唾液HRP5第17位密码子反义突变对其在毕赤酵母中表达的影响  

Effects of antonymous mutation of 17th condon of salivary HRP5 on its expression in Pichia pastoris

作　　者：金科华[1] 赵亚华[2] 罗德生[1] 蒋智勇 崔红[2] 周崎[4] 赵长臣[2] 

机构地区：[1]咸宁学院医学院生物化学与分子生物学教研室,湖北咸宁437100 [2]华南农业大学生命科学学院生物化学教研室 [3]广东省兽医研究所猪病研究室 [4]咸宁学院医学院

出　　处：《解放军医学杂志》2007年第7期707-709,共3页Medical Journal of Chinese People's Liberation Army

摘　　要：目的在毕赤酵母中表达唾液富组蛋白5(HRP5)基因原始序列hrp5及突变序列hrp5′(Lys17→Asn),比较表达量及抗白色念珠菌活性。方法选用毕赤酵母偏爱密码子,设计3′端互补、5′端带酶切位点的两对引物,PCR合成hrp5和hrp5′,克隆至pPICZα-A,转化大肠杆菌,将酶切、测序鉴定的阳性重组质粒pPICZα-A-hrp5和pPICZα-A-hrp5′经SacI线性化后电击转化GS115,筛选阳性菌落,PCR鉴定,甲醇诱导表达,比较抗菌活性,测定表达量。结果hrp5及hrp5′正确整合到GS115基因组中,突变前后表达产物与HRP5标准品活性相当,表达量分别为4μmol/L、5μmol/L。结论HRP5在毕赤酵母中成功表达,活性区Lys17突变为Asn使表达量提高25%,而对其杀菌活性无影响。Objective To induce the the 17th condon antonymous mutagenesis of salivary histatin 5 （HRP5） cDNA, express the mutant and hrp5 in Pichia pastoris, and to study the effects of mutation on expression. Methods According to the Pichia pastoris＇ codon bias, two pairs of primers （H1 and H2, H3 and H4） were designed. H1 and H2, H3 and H4 have complementary 3＇ end, and the EcoR I site was added to the 5＇ end of H1 and H3, Sal I site to H2, H4. The cDNA of hrp5 and hrp5＇ was generated with PCR by H1 and H2, H3 and H4, respectively. The secrete vector pPICZα-A, hrp5 and hrp5＇ were digested with EcoR I ＋ Sal I, linkede by T1 DNA ligase and transformed to F. coli TOP10 comptetent cell, positive colonies were screened on LB plates with Zeocin. The recombinant plasrnids pPICZα-A-hrp5 and pPICZα-A-hrp5＇ identified by digestion and DNA sequencing were amplified largely, linearized by Sac I and transformed to GS115 comptetent cell by electroporation, positive colonies were screened on YEPD plates with Zeocin, the recombinant GSl15 were confirmed by PCR, cultured and induced expression by methanol. The amount and anticandidal activity of the expressed products was compared with synthetic HRP5. Results Both hrp5 and hrpS＇ were integrated into the genome of GSl15 and expressed successfully, the anticandidal activity of the recombinant HRP5 and HRP5＇ was identical with synthetic HRP5, the amount of expressed HRP5 and HRP5＇ was 4μmol/L and 5μmol/L, respectively. Conclusion Both the recombinant HRP5 and HRP5＇ showed better anticandidal activity. The amount of expressed pmdcts increased 25% by substituting Asn for Lysl7 without changing anticandidal activity.

关 键 词：唾液富组蛋白5 念珠菌 白色 基因表达 

分 类 号：R379.4[医药卫生—病原生物学]