A kind of hepatitis B virus surface protein mutant and its application in anti-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: 2022-08-02
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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  • A kind of hepatitis B virus surface protein mutant and its application in anti-hepatitis B virus
  • A kind of hepatitis B virus surface protein mutant and its application in anti-hepatitis B virus
  • A kind of hepatitis B virus surface protein mutant and its application in anti-hepatitis B virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction and expression verification of pS-WT and mutant recombinant plasmids

[0053] In this example, the construction of the recombinant plasmid and the verification of its expression are carried out by taking the hepatitis B virus S protein as an example.

[0054] The construction of recombinant plasmid, it comprises steps:

[0055] 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 the method of enzyme cleavage and ligation, as follows: Using the pHBV B6 1.3 replicon as a template, S-WT was obtained by PCR amplification experiments and gel recovery. Gene fragment. PCR primers were S-F and S-R.

[0056] S-F: 5'-CGCGGATCCATGGAGAACATCGCATCAGGACT-3' (SEQ ID NO: 30)

[0057] S-R: 5'-ATTTGCGGCGCTTAAATGTATACCCAAAGACAA-3' (SEQ ID NO: 31)

[0058] The PCR reaction system is as follows:

[0059]

[0060] The PCR reaction conditions were as follows: 98°C fo...

Embodiment 2

[0075] Example 2: Effect of S-L13R / D / E / K mutant on the replication of HBV replicon pHBV B6 1.3 and the expression and secretion of HBsAg at the cellular level

[0076] HBV replication ability was detected by Southern blot, and HBsAg expression and secretion were detected by ELISA

[0077] 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 at 1:2, respectively, 72h after transfection, the cell supernatant and cell lysate were collected for Southern blot to detect the HBV replication ability, and ELISA (the same procedure as the detection method in Example 1) was used to detect the expression and secretion of HBsAg.

[0078] 2. Southern blot was used to detect HBV DNA in cell core granules. Methods as below:

[0079] (1) Cell lysis: The cells were lysed by the same cell lysis method as in Example 1.

[0080] (2) DNase I treatment: the cell lysate was collected and shak...

Embodiment 3

[0103]Example 3: Effect of S-L13R / D / E / K mutant on the expression and secretion of wild-type S protein at the cellular level

[0104] 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, the plasmids such as pS-WT and pS-L13R were 1:0 and 1:1, respectively. The ratios of 1, 1:2, 1:4, and 1:8 were used to co-transfect Huh7 cells. The transfection volume of pS-WT was constant, and the transfection volume of pS-L13R increased with the ratio. The total transfection was completed with pCDNA3 plasmid. quantity. 48h after transfection, the cell supernatant and lysate were collected to measure HBsAg in intracellular and supernatant by ELISA method (the steps were the same as the detection method in Example 1).

[0105] The result is as image 3 As shown, the ELISA results showed that with the increase of the transfection amount of pS-L13R, the level of HBsAg in the supernatant gr...

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Abstract

The present invention relates to a hepatitis B virus surface protein mutant, specifically an S protein mutant, which can inhibit the replication of hepatitis B virus (HBV) and / or inhibit the expression and secretion of surface antigen (HBsAg), and also relates to a mutant with the above S protein. Body-related biomaterials and their application in anti-HBV. In the in vitro test, it is found that the S-L13R / E / D / K mutant can inhibit the replication of HBV in Huh7 cells, and inhibit the expression and secretion of HBsAg; HBsAg turned negative, surface antibody HBsAb could appear in the blood, HBeAg level and serum HBV DNA level also turned negative, and there was no obvious increase in ALT during the negative process, which proved its safety. The secretion of HBsAg of different genotypes of HBV and the S protein mutants of different genotypes have significant effects on the secretion of HBsAg of the same genotype of HBV, which provides a new method for clinical treatment of chronic hepatitis B.

Description

technical field [0001] The 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, in particular to 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 chronic HBV infection in the world, and about 1 million people die of liver diseases related to HBV infection every year, such as liver cirrhosis, liver failure and liver cancer, which seriously threatens public health. "Functional cure" of chronic hepatitis B means that after treatment, serum hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are turned negative, and may be accompanied by hepatitis B surface antibody (HBsAb) and hepatitis B...

Claims

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Application Information

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Patent Type & Authority Patents(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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