Building method for autovaccine by aiming at human TNF(Tumor Necrosis Factor)-alpha molecule

Abstract

The invention discloses a building method for autovaccine in-vivo induced by aiming at human TNF(Tumor Necrosis Factor)-alpha molecule. With a step-by-step cloning method, a fusion gene of hTNF-TT830-844, hTNF-HEL46-61 and hTNF-PADRE is built; point mutation (T439-A,C440-G) is introduced into a natural human TNF gene to optimize a mRNA (Ribonucleic Acid) secondary structure; the fusion gene is cloned into a pET22b prokaryotic expression vector, and efficient expression is achieved in the bacterial strain of escherichia coli; three T accessory cell epitope peptides are introduced between the epitope peptide structure domains of hTNF by the computer-aided analysis and is fused with the hTNF-alpha to overcome the immunological tolerance of an organism for the autologous protein, and therefore the organism generates high-level humoral immune response; the generated high-level hTNF-alpha neutralizing polyclone antibody can neutralize killing activity of the hTNF-alpha on L929 cells in vitro; the hTNF-PADRE has the strongest immunogenicity; the high-level antibody can be induced under the condition of using no immunological adjuvant; and the vaccine has favorable protection and curing action on mouse models suffering from rheumatoid arthritis induced by the II-type collagen, cachexia and the like induced by LPS (lipopolysaccharide).

Description

technical field [0001] The invention belongs to the field of medical biotechnology, specifically relates to the technical fields of gene cloning, gene recombination, expression of foreign genes in prokaryotic cells, purification of target protein, etc. A method for constructing an antibody-based protein vaccine. Background technique [0002] 1. Research on TNF-α molecules and anti-TNF-α therapy [0003] 1.1 TNF-α molecule [0004] In 1975, Carswell et al. found that there was a highly active tumor cytotoxic factor in the serum of mice infected with BCG and bacterial endotoxin, which could cause hemorrhagic necrosis of tumors, but had no killing activity on normal tissue cells, and named it tumor necrosis. Factor - TNF-α. The human TNF-α gene is located on chromosome 6. The gene is 4kb long and contains 4 exons. It is linked to the MHC gene. Its precursor consists of 232 amino acids and contains a signal peptide of 76 amino acids. The mouse gene is located on chromosome 1...

AI Technical Summary

Problems solved by technology

Recently, Waterston et al. evaluated the immunogenicity and safety of TNF-α autovaccine through a phase one clinical trial. Unfortunately, although the serum antibodies induced by the vaccine can recognize denatured human TNF-α molecules, they cannot neutralize the natural state TNF-α molecules. Human TNF-α molecule, the analysis may be due to the denaturation of the antigen during the purification process of the antigen

[0031] The above studies show that although TNF-α autovaccine is also a good way to treat diseases related to the overexpression of TNF-α, however, screening for effective T cell helper epitopes, suitable epitope insertion positions and suitable purification methods are all important factors for the treatment of TNF-α. -Critical issues in the study of α autovaccine

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