心肌肌钙蛋白基因中连续AGG的同义替换与GST融合表达

Replacement consecutive AGG codons in the cDNA of cardiac troponin with the synonymous codon and its GST fusion expression

目的获得高表达、易纯化的GST-cTnT融合蛋白,初步探讨其免疫反应性。方法(1)PCR扩增心肌肌钙蛋白T基因cDNA及其连续稀有密码子的同义替换;(2)PGEX-4T-3/TnT-1、2、3、4的构建与GST-cTnT融合蛋白的表达和纯化;(3)WesternBlot与电化学发光cTnT测定法研究GST-cTnT融合蛋白的免疫反应性。结果成功构建了PGEX-4T-3/TnT-1、2、3、4的表达载体。BL21-PGEX-4T-3/TnT-2、3、4表达的目的蛋白高于BL21-PGEX-4T-3/TnT-1,通过谷胱甘肽-琼脂糖亲和层析得到高纯度的融合蛋白。WesternBlot与电化学发光cTnT测定法表明表达的融合蛋白和相应的抗体相互作用,且GST蛋白不与相应的抗体作用。结论cTnT基因上的连续稀有密码子的同义替换,可提高其在E.Coli中的表达量。GST-cTnT融合蛋白中的GST蛋白不影响cTnT的免疫反应性。

Objective To obtain higher expression, easier purified and fusio protein of GST-cTnT and preliminary study on immunoactivity of the protein. Methods （1）cTnT was amplified by PCR from cardiac cDNA and consecutive AGG codons in the cDNA of cardiac troponin was replaced with the synonymous codon. （2）expression vector of PGEX-4T-3/TnT-1,2,3 ~4 was construct- ed. IFrG induced BL21-PGEX-4T-3/TnT-1 ~2,3~4. Fusio protein of GST-cTnT was pmified by affinutt chromatographic column of glutathion Sepharose-4B. （3）Immunoreaetivity was approached by immunoblotting and eleetrochemiluminescenee of eTnT assay. suits Construction of expression vector of PGEX-4T-3/ TnT-1,2,3,4 was successful and obtained highly purified GST-cTnT fusion protein; GST-cTnT could be recognized by antibody that target cardiac troponin T, while GST didn＇t interreact with the antibody. Conclusions The increase in cTnT expression was obtained when both pair of the rare arginine codons was replaced. GST of fusion protein GST-cTnT didn＇t affect immunoreactivity of cTnT monoclonal antibody.