犬细小病毒VP2基因的克隆及其在毕赤酵母中的表达

目的表达犬细小病毒VP2蛋白（CPVVP2），用于犬细小病毒病的诊断、疫苗研制和VP2蛋白功能研究。方法采用PCR方法对CPVVP2基因进行扩增，将CPVVP2基因克隆到毕赤酵母（Pichiapastoris）分泌表达载体pPICZαA中，构建真核重组表达载体pPICZαA—VP2，将该重组质粒线性化后，转化毕赤酵母菌GS115中，在甲醇诱导下表达CPVVP2，SDS-PAGE和Western blotting鉴定表达蛋白。结果成功扩增了CPVVP2基因，构建了真核重组表达载体pPICZαA-VP2在毕赤酵母菌中表达出约64．35kD蛋白。Western blotting鉴定表明，表达VP2蛋白与犬细小病毒阳性血清有反应性。结论在毕赤酵母中成功地表达了CPVVP2蛋白，能被犬细小病毒阳性血清识别。

Cloning of VP2 Gene of Canine Parvovirus and Expression in Pichia pastoris

Objective To establish a yeast Pichia pastoris expression system expressing canine parvovirus VP2 protein （CPV VP2） to serve the diagnosis of CPV, development of CPV vaccine and research of VP2 protein function. Methods CPV VP2 gene was amplified by PCR, cloned into secretory Pichia pastoris expression vector pPICZaA, and was constructed into eukaryotie expression vector, which was named pPICZαA-VP2. After being linearized with enzyme digestion, the vector was transformed into Pichia pastoris GS115 by electroporation method. The CPV VP2 protein expressed with methanol induction. The expressed protein in yeast was analyzed by SDS-PAGE and Western blotting. Results CPV VP2 gene was successfully amplified, SDS-PAGE and Western blotting analysis of the culture superuatants showed that the VP2 protein of 64.35 kD was expressed in Pichia pastor/s, and the expressed CPV VP2 protein shared reaction with positive serum against CPV. Conclusion CPV VP2 has been successfully expressed in yeast Pichia pastoris. The expressed CPV VP2 protein shares reaction with positive serum against CPV.