The Cloning Of DNA Sequence Encoding Rat TSHβ And Its Expressing 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 ofαandβchain. A chain has 96 amino acid remnants and its sequence is similar to that of luteinizing hormone, follicle stimulating hormone and chorionic gonadotropin. Theαchain is deemed to the common chain.βchain has 121 amino acid remnants. Its sequence is different from that of the above three hormones.βchain primarily presents the biological activity of TSH. To prepare anti-TSHantibody, it is necessary to gain enough TSHβto 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 genome DNA from mouse liver to be the template, perform nest polymerase chain reaction (PCR) and overlapping extension PCR to clone the exons of mouse TSHβcoding gene, and then clone it to pGEM-T Easy. After confirmatory sequencing, we insert the aimed gene to pQE-30Xa and pGEX-SX-1, which are prokaryotic expression vectors, and then transform them respectively to E. coli M15[pREP4] and E. coli BL21 to express aimed protein. Perform SDS-PAGE to check the molecular weight and quantity of TSHβ. Operate Western-blotting to further identify. Optimize the expressing天津市重中之重项目(05YFGDSF02700) condition to express pQE-30Xa/TSHβin E. coli M15 [pREP4] in batches. TSHβis 6mg in per liter culture medium. The recombinant TSHβis 24.32% in total germ protein. The recombinant TSHβhas 6×His purification tag, so it can be seperated with Ni²⁺-IDA resin affinity chromatography via the bond between histidine and Ni²⁺. After rinsing the mixed other kinds of protein, we can wash the aimed protein from resin. By Ni²⁺-IDA resin affinity chromatography we can purify the aimed protein produced in batches.