华支睾吸虫溶血磷脂酶基因的识别、克隆和序列分析

INDENTIFICATION AND CLONING AND SEQUENCE ANALYSIS OF A NOVEL GENE ENCODING LYSOPHOSPHOLIPASE HOMOLOGUE FROM CLONORCHIASIS SINENSIS

目的 筛选cDNA文库识别华支睾吸虫新基因 ,并将所发现的华支睾吸虫溶血磷脂酶 (csLysoPLAH)基因编码区克隆到原核表达质粒PGEX 4T 1和真核表达质粒pcDNA3上 ,为进一步研究其功能奠定基础。 方法 对华支睾吸虫cDNA文库进行随机筛选并测序 ,在NCBI和ExPasy网站上进行序列分析 ,识别华支睾吸虫新基因 ,并应用Motifsan、NCBIConservedDomainSearch等程序对其进行结构域分析。根据PGEX 4T 1和 pcDNA3上的克隆位点及所发现的csLysoPLAHcDNA编码序列设计引物 ,进行PCR扩增 ,扩增产物回收纯化后克隆到原核表达载体PGEX 4T 1和真核表达载体 pcDNA3上。构建的PGEX 4T 1 csLysoPLAH、pcDNA3 csLysoPLAH重组表达质粒经PCR、双酶切及测序证实。 结果 发现csLysoPLAH基因 ,完整阅读框含 70 8个碱基 ,编码 2 3 5个氨基酸 ,理论分子质量为 2 5 .2 62 8ku ,理论pI为6.0 3。序列分析表明 ,csLysoPLAH编码氨基酸序列与其它物种有较高的同源性 ,csLysoPLAH具有磷脂酶 /羧酸酯酶保守功能域和完整的脂酶保守功能域 ,并含有丝氨酸酯酶特征性的标签肽 (即GXSXG)。所构建的重组原核和真核表达质粒经PCR、双酶切及测序证实与目标基因相符。 结论 发现华支睾吸虫溶血磷脂酶基因 ,并成功构建原核和真核重组表达质粒。

Objective To identify the novel genes of Clonorchiasis sinensis by screening the cDNA library and clone the screened gene-lysophospholipase homologue (LysoPLAH) to the prokaryotic expression vectors and eukaryotic expression vectors. Methods The cDNA library of C. sinensis were screened. The homologue of the novel sequences with a high identity was compared on amino acid and nucleotide level with blast programme on NCBI BLAST site. The motifs of the protein coded by the novel gene were searched with MotifScan in a proteins sequence on ExPASy site and NCBI Conserved Domain Search. Besides,a pair of specific oligonucleotide primers via BamHⅠ,XhoⅠ restriction sites respectively were designed and synthysed according to the coding region of LysoPLAH gene found by screening library. The coding region of LysoPLAH gene was amplified by PCR and then cloned into the prokaryotic expression vectors PGEX-4T-1 and eukaryotic expression vectors pcDNA3 via BamHⅠ and XhoⅠ restriction sites,and transformed into Escherichia coli JM109 respectively. The positive recombinant PGEX-4T-1-LysoPLAH and pcDNA3-LysoPLAH were screened and identified by endonuclease digestion,PCR and sequence. Results A novel cDNA sequence coding LysoPLAH was found from the cDNA library of C. sinensis . Translation of nucleotides sequence revealed putative open reading frame of 235 amino acids with a molecular mass of 25.262 8 ku and pI of 6.03. The predicted primary structure of LysoPLAH shares high sequence homology with other species and contain conserved domain of Phospholipase/Carboxylesterase and aconsensus signature peptide(GXSXG),which was present in all lipases. The recombinant plasmids PGEX-4T-1-LysoPLAH and pcDNA3-LysoPLAH were constructed. Conclusion A novel gene coding LysoPLAH of C. sinensis was found and cloned and its prokaryotic expression vectors and eukaryotic expression vectors were constructed successfully.