Genetic Immunization Of Mice With Human Thyrotropin Receptor Provides A Model Of Graves' Disease

Objecetive: To construct the recombinant plasmids pcDNA3.1/hTSHRf andpcDNA3.1/hTSHRe for expression of hTSHR with eukaryotic expression vectorpcDNA3.1 (D)/V5-His-TOPO, the two kinds of cDNA fragments of hTSHRextracellular domain were obtained, which were 188-403bp and 407～940bp,respectively. And the recombinant plasmids were transfected into Chinese hamsterovary (CHO) cells, and then used to establish the animal models of Graves' diseaseby genetic immunization.Methods: Extract total RNA from human normal thyroid, hTSHRf and hTSHRe wereobtained through RT-PCR. The purified cDNAs were inserted into theampicillin-resistant plasmid vector pcDNA3.1 (D)/V5-His-TOPO. The plasmidswere introduced into the competence TOP10 E.coli strain, purified, then therecombinant plasmids pcDNA3.1/hTSHRf and pcDNA3.1/hTSHRe were identifiedby restricting enzyme Hindâ…¢digestion analysis, PCR amplifying and DNAsequencing. The recombinant expression plasmids pcDNA3.1/hTSHRf andpcDNA3.1/hTSHRe, which were recombined with hTSHR extracellular domainfragments (aa29～100, aa101～278), respectively, were transfected into Chinesehamster ovary cells by Lipofectin method, with which the transcription were detectedby means of RT-PCR, human TSHR protein expression on CHO cells was detected byWestern blot analysis. BALB/c mice were given Bupivacaine Hydrochloride prior toimmunization on weeksl, 4, 7, 9 andl0 with 100μg recombinant plasmids. Controlgroup were injected with normal sodium (NS), and blood of mice were obtained fromangulus oculi medialis on weeks0, 6, 11, and obtain sera. All mice were killed and thyroids were taken, histological analysis of thyroid tissue was carried out withhematoxylin and eosin staining. The amount of TRAb in sera was assayed byenzyme-linked immunosorbent assay (ELISA). The index of TSAb and TBAb in serawas determined by radioimmunity assay (RIA). Sera T4 level was determined byradioimmunity assay (RIA).Results: 1. Restriction enzyme digestion, PCR amplifying and DNA sequencingconfirmed that recombinant plasmid pcDNA3.1/hTSHR had been constructedsuccessfully. 2. Two bands of 216bp and 534bp were amplified from CHO cellstransfected by the recombinant plasmids pcDNA3.1/hTSHRf andpcDNA3.1/hTSHRe, respectively. Western blotting analysis revealed that the lysateof CHO cells transfected by pcDNA3.1/hTSHRf and pcDNA3.1/hTSHRe had strongbands with molecular weight of about 11.94KD, 23.6KD, respectively. 3. In the miceimmunized with pcDNA3.1/hTSHRf and pcDNA3.1/hTSHRe, sera T₄, TRAb andTSAb elevated significantly (P＜0.05), there were no significant changes in seraTBAb index (P＞0.05). Sera T₄, TRAb, TSAb and TBAb of control group showed nostatistically significance (P＞0.05). Relative to controls, histopathological examinationof thyroid glands from immunized mice showed morphological alterationscharacteristic of hyperactive glands, including a great number of new follicles,follicular epithelial cells hyperplasia.Conclusion:1. The recombinant plasmids have been successfully constructed. 2. Thetranscription on CHO cells transfected by the recombinant plasmids of recombinantshave been proved by RT-PCR, eukaryotic expression was proved successfully byWestern blot analysis. 3.Animal models of Graves' disease have been madesuccessfully by genetic immunization.