Strategically modified hepatitis b core proteins and their derivatives
A core protein, strategic technology, applied in the direction of anti-animal/human immunoglobulins, microorganisms, immunoglobulins, etc., can solve the problem of amino acid side chains not showing chemical reactivity, etc., to achieve the effect of enhancing chemical reactivity
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[0094] iii. Preparation of strategically modified core protein
[0095] The strategic modification of the hepatitis B core protein is usually carried out 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 genes are then expressed in convenient systems known in the art, for example in viral cultures of infected immortalized cells.
[0096] Methods for making HBcAg proteins in general and methods for making pre-core, core and HBeAg proteins in particular are well known in the art. The same method is readily adapted to isolate the modified core protein particles of the invention. In addition, HBcAg and HBeAg can be produced by a variety of well known recombinant DNA techniques. See, eg, US Patent Nos. 4,356,270 to Itakura and 4,563,423 to Murray et al. These recombinant DNA techniques are readily adapted to methods for producing the modified core particles of the invention. The...
Embodiment 1
[0167] Example 1: Construction of modified hepatitis B core protein expression vector.
[0168] A lysine codon (TTT) was introduced between amino acids D78 and P79 of the HBc gene by site-directed mutagenesis, and EcoRI (GAATTC) and SacI (GAGCTC) restriction endonuclease sites were also introduced to facilitate the genetic insertion of other codons to generate strategically modified hybrid HBc particles. The amino acid residue sequence of such an insert is GIQKEL, where GIQ is the product of the EcoRI site and EL is the product of the SacI site. Therefore, the strategically modified hepatitis B core protein has a length of 155 amino acid residues. The construction of the pKK322-HBc155-K81 expression plasmid is described below.
[0169] The 5' end of the HBc gene (bases 1-234, amino acids 1-78) 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, an NcoI restriction site (CCATGG) was introdu...
Embodiment 2
[0171] Embodiment 2: the purification of the hepatitis B core protein particle of modification
[0172] The strategically modified HBc protein of Example 1 is expressed in E. coli, typically E. coli BLR or BL21 from Novagen (Madison, Wisconsin) or E. coli TB11 from Amersham (Arlington Heights, Illinois). The HBc155-K81 transfected with E. coli is the expressed plasmid pKK332-HBc155-K81. Strategically modified HBc particles were purified by established methods using Sepharose CL-4B chromatography. Since the particles are purified in a predictable manner, simple spectrophotometric (OD 280 ) plus monitoring particle elution by SDS-PAGE testing of the purity of the individual fractions prior to collection is sufficient to allow routine purification to yield high yields of electrophoretic pure particles (5-120 mg / L cell culture). The spherical structure of the purified strategically modified HBV core particles can be clearly seen under the electron microscope.
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