汉坦病毒部分S基因在毕赤酵母中的分泌表达

Expression of partial S gene of Hantavirus Z10 in pichia pastoris

目的构建并表达汉坦病毒（HV）Z10株（HV-Z10）S基因蛋白编码区前300bp核苷酸序列，研究该基因在毕赤酵母（Pichia pastoris）中的表达及其产物的生物活性。方法根据HV-Z10 S基因前300bp（S300）的核苷酸序列，按照编码氨基酸的密码子转换成酵母偏爱的形式，设计合成8条引物，通过连续PCR，获得人工合成Z10株S基因前300bp的基因序列SP300，经测序，SP300与S300的核苷酸相似性为76.2％，但编码的氨基酸序列完全一致。将SP300克隆到酵母穿梭载体pPICZaA，构建含α-factor分沁信号肽的重组表达载体pPICZaA-SP300。将pPICZaA-SP300、pPICZaA-S300化学法转化酵母GS115菌株，筛选重组转化子。结果重组了SP300与S300的酵母转化子经甲醇诱导，表达出重组蛋白rNP300及rN300，SDS-PAGE显示相对分子质量为12×10^3左右。经ELISA检测及Western Blot分析，表达产物能与抗汉坦病毒抗体起免疫反应。结论SP300和S300基因在毕赤酵母中获得分泌表达，使用酵母偏爱密码子的SP300基因在毕赤酵母中的表达量与S300的表达量基本一致，表达量在诱导24h后最高。

Objective The purpose of this study is to express partial S gene of Hantavirus Z10. Methods The 300 bp S gene of Z10 strain was synthesized by using a successive PCR method for the optimal expression in Pichia pastoris and subcloned into pMD19-T. The SP300 gene was constructed into pPICZaA and sequenced. The recombinant pPICZaA-SP300 and pPICZaA-S300 was transformed into Pichia with LiC1. Results The recombination Pichia were cultivate, and expressed the SP300 or 5300 gene induced in Pichia by methanol. Conclusion The nucleocapsid secreted from the Pichia can be detected by ELISA and WesternBlot.