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A kind of hbv cccDNA nucleosome assembled in vitro and preparation method thereof

A nucleosome and prokaryotic technology, which is applied in the field of preparation of HBVcccDNA nucleosomes, can solve the problems of various types of cccDNA nucleosomes, small and uneven cccDNA nucleosomes, and achieve non-digestible degradation, high stability, high yield effect

Active Publication Date: 2021-05-04
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, not only the amount of cccDNA nucleosomes isolated from cells infected with hepatitis B virus is small, but also the types of cccDNA nucleosomes are diverse and uneven, which cannot meet actual needs
Currently there is no better way to obtain or prepare HBV cccDNA nucleosomes

Method used

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  • A kind of hbv cccDNA nucleosome assembled in vitro and preparation method thereof
  • A kind of hbv cccDNA nucleosome assembled in vitro and preparation method thereof
  • A kind of hbv cccDNA nucleosome assembled in vitro and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] An HBV cccDNA nucleosome assembled in vitro, including protein complex and HBV cccDNA fragment X1 (SEQID No.1).

[0054] The preparation method of above-mentioned HBV cccDNA nucleosome comprises the following steps:

[0055] Preparation of HBV cccDNA Fragment

[0056] Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to chemically synthesize HBV cccDNA and integrate it into the pUC-D plasmid to obtain a recombinant plasmid;

[0057] Using the recombinant plasmid as a template, the X1 primer was used for PCR amplification; the PCR product was isolated and purified using a Source 15Q 4.6 / 100 PE ion column (manufacturer: General Electric Medical) to obtain the HBV cccDNA fragment X1 (SEQ ID No.1).

[0058] The sequence of the X1 primer is X1-F: 5'-AATTCTGTCGTGCTCTCCCGCAAG-3', X1-R: 5'-AGCCCCAAGCGGCCCCG-3'.

[0059] The PCR amplification conditions are as follows: 95°C for 30s; 40 cycles of 95°C for 10s, 54°C for 30s, 72°C for 15s; 72°C for 10min.

[0060] Preparation o...

Embodiment 2

[0073] Compared with Example 1, the difference of Example 2 is that the primers amplified by PCR are X2 primers, and the obtained HBVcccDNA fragment is X2 (SEQ ID No.2);

[0074] The sequence of the X2 primer is X2-F: 5'-CTCCGCCTCTTGTACCGACCG-3', X2-R: 5'-CAGGTTCCTGTGGGCGTTCAC-3';

[0075] The PCR amplification conditions are: 95°C for 30s; 40 cycles of 95°C for 10s, 57°C for 30s, 72°C for 15s; 72°C for 10min.

Embodiment 3

[0077] Compared with Example 1, the difference of Example 3 is that the primers amplified by PCR are PreX primers, and the HBVcccDNA fragment obtained is PreX (SEQ ID No.3);

[0078] The sequence of the PreX primer is PreX-F: 5'-GGAACATATTGTAAGAAAAATCAAAAT G-3', PreX-R: 5'-AAGGCATTAAAGCAGGATATCCAC-3';

[0079] The PCR amplification conditions are as follows: 95°C for 30s; 40 cycles of 95°C for 10s, 48°C for 30s, 72°C for 15s; 72°C for 10min.

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Abstract

The invention belongs to the field of biotechnology, and discloses a HBV cccDNA nucleosome assembled in vitro, including HBV cccDNA fragments and histones. The HBV cccDNA fragments are selected from X1 as shown in SEQ ID No.1, such as SEQ ID No. .2 at least one of X2 shown in 2, PreX shown in SEQ ID No.3 or C1 shown in SEQ ID No.4. The invention also discloses a method for preparing the HBV cccDNA nucleosome assembled in vitro, which includes the following steps: S1: obtaining HBV cccDNA fragments through PCR amplification or prokaryotic amplification; S2: prokaryotic expression of histones, and the obtained histones The protein is recombined in vitro to obtain a protein complex; S3: the HBV cccDNA fragment and the protein complex are assembled in vitro to obtain the HBV cccDNA nucleosome. The preparation method can stably produce a sufficient amount of cccDNA nucleosomes with a highly uniform structure, and can meet the requirements of relevant functional experiments such as disease drug research.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a preparation method of HBV cccDNA nucleosome assembled in vitro. Background technique [0002] Hepatitis B virus (Hepatitis B virus, HBV) is referred to as hepatitis B virus for short. The chronic infection of this virus is a major global public health problem including my country. Hepatitis B virus is also one of the important factors leading to the occurrence of primary hepatocellular carcinoma in East Asia, which poses a serious threat to people's health. [0003] At present, there are two main types of drugs used in the clinical treatment of hepatitis B virus: (1) Interferon drugs, including IFNα-2b, PEG-IFNα-2a, etc., can enhance the body’s anti-hepatitis B immunity by regulating the host’s immune response against the virus. Virus clearance; (2) Nucleotide analogs, including lamivudine (lamivudine), adefovir (Adefovir), entecavir (Entecavir), telbivudine (telbivudine), tenofov...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04
CPCC12N7/00C12N2730/10121C12N2730/10151
Inventor 陈守登周子亮陈小雪黄钊霞
Owner THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV