Designing, Cloning, and Expression of Protein G Coding Gene and Evaluating Its Function in Laboratory Conditions

Abstract

Background and Purpose: We can obtain accurate information about a specific antigen using immunoglobulins. The absorption chromatography method is used for specific isolation of a class of immunoglobulin. The present study aims to prepare the recombinant Protein G of Streptococcus bacteria, used in absorption chromatography to separate serum IgG.

Methods: Genomic DNA of Streptococcus bacteria was extracted and a PCR reaction was performed using primers designed based on the Protein G gene, and PCR product was cloned in PET28a plasmid. To isolate the mentioned gene, the recombinant plasmid was digested with xho I and Nco I enzymes. The Protein G gene was purified and subcloned in the expression plasmid. The Protein G gene was amplified by PCR. To examine the size of the recombinant protein, a 12% SDS page was loaded, and protein performance was examined in the ELISA test.

Results: The PCR product, which has 641 nucleotide pairs, was successfully cloned and confirmed in the expression plasmid. The recombinant protein G is ready to be used in an absorption chromatography column for the isolation of serum IgG. Based on ELISA tests, protein G could identify mouse IgG and rabbit IgG and did not react with chicken IgY.

Conclusion: This recombinant protein is biologically active and can be used at least for some animal species in immunology laboratories.

Keywords: Protein G, Streptococcus, Cloning, Immunoglobulin