摘要
利用重组毕赤酵母表达四联鲎抗菌肽Tachyplesin Ⅱ以实现高效制备。根据鲎抗菌肽Tachyplesin Ⅱ的氨基酸序列,以及胰弹性蛋白酶和羧肽酶A的特异性裂解位点,设计四联鲎抗菌肽(4Tp Ⅱ)序列,按照毕赤酵母密码子偏好性进行优化,经EcoR Ⅰ和Not Ⅰ双酶切后构建重组质粒pPICZαA-4Tp Ⅱ,电转至毕赤酵母X-33,利用Zeocin抗性筛选阳性转化子,经甲醇诱导表达后SDS-PAGE分析。成功构建重组质粒pPICZαA-4Tp Ⅱ,并筛选出阳性转化子,经30℃、250 r/min、1%甲醇诱导表达5 d后,获得重组蛋白,可为高效制备鲎抗菌肽Tachyplesin Ⅱ奠定基础。
The recombinant Pichia pastoris was used to express quadruple Limulus antimicrobial peptide,Tachyplesin Ⅱ,to achieve efficient preparation.According to the amino acid sequence of Tachyplesin Ⅱ and the specific cleavage sites of pancreatic elastase and carboxypeptidase A,the quadruple(4TpⅡ) sequence was designed and optimized according to the codon preference of Pichia pastoris.After digested by EcoR Ⅰ and Not Ⅰ,the recombinant plasmid pPICZαA-4Tp Ⅱ was constructed and electroporated to Pichia pastoris X-33.The positive transformants were screened by Zeocin resistance and the expression was analyzed by SDS-PAGE.The recombinant plasmid pPICZa A-4Tp Ⅱ was successfully constructed,and the positive transformants were screened.After induced expression at 30 ℃,250 r/min and 1% methanol for 5 days,the recombinant protein was obtained.This study can lay a foundation for the efficient preparation of Limulus antimicrobial peptide Tachyplesin Ⅱ.
出处
《科技创新与应用》
2024年第7期47-50,共4页
Technology Innovation and Application
基金
福建省自然科学基金青创项目(2022J05322)
福建省中青年教师教育科研项目(JAT210492)
厦门医学院大学生创新创业训练计划项目(S202112631012,202212631005,202212631006)。
