中国生物工程杂志

AIM：in oder to clone endothelin 1(ET1) gene, express ET1 fusion protein , induce organism producted ET1 antibody, and neutralize superfluous ET1 in patients. METHODS：The gene of human ET1 was synthesized according to the preferential codons of E. coli, cloned into the EcoRI and SalI sites of vector pThioHisA. The recombinant plasmid pThioHisA-ET1 was constructed , sequenced and transformed into E.coli TOP10. Induced and expressed fusion protein were identified and analysed by 12% SDS-PAGE and densitometry analyses. After the elution, denature and renature, the fusion protein Thioredoxin-ET1 was obtained by ProBondTM chromatogragraphy. The purity of Thioredoxin-ET1 was detected by HPLC. Inoculate Thioredoxin-ET1 once per mouse every 2 weeks in 25, 50 and 100μg separately on 3 groups for 4 times. 10 days after last inoculation, we obtained venipuncture blood. RESULTS：SDS-PAGE and densitometry analyses showed the expressed fusion protein Thioredoxin-ET1,with a molecular weight of about 19 kD, was about 50% of total bacterial protein after induction at 37℃ by 1mmol/LIPTG for 4 hours. The purity of purefied Thioredoxin-ET1 is 90%. The results of Western blot and ELISA indicated that Thioredoxin-ET1 fusion protein has immunogenicity of ET1 and the titer of ET1 was about 104 in anti-serum. CONCLUSION： Thioredoxin-ET1 fusion protein could induce ET1 antibody production in mouse. And it could be used to explore mechanisms of ET1 over-expression in patients and provided a new strategy to immunotherapy.