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目的: 克隆、分析泡球蚴 18 (Em18)抗原基因,构建 pET41a Em18 原核表达质粒,并初步诱导表达Em18重组蛋白。方法: DNAman软件设计引物,RT PCR法克隆 Em18 cDNA并构建 pMD18 T/Em18 质粒,测序确定序列,利用DNAman和BLAST软件,对其进行基因序列分析。构建 pET41a Em18 原核表达质粒,测序鉴定插入序列正确性。IPTG初步诱导和表达 rEm18 GST重组蛋白,SDS PAGE电泳检测。结果: 测序结果显示Em18抗原基因长度为 486 bp,编码 161 个氨基酸。BLAST 比对分析表明为一新序列并被 GenBank 收录(AY513691)。构建的 pET41a Em18原核表达质粒,经 IPTG诱导后,SDS PAGE检测表明 rEm18 GST重组蛋白得到成功表达,在相对分子量为50 KDa处有表达条带。结论: 成功克隆并构建了 pET41a Em18 原核表达质粒,初步诱导表达出 rEm18 GST重组蛋白,为新一代包虫病诊断试剂盒的研制奠定基础。
OBJECTIVE: To clone and analyze the antigen of Em 18 antigen and construct the prokaryotic expression vector pET41a Em18 and induce the expression of Em18 recombinant protein. Methods: Primer was designed by DNAman software. The cDNA of Em18 was cloned by RT PCR and the pMD18 T / Em18 plasmid was constructed. Sequences were identified by sequencing. DNA sequence analysis was performed by DNAman and BLAST software. The prokaryotic expression plasmid pET41a Em18 was constructed and sequenced to confirm the correctness of the inserted sequence. IPTG primary induction and expression rEm18 GST recombinant protein, SDS PAGE electrophoresis. Results: The sequencing results showed that the length of Em18 antigen gene was 486 bp, encoding 161 amino acids. BLAST analysis showed a new sequence and was included in GenBank (AY513691). The constructed prokaryotic expression vector pET41a Em18, induced by IPTG, SDS PAGE showed rEm18 GST recombinant protein was successfully expressed in the relative molecular weight of 50 KDa at the expression band. Conclusion: The prokaryotic expression vector pET41a Em18 was successfully cloned and constructed. The rEm18 GST recombinant protein was induced initially, which laid the foundation for the development of a new generation of diagnostic kit for hydatid disease.