Monthly Archives: June 2015

Something You do not Know About Chimeric Antibody

Something You do not Know About Chimeric Antibody

Summary

In the antibody field, what familiar to us are the Genetically engineered antibodies and Monoclonal antibody. However, one of the most important antibody we do not know is the chimeric antibody. In this article I will introduce some knowledge about this antibody.chimeric antibody

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Definition

Chimeric antibody was the first success in the preparation of genetically engineered antibody. It is composed of V region gene and gene-splicing Cregion of a human antibody. And then it is inserted into a vector, transfection of antibody molecules myeloma tissue expression.because it reduces the murine components, thereby reducing the adverse effects caused by murine antibody, and helps improve the outcome.

The features of chimeric antibody

  • It not only retains the parent murine antibodies with high specificity and affinity, but also reduces the murine component about 70%.
  • The human Fc portion can effectively mediate biological effector functions, such as antibody-dependent cellular cytotoxicity mediated (Antibody dependent cell-medi-ated cytotoxicity, ADCC), complement-dependent cytotoxicity (Complement dependent cytotoxicity, CDC ), etc.
  • It is free to choose the type of antibody, subtypes, classes, subclasses, size, domain and add glycosylation sites, etc., in order to take advantage of their different physical and chemical properties and biological characteristics;
  • Chimeric antibodies Expression “box”, which is produced by efficient eukaryotic expression vector and a human antibody, allows the insertion of different variable region of the murine monoclonal antibody, shortens the operation and development cycle;

The steps to produce chimeric antibody

Production of chimeric antibodies, the first step is the key step is to obtain from hybridoma cell McAb specific variable region gene, and then connect with the human antibody constant region genes, and finally to build a good human – mouse chimeric antibody gene expression in myeloma cells. The main steps include murine antibody variable region gene cloning, fishing human antibody constant region gene extraction, vectors and host cells are constructed gene splicing human antibody constant region gene of murine antibody variable region and as well as efficient expression screening positive cells and expression identification products

  • Cloning of murine antibody variable region gene
  • Human antibody constant region gene angling
  • Human – mouse chimeric antibody expression vector
  • Efficient expression vectors and host cells Construction
  • Human – mouse chimeric antibody expression and identification

Chimeric antibody, as the first born of genetically engineered antibody, has its unique advantages: retaining parental murine antibodies with high specificity and affinity; having a longer half-life in the body; playing a variety of biological functions effectively; with easy operation and low cost and great prospects for clinical application etc.

A Brief Look on Protein Sequencing

A Brief Look on Protein Sequencing

Tags: protein sequencing

protein sequencingSummary

We talk about the protein sequencing is mainly about the primari structure of protein sequencing. This primary structure includes the the number of polypeptide chains in protein. And in many cases, polypeptides and protein can be used as the one. The oder of the acid amino in polypeptide is the bio-functional bases of protein.

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Requirment of the sequencing

Sample purity(over 97%)

Accurate molecular weight of proteins

Accurate number of subunits

Determination of amino acid composition of the protein; and calculate the number of each amino acid in the molecular weight.

Determination of the amount of ammonia in the hydrolysis, calculated amide content

Determination of protein and amino acid sequence of a polypeptide

Open and isolation of peptide chain

The number determination of protein molecules in the polypeptide chain

Break of disulfide bonds

Determination of the amino acid composition of each polypeptide chain, and calculate the molecular ratio of amino acid components

Determination of the N-terminal, C terminal

Polypeptide chain scission

Determination of the amino acid sequence of each peptide.

The steps of sequencing

  1. Split the polypeptide chain

Protein molecules are composed of multiple polypeptide chains, it must be firstly splited. Several polypeptide chains are linked together with the help of non-covalently, which is known as oligomeric proteins.

  1. To determine the number of the polypeptides in protein.

Determination of the number of the polypeptide chain in protein molecules. Through the determination of amino acid residues at the end of the number of moles and the relationship between the protein molecular weight, can determine the number of the polypeptide chain..

  1. Disulfide bond rupture.

Several polypeptide chain through disulfide bond together, can be in 8 mol/L urea or, in the presence of 6 mol/L guanidine hydrochloride with excessive  – mercaptoethanol processing, make the disulfide bond reduction of sulphur, and then use generated thiol alkylating reagent protection, to prevent the oxidation of the it to be.

  1. To determine the amino acid composition, each of the polypeptide chain and calculate the amino acid composition of molecular ratio
  2. Analyzing the N – end of a polypeptide chain and C – end
  3. Polypeptide chain rupture into multiple peptides. Two or more different fracture method can be used to peptide broken into two or more sets of peptides or peptide fragments, and the separation
  4. The determination of each amino acid sequence of peptides
  5. Determine the peptides sequence in the polypeptide chain. To use two or more sets of amino acid sequence of peptides crisscross overlap each other, piecing together the whole amino acid sequence of a polypeptide chain.
  6. To determine the location of disulfide bond in the polypeptide chain

Those are the steps of protein sequencing. And you can take those tips to achieve your goals.