The Expression And Purification Of Thyroid Peroxidase Peptide Phe⁴⁷³-Ala⁸⁰⁰

Autoimmune thyroidits also named Hashimoto's thyoiditis (HT) is a common organ specific autoimmune disease. Thyroid peroxidase (TPO) is a key catalase on the catalytic reaction of the synthesis of thyroid hormone. And it is also an autoantigen in experimental autoimmune thyroiditis (EAT). EAT has the similar characterisics with autoimmune thyroiditis. So it is usually used to study the pathogenesis of HT. H-2k and H-2s mice have severous infiltration and are the most used animals for EAT model.TPO is a kind of glycosylated hemoglobin, expressing on the thyroid cell surface. It is a critical catalase on the synthesis of thyroid hormones, also it is a major thyroid autoantigen. Expression disorder of epitopes of TPO or immune responses to TPO and TPOAb ususlly became the immportant damage mechanism of thyrocyte of patients with autoimmune thyroidits.This study focuses on the expression and purification of mouse thyroid peroxidase peptide Phe473-Ala800 (mTPO^F473-A800).Objective:Constructed the recombinant pGEX-6p-1-mTPO^F473-A800 prokaryotic expressing plasmid, expressed it in E. coli strain Rosseta, and purified the expressed peptide.Method:1 Extraction of the total RNA. The thyroid glands of 6 C57BL/6 mice were removed and grinded in liquid nitrogen for the RNA.2 Amplification of mTPO^F473-A800 gene. The mTPO^F473-A800 gene was amplified by RT-PCR.3 Construction of recombinant cloning vector. mTPO^F473-A800 gene was induced into pEASY-T3 vector. The productions were induced into DH5a competent cells ,cultivation at 37℃for 12 hours.The next day, screening the \successfully induced pEASY-T3- mTPO^F473-A800 plasmid by PCR.To choose the positive clones to extract the recombinant plasmids and to identify the correct recombinant plasmid pEASY-T3- mTPO^F473-A800 by double enzyme digestion of EcRoI/XhoI. Send out 10ul of recombinant plasmids for sequencing.4 Construction of recombinant expression plasmid. pEASY-T3- mTPO^F473-A800 plasmid togethe with pET28a, pET32a and pGEX-6p-1 were digested by EcRoI/XhoI. Then induce mTPO^F473-A800 into pET28a, pET32a and pGEX-6p-1 by DNA ligase. The productions were induced into DH5a competent cells, cultivation at 37℃for 12 hours. The next day, screen the successfully induced pEASY-T3- mTPO^F473-A800 plasmids by PCR. Choosed the positive clones to extract recombinant plasmids and to identify the correct recombinant expression plasmids by double enzyme digestion of EcRoI/XhoI. Then the positive recombinant plasmids were induced into E.coli Rosseta competent cells. Optimised the expression conditions and screened the best expressed plasmid.5 The expression of pGEX-6p-1- mTPO^F473-A800and purification of GST- mTPO^F473-A800 recombinant peptides. Express GST-mTPO^F473-A800 recombinant protein at optimum conditions and obtain mTPO^F473-A800 peptide by GST column.Results:1 Extracted the total RNA of mouse thyroid peroxidase. The mTPO^F473-A800 gene was induced into pEASY-T3 vector, and the sequencing results shown that we got the correct gene.2 Constructing recombinant expression vectors and optimizating expression conditions. After mTPO^F473-A800 gene was induced into 3 vectors successfully identified by double enzyme digestion, Rosseta/pGEX-6p-1- mTPO^F473-A800 were induced by 1mM IPTG at 30℃SDS-PAGE electrophoresis showed a great production and high solubility under this optumized conditions. 3 Expression and purification of pGEX-6p-1- mTPO^F473-A800 recombinant peptides. Rosseta/pGEX-6p-1- mTPO^F473-A800 was nduced by 1mM IPTG at 30℃. Then the bacteia were collected and the products were analyzed by SDS-PAGE and Western blotting. The mTPO^F473-A800 protein was purified by GST column.Conclution:Recombinant vector pGEX-6p-1-mTPO^F473-A800 was etablished successfully. Large scale expression was made under optimum conditions and to purified mTPO^F473-A800 peptides were obtained in GST column.