Expression And Purification Of The Second Extracellular Loop Of Angiotensin Ⅱ Receptor Subtype 1A (AT_1A) In E.coli

ObjectiveRecently, some studies demonstrated that in the sera of preeclamptic patients, primary hypertensive, secondary hypertensive and malignant hypertensive patients, autoantibodies against the peptide corresponding to the amino acids (165-191) of the second extracellular loop of AT1A receptor were detected. These autoantibodies were able to identify AT1A receptor and showed agonist-like activity. This effect could be blocked by specific AT1 receptor antagonist (Losartan). However, the mechanism for the pathophysiological role of AT1A receptor autoantibody was unclear and needs to be further clarified, which may be important for creating new medicine to hypertension. No monoclonal antibody or polyclonal antibody against the peptide corresponding to the amino acids of the second extracellular loop of AT1 A receptor is available yet. In order to obtain enough polyclonal sera by immunologic method, a large quantity of peptides corresponding to the amino acids of second the extracellular loop of AT1A receptor are required as antigen. Therefore, in the present study, we used gene engineering technology to acquire such antigen.Materials and methodsThe genomic DNA was derived from rat heart. The gene fragment was amplified in vitro and was recombmed with express vectors After PCR screening, recombmed vectors were transformed into E.coli BL21 (DE3 ) The fusion protein was expressed by 1PTG induction. The sequence of recombined vector which expressed soluble fusion protein was identified. Glutathione Sepharose 4B was used to purify fusion protein. The objective peptides were separated effectively through cutting off GST from fusion protein with Thrombin. ResultsL The prokaryotic expression vectors of pGEX4T-1-AT1A, pET22b-AT1A, pQE31-AT1A and pGEX6P-1-AT1A were successfully constructed.2 Soluble GST fusion protein was expressed in E.coli BL21 (DE3) harboring pGEX4T-1-AT1A by IPTG inducing.3 The fusion protein was purified by Glutathione Sepharose 4B chromatography.4 The objective peptides were separated effectively through cutting off GST from fusion protein with Thrombin The yield of purified peptides was 2.0 mg/L.ConclusionThis experiment obtained peptides corresponding to the amino acids of the second extracellular loop of AT1A receptor and thus may provide large quantity of antigen for production of enough polyclonal antibodies against AT1A receptor by immunologic method.