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Hepatitis B virus surface protein mutant and application thereof in resisting hepatitis B virus

A protein mutant, hepatitis B virus technology, applied in the field of biomedicine, can solve the problems of vaccine immune escape and HBsAg detection failure

Active Publication Date: 2021-08-03
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mutation of S protein is mainly manifested as the change of the epitope that is crucial to the host's immune recognition, such as the mutation of some sites in the main hydrophilic region, which can cause immune escape from the vaccine and the failure of HBsAg detection
In addition, because the PreS / S gene overlaps with the P gene encoding HBV polymerase, the mutation of the PreS / S gene may also cause a mutation of the P gene, resulting in drug resistance mutations that are insensitive to direct antiviral drugs

Method used

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  • Hepatitis B virus surface protein mutant and application thereof in resisting hepatitis B virus
  • Hepatitis B virus surface protein mutant and application thereof in resisting hepatitis B virus
  • Hepatitis B virus surface protein mutant and application thereof in resisting hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction and expression verification of pS-WT and mutant recombinant plasmids

[0054] In this example, the S protein of hepatitis B virus was taken as an example to carry out the construction and expression verification of its recombinant plasmid.

[0055] The construction of recombinant plasmid, it comprises steps:

[0056] 1. Construct pS-WT recombinant plasmid on the backbone of pCDNA3 vector. The S gene of the HBV B6 strain was inserted between BamHI and NotI by enzyme digestion and ligation, as follows: the pHBV B6 1.3 replicon was used as a template, and S-WT was obtained by PCR amplification experiment and gel recovery. Gene fragment. PCR primers are S-F and S-R.

[0057] S-F: 5'-CGCGGATCCATGGAGAACATCGCATCAGGACT-3' (SEQ ID NO: 30)

[0058] S-R: 5'-ATTTGCGGCCGCTTAAATGTATACCCAAAAGACAA-3' (SEQ ID NO: 31)

[0059] The PCR reaction system is as follows:

[0060]

[0061] The PCR reaction conditions are as follows: 98°C for 3min; 98°C for 1...

Embodiment 2

[0077] Example 2: Effects of the S-L13R / D / E / K mutant on the replication of the HBV replicon pHBV B6 1.3 and the expression and secretion of HBsAg at the cellular level

[0078] Use Southern blot to detect HBV replication ability, use ELISA to detect the expression and secretion of HBsAg

[0079] 1. Co-transfect Huh7 cells with pHBV B6 1.3 and pS-WT, pS-L13R, pS-L13K, pS-L13E, pS-L13D and pCDNA3 (pCtrl) constructed according to the method of Example 1 in a ratio of 1:2, After 72 hours of transfection, the cell supernatant and cell lysate were collected to detect HBV replication ability by Southern blot, and the expression and secretion of HBsAg were detected by ELISA (the steps were the same as those in Example 1).

[0080] 2. The Southern blot method was used to detect HBV DNA in the core granules of cells. Methods as below:

[0081] (1) Cell lysis: the cells were lysed by the same cell lysis method as in Example 1.

[0082] (2) DNase I treatment: collect the cell lysate ...

Embodiment 3

[0105] Example 3: Effects of S-L13R / D / E / K mutants on the expression and secretion of wild-type S protein at the cellular level

[0106] In order to verify whether the S-L13R / D / E / K mutant has a direct inhibitory effect on the expression and secretion of the wild-type S protein, in this example, plasmids such as pS-WT and pS-L13R were divided into 1:0 and 1: 1. Co-transfect Huh7 cells at the ratio of 1:2, 1:4, and 1:8, in which the transfection amount of pS-WT is constant, and the transfection amount of pS-L13R increases with the ratio, and the total transfection is completed with pCDNA3 plasmid quantity. After 48 hours of transfection, the cell supernatant and lysate were collected, and the HBsAg in the intracellular and supernatant were determined by ELISA method (the procedure was the same as the detection method in Example 1).

[0107] The result is as image 3 As shown, the ELISA results showed that with the increase of pS-L13R transfection amount, the level of HBsAg in t...

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Abstract

The invention relates to a hepatitis B virus surface protein mutant, in particular, relates to an S protein mutant which can inhibit hepatitis B virus (HBV) replication and / or inhibit surface antigen (HBsAg) expression and secretion, and further relates to a biological material related to the S protein mutant and an application of the S protein mutant in HBV resistance. In-vitro tests show that an S-L13R / E / D / K mutant can be used for inhibiting the replication of HBV in Huh7 cells and inhibiting the expression and secretion of HBsAg; in an Adv / prcccDNA hepatitis B chronic mouse model, blood HBsAg is converted into negative, surface antibody HBsAb can appear in blood, HBeAg level and HBV DNA level in serum are also converted into negative, and no obvious ALT rise is caused in the negative conversion process, so that the safety of the antibody is proved, the S protein mutant has obvious influence on the secretion of HBsAg of different genotypes of HBV, and the S protein mutant of different genotypes has obvious influence on the secretion of HBsAg of the same genotype of HBV, so that a new method is provided for clinically treating chronic hepatitis B.

Description

technical field [0001] The present invention relates to the technical field of biomedicine, in particular to a hepatitis B virus surface protein mutant and its application in anti-hepatitis B virus, specifically a hepatitis B virus S protein mutant, which can inhibit the replication of human hepatitis B virus and inhibit the hepatitis B surface antigen expression and secretion. Background technique [0002] Human hepatitis B virus (HBV) is the causative agent of chronic hepatitis B (CHB). There are 270 million people with HBV chronic infection in the world, and about 1 million people die of liver diseases related to HBV infection every year, such as cirrhosis, liver failure and liver cancer, which seriously threaten public health. The "functional cure" of chronic hepatitis B refers to that after treatment, the serum hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are both negative, and may be accompanied by hepatitis B surface antibody (HBsAb) and hepa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/02C12N15/51C12N15/11A61K39/29A61P31/20
CPCC07K14/005A61K39/12A61P31/20C12N2730/10122C12N2730/10134
Inventor 谢幼华郭慧敏王葳刘晶高子翔邓强
Owner FUDAN UNIV
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