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Recombinant human hepatitis B virus core protein fused protein

A hepatitis B virus and fusion protein technology, applied in the field of molecular biology, can solve the problems of inability to form core particles and maintain the normal function of HBc, etc., and achieve the effect of broad application prospects

Active Publication Date: 2015-06-10
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Weigand et al. (J Gen Virol, 2009) fused GFP to HBc and found that the fusion protein can maintain the normal function of GFP (green fluorescence), but cannot maintain the normal function of HBc, that is, it cannot form core particles

Method used

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  • Recombinant human hepatitis B virus core protein fused protein
  • Recombinant human hepatitis B virus core protein fused protein
  • Recombinant human hepatitis B virus core protein fused protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, HBV1.1c- and HBc expression vector construction

[0046] The successful replication of HBV DNA requires the joint action of core protein gene, HBV polymerase (Pol) gene and pregenomic RNA (pgRNA).

[0047] First construct the HBc plasmid containing HBV core protein gene as the basis of HBc transformation. The constructed HBc plasmid only expresses the HBc protein encoded by the Core gene in HBV, and does not express the Pol gene and pgRNA.

[0048]In order to test the function of the transformed HBc, it is also necessary to construct the plasmid HBV1.1c- which lacks the expression of HBc. Plasmid HBV1.1c- cannot express HBV core protein gene, but can express HBV polymerase (Pol) gene and pre-genomic RNA (pgRNA); HBc can provide HBV core protein in trans and support HBV DNA replication. HBV DNA replication can be restored when there are functionally normal HBc and HBV1.1c- present at the same time.

[0049] The method for constructing HBc is to remove mo...

Embodiment 2

[0065] Example 2: Construction of recombinant HBc with glycine-serine linkers of different lengths fused to the N-terminus

[0066] Recombinant HBc expression vectors with glycine-serine linkers with N-terminal fusion lengths of 47aa, 92aa, and 186aa were constructed.

[0067] 1. Construction of 12G4S-HBc

[0068] On the basis of the aforementioned HBc plasmid, construct 12G4S-HBc with 12 amino acid linkers fused to the N-terminus of HBc. The amino acid sequence of the 12G4S linker is: GSGGGGSGGGGS, and the nucleotide sequence of the 12G4S linker is: GGTTCAG GAGGTGGTGG ATCTGGAGGA GGTGGATCT. The specific construction method of 12G4S-HBC is:

[0069] (1) Use the upstream primer F12G4S: 5'-GCTTAGCCTTGGGTGGCTTTGGGGCATGGGTTCAGGAGGTGGTGGATCTGGAGGAGGTGGATCTGACATCGACCCTTAAAGA-3', and the downstream primer R1798: 5'-CCAATTTATGCCTACAGCCT-3' to amplify the HBc plasmid. Template, 0.5ul PrimeSTAR HS polymerase, 5×PrimeSTAR HS buffer 10μl, dNTP 4μl, and sterilized ultrapure water to make ...

Embodiment 3

[0116] Example 3. Verification of the influence of the fusion of G4S linkers of different lengths on the normal function of HBc

[0117] The 12G4S-HBc, 47G4S-HBc, 92G4S-HBc and 186G4S-HBc constructed above were co-transfected with HBV1.1c- into HEK293 cells, and the cells were collected after 4 days, and the HBV DNA of the intracellular core particles was extracted, and then detected by Southern blot. The result is as image 3 As shown, when the above four kinds of glycine-serine fusion HBc proteins are co-transfected with HBV1.1c-, they can all support HBV replication, and it can be seen that the intermediate rcDNA and ssDNA are obviously replicated, and 47G4S-HBc even shows a significantly stronger replication level than HBc , while the HBV replication level of 92G4S-HBc and 186G4S-HBc is slightly lower than that of HBc, indicating that the insertion of long polyglycine sequences has less interference with HBc function, and even enhances HBc function in some cases.

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Abstract

The invention discloses a recombinant human hepatitis B virus core protein fused protein. The fused protein comprises a protein (X), a linker peptide (L) and a hepatitis B virus core protein (HBc) from the end N to the end C in sequence; the linker peptide (L) has the amino acid sequence of Gly-Ser-(Gly-Gly-Gly-Gly-Ser)n, and n is an integer between 2 and 20 and is 9 or 18, particularly; the end C of the linker peptide (L) is connected with the end N of the hepatitis B virus core protein (HBc); the end C of the protein (X) is connected with the end N of the linker peptide (L); and the protein (X) is a red fluorescent protein or vesicular stomatitis virus G glycoprotein. The hepatitis B virus core protein (HBc) is connected with the functional protein, and the functions of the proteins on two ends of the linker peptide (L) can be both guranteed. The problem in the prior art that the functions of the hepatitis B virus core protein (HBc) and the functions of the functional protein can not be both guaranteed after the hepatitis B virus core protein (HBc) is fused with the functional protein is solved. The fused protein is of great importance to the research on the hepatitis B virus (HBV).

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a fusion protein, in particular to a recombinant human hepatitis B virus core protein fusion protein. Background technique [0002] Fusion protein refers to the use of genetic engineering technology to fuse two or more proteins that are separated from each other in the natural state. Each part of the fusion is on the same amino acid chain and expressed as a whole. Fusion proteins are widely used in life science-related research fields and biological industries. [0003] The usual method for fusion expression of two proteins is to link the DNA sequences encoding the two proteins together in order to form a fused DNA molecule, and after the DNA molecule is transferred into cells, it is driven by a eukaryotic promoter , the corresponding mRNAs can be transcribed, and then these mRNAs are used as templates to produce fusion proteins using the translation machinery of the cell. [0004...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63
Inventor 胡接力黄爱龙陈江燕
Owner CHONGQING MEDICAL UNIVERSITY
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