfection amiRNA supernatant.
The series connection amiRNA of embodiment three target HBV in the mouse model body to the restraining effect of HBV.
Materials and methods
1. animal model
SPF level C57 mouse, 16 ± 18g is available from Military Medical Science Institute's experimentation on animals center.Mode modeling with the large content volume plasmid of mouse tail vein fast injection, amiRNA expression plasmid and 6 μ gpHBV1.18 (containing 1.18 copy HBV DNA) with 6 μ g are dissolved in the PBS of Mouse Weight 1/10 volume altogether respectively, and 5-8 at the uniform velocity injects complete in second.
2.HBsAg, HBeAg detects
After modeling, the 2nd, 5,9 and 14 day mouse tail vein got blood, and after the proper ratio dilution, HBsAg and HBeAg detect according to ELISA immue quantitative detection reagent box (AUTOBIO DIAGNOSTICS CO., LTD) specification sheets operation steps.
Result
Result such as Fig. 4, shown in Figure 5.As seen from the figure, all can significantly suppress the expression level of HBsAg and HBeAg in the series connection ami rna expression plasmid Mice Body that the present invention builds.
Claims (5)
1. suppress the amiRNA expressed sequence of HBV, this sequence is selected from:
1.1SEQ the sequence shown in any one in ID NO:1-17, perhaps
1.2 with 1.1 in sequence (preferably at least 80%, 85%, 90%, 95%, 98% or higher) the conforming sequence that has at least 70%, perhaps
1.3 the fragment of above-mentioned sequence or complementary sequence.
2. suppress the amiRNA expressed sequence combination of HBV, it is characterized in that described combination contains amiRNA expressed sequence claimed in claim 1.
3. gene therapy vector, this carrier can be plasmid vector or virus vector, it is characterized in that described carrier contains claim 1 and amiRNA expressed sequence claimed in claim 2 and combination thereof.
4. the cell of a transformation is characterized in that this cell can express or contain claim 1 and amiRNA expressed sequence claimed in claim 2 and combination thereof or contain the gene therapy vector described in claim 3.
5. medicine and a composition thereof for the treatment of the hepatitis B virus infection relative disease, is characterized in that containing the gene therapy vector described in claim 1 and amiRNA expressed sequence claimed in claim 2 and combination or claim 3 or the cell of a kind of transformation described in claim 4.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11535851B2 (en) | 2016-05-05 | 2022-12-27 | Benitec IP Holdings Inc. | Reagents for treatment of hepatitis B virus (HBV) infection and use thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212555A (en) * | 2011-04-12 | 2011-10-12 | 武汉大学 | Method for constructing pSU6-miR-122 vector expressing miR-122 and application of pSU6-miR-122 vector |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102212555A (en) * | 2011-04-12 | 2011-10-12 | 武汉大学 | Method for constructing pSU6-miR-122 vector expressing miR-122 and application of pSU6-miR-122 vector |
Non-Patent Citations (2)
| Title |
|---|
| 叶海霞等: "amiRNA分子设计及其离体合成策略", 《中国生物工程杂志》 * |
| 赵海峰等: "人工microRNA 抗乙型肝炎病毒效果初探", 《生物技术通讯》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11535851B2 (en) | 2016-05-05 | 2022-12-27 | Benitec IP Holdings Inc. | Reagents for treatment of hepatitis B virus (HBV) infection and use thereof |
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