Anti-TNF-[alpha] nanobody and genetic engineering expression thereof

Abstract

In the present invention, the sequences of the camel-derived anti-TNF-alpha and anti-human serum albumin (HSA) nanobodies are fused together through a flexible linker peptide to form an anti-TNF-alpha nanobody (TNF60) with stronger biological activity , the coding gene was connected to the expression plasmid of Escherichia coli, and the prokaryotic expression plasmid of TNF60 was constructed, after transformation of Escherichia coli, two kinds of genetically engineered expression bacteria of TNF60 were obtained by screening, and the optimal expression of TNF60 was determined by response surface method optimization conditions, using solid-phase affinity chromatography to establish a purity process, the purity of the obtained target protein can reach 95%, and analysis of the recombinant TNF60 protein sequence by mass spectrometry, Western blot (Western blot), mouse paw swelling and inflammation model experiments shows that the sequence is correct and It has good antigen binding and anti-inflammatory activity in vivo. The antibody of the present invention can be used for the treatment of rheumatoid arthritis, ankylosing spondylitis and other diseases.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to an anti-TNF-α nanometer antibody and its genetic engineering expression. Background technique [0002] Tumor necrosis factor alpha (TNFα) is a multifunctional cytokine that participates in important physiological processes such as cell apoptosis and survival, inflammatory response, and immune response. ), Crohn's disease (Crohn's disease) and psoriatic (psoriatic) and other diseases play an important role in the occurrence and development. Therefore, TNFα is considered to be a very important drug development target for the treatment of the above-mentioned related diseases. Infliximab (infliximab), adalimumab (adalimumab), golimumab (golimumab) are therapeutic anti-TNFα antibodies, which have shown significant efficacy and safety in the treatment of RA and other diseases. It has become one of the best-selling blockbuster drugs in the world, and the research and development of other ther...

AI Technical Summary

Problems solved by technology

[0003] In the early 1990s, the Hamers research group found that camelids can simultaneously process two different types of immunoglobulins, one is a classic antibody consisting of a light chain with two domains and a heavy chain with four domains, and the other One is an antibody that naturally lacks the light chain. This antibody only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not like an artificially engineered single-chain antibody fragment (scFv) It's easy to stick to each other, and even gather into blocks

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