论文部分内容阅读
目的 克隆并在巴斯德毕赤酵母中表达猪囊尾蚴抗原cC1。方法 用PCR法在cC1cDNA5′端引入所需限制酶位点和酵母分泌信号肽 ,与酵母表达载体pPIC9k重组 ,构建表达质粒 pPIC9k -cC1。采用电穿孔法转化酵母菌SMD116 8,在MD平板上筛选重组克隆 ,用G4 18快速筛选高拷贝转化子 ,阳性克隆经甲醇诱导表达后 ,培养上清SDS -PAGE和Westernblot鉴定。结果 PCR产物经测序无误 ,pPIC9-cC1和 pPIC9k -cC1酶切分析与预期相符 ,获取 86 3个酵母阳性重组克隆及9个高拷贝转化子。表达产物cC1的分子量约 4 2kDa ,占分泌总蛋白 80 %以上 ,产物浓度为 2 0 0 - 35 0mg/L。Westernblot证实表达产物具有天然cC1分子的免疫原性。结论 在巴斯德毕赤酵母中成功表达了猪囊尾蚴cC1抗原 ,为进一步研究打下基础
Objective To clone and express Cysticercus cellulosae cC1 in Pichia pastoris. Methods The restriction endonuclease site and yeast secretion signal peptide were introduced into the 5 ’end of cC1 cDNA by PCR and recombined with the yeast expression vector pPIC9k to construct the expression plasmid pPIC9k-cC1. The electroporation method was used to transform Saccharomyces cerevisiae SMD116 8. The recombinant clones were screened on MD plates and high-copy transformants were screened by G418. The positive clones were induced by methanol and identified by SDS-PAGE and Western blot. Results The PCR products were sequenced without error. The restriction analysis of pPIC9-cC1 and pPIC9k-cC1 was in accordance with the expectation, and 863 positive yeast clones and 9 high-copy transformants were obtained. The expression product of cC1 molecular weight of about 42kDa, accounting for more than 80% of the total secreted protein, the product concentration of 200 ~ 35 0mg / L. Western blot confirmed that the expressed product possessed the immunogenicity of the native cC1 molecule. Conclusion The cCl antigen of Cysticercus cellulosae was successfully expressed in Pichia pastoris, laying a foundation for further study