中华眼镜蛇心脏毒素(CardiotoxinⅢ)的克隆及其在大肠杆菌和酵母中的表达

心脏毒素cardiotoxinⅢ（CTXⅢ）是中华眼镜蛇毒主要组分之一，根据Genbank中已有的CTXs序列设计特异性引物，从中华眼镜蛇毒中克隆出208bp的CTXⅢ片段，并将该基因片段分别克隆至大肠杆菌His—patch ThioredoxinB载体中，重组CTXⅢ经渗透休克分泌至胞外。将CTXⅢ基因插入酵母pPIC9K分泌型表达载体重组表达，N端带有3个氨基酸（GYT）残基的重组CTXⅢ（rCTXⅢ）分泌量达9．5mg／L，经一步凝胶过滤纯化，其纯度达90％以上，纯化率达65％。经细胞毒性实验检测，纯化的rCTXⅢ 12h的IC50为4．66μg／ml，证实重组蛋白具有良好的生物学活性。

Molecular Cloning and Expression of Cardiotoxin Ⅲ from Naja naja atra in E.coli and Yeast Pichia pastoris

Chinese cobra (Naja naja atra) cardiotoxins are three-fingered family with 60～62 amino acids bind by four disulfide bonds. CardiotoxinⅢ (CTXⅢ) is one of the major toxic component which can cause hemolysis and cytotoxicity. However, there is no report on the fusion expression of CTXⅢ in soluble form so far. The cloning, expression and purification of recombinant CTX Ⅲ (rCTXⅢ) from Naja naja atra in E. coli and in yeast Pichia pastoris were reported here. CTXⅢ gene, fused with enterokinase in E.coli His-patch Thioredoxin expression system, were expressed in soluble form and released by osmotic-shock treatment. CTX Ⅲ gene was also cloned and expressed in the methylotropic yeast Pichia pastoris pPIC9K expression vector in the first time. The yield of the secretion level was 9.5 mg/L. Using straightforward one-step chromatography procedure, the rCTXⅢ, with three additional amino acids (GYT) at the N-terminal site, was purified to a purity of more than 90% and recovery yield of 65%. The purified rCTX Ⅲ was further characterized by cytotoxic assay with IC50 4.66μg/ml. An effective expression and purification system for recombinant CTXs in P. pastoris was developed, this system will permit us the ready isolation of active cardiotoxins. This protocol can also be easily used for the production of the toxin in a larger scale with low cost.