The Expression Of Thyroglobulin Epitopes Gene In Prokaryotic And Eukaryotic System, The Establishment Of ELISA For Detecting TgAb Using These Recombinant Proteins,and Their Preliminary Clinical Application

Human thyroid thyroglobulin (Tg) is a kind of the glucoproteins in the thyroid gland. As Tg is a major antigen of the thyroid gland, the TgAb detection has an important value for the diagnosis of Hashimoto' thyroiditis (HT) . Up to now, the source of Tg is limited, since it is extracted from human thyriod tissues. There is no report about the TgAb assay using the recombinant Tg as antigen.Objective: The Tg epitope gene were expressed in E.coli BL21 Star^TM(DE3) and Chinese Hamster Ovary (CHO) cell respectively. The expression products that prokaryotic protein or eukaryotic protein had immune activity were applicated in ELISA respectively. Then ELISA was in clinical study primarily.Methods: Total RNAs were extracted from the human thyroid patient with Graves' disease (GD) by TRIzol reagent. The Tg epitope gene was amplified by RT—PCR. The target gene were inserted into the expressive vectors of pET102/D-T0P0 and pcDNA?3. lD/V5-His-T0P0? and then sequenced respectively. The recombinant prokaryotic expressive plasmid Tg - pET102/D-T0P0 was transformed into E. coli BL21 Star^TM(DE3). IPTG was used as inductor. The recombinant Tg prokaryotic protein was purified by Ni ? NTA His ? Bind resin. The molecule weight, immune activity and quantity of it was identified by SDS—PAGE, ELISA and the method of Comasse Brilliant blue respectively. The recombinant eukaryotic expressive plasmid Tg - pcDNA^TM3.1D/V5-His-TOPO^? was transfected into CHO cell. Test a range of concentration to ensure that a stable cell line was obtained. The recombinant Tg eukaryotic proteinwas purified by Ni ? NTA His ? Bind resin. The molecule weight, immune activity and quantity of it was identified as the same as above. Tg prokaryotic fusion protein and eukaryotic fusion protein were used as antigens in establishing ELISA respectively. Then the methods were applied in the detection of TgAb in HT.Results: The Tg epitope gene has been inserted into the expressive vectors of pET102/D-T0P0 and pcDNA?3. lD/V5-His-TOPO? respectively. The sequences are the same as reported by Genebank. The optimized expressive condition of E.coli BL21 Star?(DE3) transformed Tg— pET102/D-T0P0 was 6 hours at 25°C by lmM IPTG. The production rate was 1. 36mg/L media after purified by His ? Tag prification system. Its molecule weight of Tg prokaryotic -protein was 73. 47kD, which is 3. 77 % above the true evaluation. Its immune activity was identified by ELISA. After Tg-pcDNA?3. lD/V5-His-T0P0? was trandfected into CHO cell, the optimized concentration of G418 is 1000 u g/ml. The production was purified by His *Tag prification system as well. The molecule weight of Tg eukaryotic protein and dimmer were 58. 30kD and 115. 21kD which are 1.19% and 2.36% below true evaluation. Their immune activity were identified by ELISA. The correlation between two ELISAs and RIA method for detecting TgAb were all significant 0=0. 792, RO. 01 and r=0. 859 KO. 01). The inter—assay coefficient of variation (CV) were 5.09% and 7. 56%, the intra—assay CV were 12. 94% and 17.65%. The serum level of TgAb in normal group were 0. 486±0.184 and 0. 422 + 0. 159 (3c±s) . The positive rate were 64. 71% (11/17) and65.22% (15/23) in HT separately. Conclusions:1.Successful cloning the Tg epitope gene and construction the prokaryotic expressive plasmid and eukaryotic expressive plasmid.