A kind of siRNA molecule that interferes with hbv gene and its application

Abstract

The invention relates to an siRNA molecule interfering an HBV gene and application thereof. In the siRNA molecule, a positive-sense strand is shown in SEQ ID NO:3, and an antisense strand is shown in SEQ ID NO:4, wherein the antisense strand can be specifically combined with mRNA of an HBV polymerase gene to degrade the mRNA, so that the translation process after transcription is interfered to fulfill the aim of resisting hepatitis B virus.

Description

technical field

[0001] The invention relates to a siRNA molecule interfering with HBV gene and application thereof. Background technique

[0002] Hepatitis, that is, inflammation of the liver, has a variety of pathogenic factors - such as viruses, bacteria, parasites, chemical poisons, drugs and poisons, alcohol, etc., which attack the liver, destroying the cells of the liver and impairing the function of the liver. It can cause A series of symptoms of discomfort in the body, as well as abnormalities in liver function indicators. In most cases, hepatitis refers to viral hepatitis caused by hepatitis A, B, C, D, E and other hepatitis viruses.

[0003] Hepatitis B caused by hepatitis B virus (Hepatitis B virus, HBV) infection is an infectious disease that seriously threatens global human health, and is also the most serious type of viral hepatitis. It can cause chronic liver disease, and patients die of The risk of cirrhosis and liver cancer is extremely high.

[0004] HBV ...

Examples

preparation example Construction

[0030] Various methods can be used to prepare siRNA, such as: chemical synthesis, in vitro transcription, long-chain dsRNA enzyme digestion, vector expression of siRNA, PCR synthesis of siRNA expression elements, etc. The emergence of these methods provides researchers with options. Better access to gene silencing efficiency.

[0031] The siRNA molecule of the present invention can be used as an active ingredient of an anti-HBV medicine.

[0032] As another expression form of the siRNA molecule, it can be prepared in the form of a DNA expression cassette, such as: U6 promoter-siRNA transcription template-H1 promoter.

[0033] In short, "U6 promoter-siRNA transcription template-H1 promoter" is abbreviated as "U6-siRNA transcription template-H1" or "U6-siRNA-H1" hereinafter, which have the same meaning and scope.

[0034] For the purpose of application, the siRNA molecule, the DNA expression cassette expressing the siRNA molecule, or the plasmid containing the siRNA molecule ex...

Embodiment 1

[0044] Preparation of siRNA all-site molecular library

[0045] 1. Main instruments, reagents and materials

[0046] 1.1 Instruments: PCR instrument (ABI Company); electrotransfer instrument (Bio-Rad Company); centrifuge (Eppendorf Company), long-wavelength ultraviolet lamp, etc.

[0047] 1.2 Materials and reagents: HepG22.2.15 cells (preserved by Biomec); 1kb plus DNA Ladder (invitrogen); DNase I (Roche); MnCl 2 (BBI Company); Phosphate linker (Sigma-aldrich Company); ATP (BBI Company); BSA (NEB Company); BmsbI (NEB Company); T4 DNA ligase (NEB Company); Tag DNA polymerase (Biometics ); Agarose (BBI company); dNTP (Shanghai Sangong); Phenol chloroform extraction reagent (Shanghai Sangong); Low molecular weight DNA Ladder (NEB company); EcoP15I (NEB company); T4DNA polymerase (NEB company); FokI enzyme (NEB company); SfiI enzyme (NEB company); Competent cells (invitrogen company); pU6H1-GFP expression vector (NT Oimcs company). Gel extraction kit: QIAEX II Gel Extraction Ki...

Embodiment 2

[0103] Preparation of siRNA expression cassette and screening of target sites

[0104] 1. Main instruments, reagents and materials

[0105] 1.1 Instruments: PCR instrument (ABI Company); real-time quantitative PCR instrument (Bio-Rad Company); gel electrophoresis equipment (Beijing Liuyi); long-wavelength ultraviolet lamp; cell incubator (Thermo Company), etc.

[0106] 1.2 Materials and reagents: 1kb plus DNA Ladder (Invitrogen); Pfu DNA polymerase (Biometics); agarose (BBI); dNTP (Shanghai Sangong); Maike Corporation), Lipofectamin TM 2000 (invitrogen company), DMEM medium (Gibco company), TurboCapturemRNA kit (QIAGEN company), SensiMix TM One-Step Kit (Quantace Company), etc. Other biochemical reagents were purchased from Shanghai Sangong.

