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Strategically modified hepatitis b core proteins and their derivatives

A core protein and strategic technology, applied in anti-animal/human immunoglobulins, microorganisms, immunoglobulins, etc., can solve the problem that amino acid side chains do not show chemical reactivity, and achieve the effect of enhancing chemical reactivity

Inactive Publication Date: 2001-06-20
IMMUNE COMPLEX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] However, the amino acid side chains do not show obvious chemical reactivity in the natural sequence of the natural unmodified HBV core protein particle

Method used

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  • Strategically modified hepatitis b core proteins and their derivatives
  • Strategically modified hepatitis b core proteins and their derivatives

Examples

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Effect test

preparation example Construction

[0092] Ⅲ. Preparation of strategically modified core protein

[0093] The strategic modification of hepatitis B core protein is usually performed at the DNA level by methods known in the art, for example, by inserting codons corresponding to the amino acid sequence to be inserted. The manipulated gene is then expressed in a convenient system known in the art, for example in a viral culture of infected immortalized cells.

[0094] The method of preparing HBcAg protein in general and the method of preparing pre-core, core and HBeAg protein in particular are well known in the art. The same method can easily be modified into a method for separating the modified core protein particles of the present invention. In addition, HBcAg and HBeAg can be produced by a variety of well-known recombinant DNA techniques. See, for example, U.S. Patent No. 4,356,270 to Itakura and 4,563,423 to Murray et al. These recombinant DNA technologies can easily be adapted into methods for producing the modifi...

Embodiment 1

[0161] Example 1: Construction of modified hepatitis B core protein expression vector.

[0162] Site-directed mutagenesis introduced a lysine codon (TTT) between amino acids D78 and P79 of the HBc gene, and also introduced EcoRI (GAATTC) and SacⅠ (GAGCTC) restriction endonuclease sites to facilitate genetic insertion of other codons To produce strategically modified hybrid HBc particles. The amino acid residue sequence of such an inserted sequence is GIQKEL, where GIQ is the product of EcoRI site and EL is the product of SacI site. Therefore, the length of the strategically modified hepatitis B core protein is 155 amino acid residues. The construction of pKK322-HBc155-K81 expression plasmid is described below.

[0163] The 5'end (bases 1-234, amino acids 1-78) of the HBc gene was amplified with oligonucleotide primers P1F (SEQ ID NO: 17) and P1R (SEQ ID NO: 18, on the complementary strand) and At the same time, a NcoI restriction site (CCATGG) was introduced at the 5'end of the am...

Embodiment 2

[0165] Example 2: Purification of modified hepatitis B core protein particles

[0166] E. coli, usually E. coli BLR or BL21 from Novagen (Madison, Wisconsin) or E. coli TB11 from Amersham (Arlington Heights, Illinois), expresses the strategically modified HBc protein of Example 1. HBc155-K81 transfected with E. coli is the expression plasmid pKK332-HBc155-K81. The strategically modified HBc particles were purified by an established method using Sepharose CL-4B chromatography. Since the particles are purified in a predictable manner, simple spectrophotometry (OD 280 ) Plus the use of SDS-PAGE to detect the purity of each fraction before collection to monitor the elution of particles is sufficient to allow conventional purification to obtain high-yield electrophoretic pure particles (5-120mg / L cell culture). The spherical structure of the purified strategically modified hepatitis B core particles can be clearly seen under the electron microscope.

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Abstract

A strategically modified hepatitis B core protein is described, where an insert is provided, preferably in an immunodominant region of the nucleocapsid protein, containing a chemically reactive amino acid residue. The modified hepatitis B core protein or its aggregated nucleocapsid protein particles can be pendently linked to a hapten to form a modified nucleocapsid conjugate. Such a conjugate is useful in the preparation of vaccines or antibodies. The modified hepatitis B core protein can also be modified to include a T cell epitope.

Description

Technical field [0001] The present invention relates to the cross-fields of immunology and protein engineering, in particular to carrier proteins, and more specifically to hepatotropic DNA virus nucleocapsid proteins that are strategically modified by inserting chemically reactive amino acid residues, Pendently linked hapten conjugates of the hepatic DNA virus nucleocapsid protein, and related immunogenic particles composed of these conjugates. Background of the invention [0002] It is known that antibodies against small molecules can be produced by using large immunogenic protein molecules as carriers. Small molecules whose immunogenicity is enhanced by being linked to a carrier are called haptens. The phenomenon that relatively large molecules enhance the immunogenicity of small molecule entities is called "carrier effect" in the art. [0003] The part of the immunogen recognized by helper T cells (Th cells) are T cell determinants or epitopes. The part of the immunogen bound ...

Claims

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Application Information

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IPC IPC(8): C12N15/09A61K39/00A61K39/29A61K39/38A61K39/39A61P31/04A61P31/20C07K7/00C07K14/02C07K16/00C12N9/02
CPCC07K2319/00Y10S977/894Y10S977/795C12N2730/10122Y10S977/917C12N9/0077A61K39/00C07K14/005A61P31/04A61P31/20Y02A50/30C07K14/02
Inventor A·J·比尔克特
Owner IMMUNE COMPLEX
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