Molecular cloning of antigens to thyroid autoantibodies using the expression vector lambda gt11

Area of Science:

• Immunology
• Molecular Biology
• Endocrinology

Background:

• Autoimmune thyroid diseases (AITD) pathogenesis understanding requires molecular cloning of autoantigens.
• Purification of key antigens like TSH receptor and microsomal antigen is challenging.
• Traditional cloning methods necessitate purified proteins, which are often unavailable.

Purpose of the Study:

• To develop and evaluate an alternative molecular cloning strategy for thyroid autoantigens.
• To identify previously unknown antigens involved in autoimmune thyroid disease.
• To assess the feasibility of using antibody screening of cDNA expression libraries.

Main Methods:

• Construction of cDNA expression libraries in the gt11 vector using mRNA from human, pig, and rat thyroid cells.
• Screening of these libraries with antibodies as probes to identify autoantigens.
• Analysis of advantages and disadvantages of the expression library system.

Main Results:

• Demonstrated the successful construction and screening of thyroid cDNA expression libraries.
• Highlighted that protein purification is not required, enabling identification of novel antigens.
• Identified limitations including potential loss of antigenicity in fusion proteins and issues with reading frame and glycosylation.

Conclusions:

• cDNA expression libraries offer a viable alternative for cloning autoantigens when purification is not feasible.
• This method facilitates the identification of new antigens in autoimmune thyroid disease.
• Careful consideration of antibody selection and potential alterations in antigen structure is necessary for successful application.