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目的对人含前导肽的β-NGF基因序列进行体外扩增、克隆及分析鉴定,并将其插入Pichiapastoris酵母分泌型表达载体pPIC9K中进行表达及鉴定。方法采用PCR技术,从人胎盘DNA基因组中扩增出含前导及信号肽的β-NGF基因序列;用A-T克隆法,与pGEM-T载体连接,经筛选得到重组质粒pGEM-Tβ-NGF,用酶切、PCR法及序列分析进行鉴定;将β-NGF插入酵母表达载体pPIC9K,用脂质体法导入GS115中,甲醇诱导分泌蛋白。结果亚克隆中,1.2%的琼脂糖凝胶电泳显示在748bp位置出现阳性条带;发酵液中蛋白SDS-PAGE电泳在14.0KD附近有特异表达带;蛋白表达量占菌体蛋白的30%;Westernblotting检测表明此带有特异活性。结论重组质粒pGEM-Tβ-NGF蛋白产量高,具有免疫活性。
OBJECTIVE: To amplify, clone, and identify the β-NGF gene sequence containing human leader peptide and insert it into the pichia pastoris yeast expression vector pPIC9K for expression and identification. Methods The β-NGF gene sequence containing the leader and signal peptide was amplified by PCR from the human placenta DNA genome. The recombinant plasmid pGEM-Tβ-NGF was cloned by using the AT cloning method and pGEM-T vector. Enzyme digestion, PCR and sequence analysis. Β-NGF was inserted into yeast expression vector pPIC9K and introduced into GS115 by liposome. Methanol induced secretion of protein. Results In the subclone, 1.2% agarose gel electrophoresis showed a positive band at 748bp. SDS-PAGE of the protein in the fermentation broth showed a specific band around 14.0KD. The protein expression accounted for 30% of the bacterial protein. Westernblotting test showed that this with specific activity. Conclusion The recombinant plasmid pGEM-Tβ-NGF has high yield and immunogenicity.