[0107] 1.3PCR primers (synthesized by Biometics):

[0108] 5'U6 promoter primer: 5'-AAGGTCGGGCAGGAAGAGGGC-3'

[0109] 3'H1 promoter primer: 5'-TATTTGCATGTCGCTATGTGTTCT-3'

[0110] HBV polymerase forward primer: 5'-TGTGGTTATCCTGCG...

technical field

[0001] The invention relates to a siRNA molecule interfering with HBV gene and application thereof. Background technique

[0002] Hepatitis, that is, inflammation of the liver, has a variety of pathogenic factors - such as viruses, bacteria, parasites, chemical poisons, drugs and poisons, alcohol, etc., which attack the liver, destroying the cells of the liver and impairing the function of the liver. It can cause A series of symptoms of discomfort in the body, as well as abnormalities in liver function indicators. In most cases, hepatitis refers to viral hepatitis caused by hepatitis A, B, C, D, E and other hepatitis viruses.

[0003] Hepatitis B caused by hepatitis B virus (Hepatitis B virus, HBV) infection is an infectious disease that seriously threatens global human health, and is also the most serious type of viral hepatitis. It can cause chronic liver disease, and patients die of The risk of cirrhosis and liver cancer is extremely high.

[0004] HBV ...

Examples

preparation example Construction

[0030] Various methods can be used to prepare siRNA, such as: chemical synthesis, in vitro transcription, long-chain dsRNA enzyme digestion, vector expression of siRNA, PCR synthesis of siRNA expression elements, etc. The emergence of these methods provides researchers with options. Better access to gene silencing efficiency.

[0031] The siRNA molecule of the present invention can be used as an active ingredient of an anti-HBV medicine.

[0032] As another expression form of the siRNA molecule, it can be prepared in the form of a DNA expression cassette, such as: U6 promoter-siRNA transcription template-H1 promoter.

[0033] In short, "U6 promoter-siRNA transcription template-H1 promoter" is abbreviated as "U6-siRNA transcription template-H1" or "U6-siRNA-H1" hereinafter, which have the same meaning and scope.

[0034] For the purpose of application, the siRNA molecule, the DNA expression cassette expressing the siRNA molecule, or the plasmid containing the siRNA molecule ex...

Embodiment 1

[0044] Preparation of siRNA all-site molecular library

[0045] 1. Main instruments, reagents and materials

[0046] 1.1 Instruments: PCR instrument (ABI Company); electrotransfer instrument (Bio-Rad Company); centrifuge (Eppendorf Company), long-wavelength ultraviolet lamp, etc.

[0047] 1.2 Materials and reagents: HepG22.2.15 cells (preserved by Biomec); 1kb plus DNA Ladder (invitrogen); DNase I (Roche); MnCl 2 (BBI Company); Phosphate linker (Sigma-aldrich Company); ATP (BBI Company); BSA (NEB Company); BmsbI (NEB Company); T4 DNA ligase (NEB Company); Tag DNA polymerase (Biometics ); Agarose (BBI company); dNTP (Shanghai Sangong); Phenol chloroform extraction reagent (Shanghai Sangong); Low molecular weight DNA Ladder (NEB company); EcoP15I (NEB company); T4DNA polymerase (NEB company); FokI enzyme (NEB company); SfiI enzyme (NEB company); Competent cells (invitrogen company); pU6H1-GFP expression vector (NT Oimcs company). Gel extraction kit: QIAEX II Gel Extraction Ki...

Embodiment 2

[0103] Preparation of siRNA expression cassette and screening of target sites

[0104] 1. Main instruments, reagents and materials

[0105] 1.1 Instruments: PCR instrument (ABI Company); real-time quantitative PCR instrument (Bio-Rad Company); gel electrophoresis equipment (Beijing Liuyi); long-wavelength ultraviolet lamp; cell incubator (Thermo Company), etc.

[0106] 1.2 Materials and reagents: 1kb plus DNA Ladder (Invitrogen); Pfu DNA polymerase (Biometics); agarose (BBI); dNTP (Shanghai Sangong); Maike Corporation), Lipofectamin TM 2000 (invitrogen company), DMEM medium (Gibco company), TurboCapturemRNA kit (QIAGEN company), SensiMix TM One-Step Kit (Quantace Company), etc. Other biochemical reagents were purchased from Shanghai Sangong.

[0107] 1.3PCR primers (synthesized by Biometics):

[0108] 5'U6 promoter primer: 5'-AAGGTCGGGCAGGAAGAGGGC-3'

[0109] 3'H1 promoter primer: 5'-TATTTGCATGTCGCTATGTGTTCT-3'

[0110] HBV polymerase forward primer: 5'-TGTGGTTATCCTGCG...

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