日本血吸虫外分泌蛋白、跨膜蛋白基因筛选与无缝克隆

Bioinformatics Analysis and Seamless Cloning of Schistosoma Japonicum Genes Coding Signal Peptide and Transmembrane Domain

目的:探讨无缝克隆技术在构建日本血吸虫基因重组质粒中的应用价值。方法:从血吸虫基因组数据库中筛选含有信号肽或跨膜结构域的基因,截取胞外段大于110个氨基酸残基的基因片段。根据无缝克隆技术原理,设计引物。以日本血吸虫基因组DNA为模板,进行PCR扩增。纯化后的PCR产物与线性化载体p ET32c(+)/Bam HⅠ+XhoⅠ以摩尔比6∶1混和,利用Seamless cloning enzyme在25℃反应30 min。化学法将连接产物转至大肠杆菌Trans 5α,菌落PCR筛选阳性克隆。提取质粒,Bam HⅠ和XhoⅠ双酶切验证后送测序。测序结果用DNAStar进行序列分析。结果:通过生物信息学软件分析,在日本血吸虫基因组数据库中共筛选到278个含有信号肽或跨膜结构域且胞外段大于110个氨基酸残基的目标基因。对其中41个单一外显子的基因进行了无缝克隆引物设计和合成。PCR成功扩增到33个基因片段,并与线性化的p ET32c(+)进行无缝克隆连接。经菌落PCR和酶切验证获得28个阳性重组质粒。测序显示28个插入基因片段序列正确。结论:利用Seamless克隆技术成功获取了一批含有日本血吸虫基因或基因片段的重组质粒,省去了传统的酶切、连接、去磷酸化等手段,节省了时间,提高了效率,为后续的日本血吸虫免疫组学的高通量抗原筛选和鉴定奠定了基础。

Objective To explore the potential value of seamless cloning for construction of the recombinant plasmid including traget genes from Schistosoma japonicum genome. Methods The genes including singal peptide and transmembrane domain were screened from Schistosoma japonicum genome dataset,the extracellular region of traget genes with more than 110amino-acid residues were captured. Based on the principle of seamless cloning,the pair-primers were desgined. The target genes were ampilified by PCR with S. japonicum genome DNA as template. The purified PCR product and linear vector p ET32c（ ＋） / Bam H Ⅰ ＋ Xho Ⅰ was mixed with a molar ratio 6： 1,and then the mixture was incubated with seamless cloning enzyme at 25 ℃,30 min. The ligation products were transformed into Escherichia coli Trans 5α by chemical transformation method with Ca Cl2 and screened by bacterial colony PCR. The positive recombinant plasmids were digested by Bam H Ⅰ＋ Xho Ⅰ and sent to company for sequencing. Results According to the bioinformatic analysis,total 278 target genes with a signal peptide or transmembrane domain（ s）,containing much more 110 amino acids in extracellular domain were successfully screened from S. japonicum genome. The 41 single-exon genes were selected and seamless cloning primers were synthetized. The 33 gene fragments were amplified and liagated with linearized p ET32c（ ＋） via seamless cloning technique.The 28 positive recombinant plasmids were screened and identified by bacterial colony PCR and digestion,respectively. The sequencing results showed that the inserted sequenceing of target genes were corrected. Conclusion With the seamless cloning technique,a batch of target genes or their fragments in S. japonicum genome are successfully cloned into vector. It saves the time of digestion,ligation and dephosphorylation and supplies an unprecedented opportunity for immunoscreening and identification of S. japonicum antigens via a high throughput manner.