蓝氏贾第鞭毛虫丙酮酸激酶特异性锤头状核酶-GCV重组载体的构建

目的构建蓝氏贾第鞭毛虫丙酮酸激酶(pyruvate kinase,PK)特异性锤头状核酶-犬贾第虫病毒(Giardia canis virus,GCV)重组载体。方法采用RNAdraw软件分析贾第虫编码丙酮酸激酶的基因序列,并设计特异性反义锤头状核酶(PKH)序列,将其与犬贾第虫病毒(GCV)连接,构建重组载体pGCV-PKH。将重组载体线性化体外转录产物,分别进行贾第虫细胞外、细胞内目的mRNA切割实验,采用荧光显微镜观察转染后24h的各组虫体。采用实时PCR对切割产物进行相对定量分析。结果构建了载有蓝氏贾第鞭毛虫丙酮酸激酶特异性锤头状核酶的犬贾第虫病毒重组载体pGCV-PKH。细胞内切割实验结果表明,荧光显微镜下只有pGCV-GFP转染组虫体显示绿色荧光,pGCV-PKH转染组丙酮酸激酶mRNA的相对含量约为正常对照组的33.14%。细胞外切割实验结果表明,该组载体能在细胞外有效切割丙酮酸激酶mRNA,在设定条件下,其切割效率为58.5%。结论重组载体pGCV-PKH能有效转染蓝氏贾第鞭毛虫细胞,并能在其细胞内外对丙酮酸激酶mRNA进行有效切割。

Construction of GCV-Specific Hammerhead Ribozyme Recombinant Vector of Pyruvate Kinase in Giardia lamblia

Objective To construct a recombinant vector of Giardia canis virus (GCV)-specific hammerhead ribozyme of pyruvate kinase (PK) in Giardia lamblia. Methods Using RNA draw software, the secondary structure of PK gene in Giardia was predicted and the cleavage site of ribozyme was selected. The antisense-specific hammerhead ribozyme (PKH) targeting this site was designed. The DNA encoding the ribozyme was synthesized and the recombinant vector pGCV-PKH was constructed. The extracellular and intracellular cleavag...