The Cloning Of Rat Tshα Cdna And Its Expression In E.coli

TRAb is an auto-antibody found in Graves' patients of autoimmune thyroid diseases. It can bond TSHR as TSH does and cause a series of biological reaction. To study its origin, it is necessary to prepare anti-TSH antibody. TSH is a glycoprotein hormone of molecular weight 28000, and consist of a andβchain.βchain has 121 amino acid remnants. Its sequence is different from that of the luteinizing hormone, follicle stimulating hormone and chorionic gonadotropin. A chain has 96 amino acid remnants and its sequence is similar to that of the above three hormones. The a chain is deemed to the common chain.βchain primarily presents the biological activity of TSH. To prepare anti-TSH antibody, it is necessary to gain enough TSH a to immune other animals.In most research, we choose rat to build animal models of hyperthyroid and hypothyroid. Examine levels of the serum thyroid hormones and TSH to confirm the success of animal models. At present we use human TSH quantitative examining kit to examine the level of mouse serum TSH. Therefore it is valuable to acquire enough mouse TSH to develop mouse TSH quantitative examining kit.In this research we extract RNA from mouse appendices suprasphenoidalis to be the template, perform nest polymerase chain reaction (PCR) and RT-PCR to clone the exons of mouse TSH a coding gene, and then clone it to pGEM-T Easy. After confirmatory sequencing, we insert the aimed gene to pGEX-3X, which are prokaryotic expression vectors, and then transform them respectively to E.coli DH5 a [pREP4] to express aimed protein. Perform SDS-PAGE to check the molecular weight and quantity of TSHα. Operate Western-blotting to further identify. Optimize the expressing condition to express pGEX-3X /TSH a in E.coli DH5 a [pREP4] in batches. TSH a is 6mg in per liter culture medium. The recombinant TSHβis 24.32% in total germ protein. The recombinant TSH a has GST purification tag, so it can be seperated with GST resin affinity chromatography. After rinsing the mixed other kinds of protein, we can wash the aimed protein from resin. By affinity chromatography we can purify the aimed protein produced in batches.