内皮素1的克隆表达及其诱发抗体的初步研究

目的：克隆内皮素1（endothelin 1,ET1）基因、表达ET1融合蛋白，以期诱导机体产生ET1抗体，中和患者体内过量的ET1。方法：根据人ET1的多肽序列合成ET1基因，将其插入到pThioHisA的EcoRI和 SalI位点，重组质粒pThioHisA-ET1进行酶切鉴定及序列测定验证后转化TOP10，IPTG诱导的重组菌经SDS-PAGE检测融合蛋白Thioredoxin-ET1的表达量；表达的融合蛋白用ProBond亲合层析纯化并经HPLC鉴测其纯度；每只小鼠按25、50、100ug/次剂量的Thioredoxin-ET1每两周免疫一次，共4次，最后一次免疫10d后制备抗血清，经Western blot和ELISA检测证明Thioredoxin-ET1融合蛋白具有ET1免疫反应原性。结果：经过质粒酶切鉴定及序列分析表明，构建了正确的融合表达载体pThioHisA-ET1，用1mmol IPTG诱导工程菌，融合蛋白Thioredoxin-ET1的表达量占菌体总蛋白的50%，多以包涵体的形式存在，包涵体经洗涤，变性和复性后，用ProBond亲合层析纯化得到了Thioredoxin-ET1融合蛋白，经HPLC鉴测其纯度为90%。制备的抗血清中ET1抗体的效价可达104。结论：应用Thioredoxin-ET1融合蛋白可以诱发机体产生ET1抗体，为研究ET1在患者体内的过量表达及对患者的免疫治疗开辟了新的途径。

Construction and Expression of Recombinant Human Endothelin 1 in E coli and Induction Its Autoimmunity Antibody in Mice

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.