细粒棘球蚴EgG1Y162基因进化分析、表达及鉴定

Analysis of Gene Evolution,Protein Expression and Identification of Echinococcus granulosus EgG1Y162

目的：克隆和鉴定细粒棘球蚴的Eg G1Y162基因,分析其蛋白表达和适应性进化并鉴定抗原性。方法：根据em Y162基因序列设计引物,分别从细粒棘球绦虫原头蚴、囊壁生发层、成虫和虫卵四个发育阶段,提取基因组DNA和总RNA,mRNA反转录为c DNA,利用PCR的方法以基因组DNA和c DNA为模板扩增Eg G1Y162基因;构建PUCm-T/Eg G1Y162重组质粒,经PCR、酶切及测序鉴定后,测序确定其正确性。利用DNAman软件与MEGA4软件对Eg G1Y162基因特点进行分析并构建Eg G1Y162核酸序列的进化树进一步探讨其同源性。荧光定量PCR检测Eg G1Y162基因在细粒棘球绦虫原头蚴、囊壁生发层、成虫和虫卵四个不同发育阶段的表达情况。利用定向克隆技术将Eg G1Y162抗原基因片段克隆至原核表达质粒PET-41a上,通过酶切分析和PCR鉴定筛选出阳性克隆,测序确定序列。IPTG初步诱导和表达Eg G1Y162-GST重组蛋白,通过SDS-PAGE电泳和Western blot试验分析鉴定。结果：从细粒棘球绦虫的两个不同发育阶段均克隆出Eg G1Y162基因,从总DNA克隆得到片段长度1 680bp,从c DNA克隆得到片段长度459bp。相似性比较表明,Eg G1Y162基因序列与em Y162相似性为91%,而Eg G1Y162基因c DNA与em Y162相似性为95%。进一步分析显示,Eg G1Y162基因序列由3个外显子和2个内含子组成,外显子区域分别为1～70,1 064～1 380和1 577～1 648。位于疏水端1～16位氨基酸构成Eg G1Y162信号肽序列,35～115位氨基酸形成一个大的纤黏连蛋白剪接体FN3,133～152位氨基酸构成羧基端跨膜区域。测序结果显示Eg G1Y162抗原基因长度为360bp,编码120个氨基酸。通过荧光定量PCR检测,发现Eg G1Y162在成虫、生发层阶段、原头蚴和虫卵阶段均有不同程度的表达。但是Eg G1Y162在成虫中的表达量最多,相对值为19.526,差异有统计学意义（P〈0.01）,其次生发层阶段,为5.122,再次在原头蚴阶段,相对值为5.083,而在虫卵阶段最少,为1.6588。构建的PET-41a/Eg G1Y162原核表达质粒,经IPTG诱导后,SDS-PAGE检测表明Eg G1Y162-GST重组蛋白得到成功表达,在相对分子量为44k Da处有表达条带。Western blot分析显示阳性印迹条带,分子量为44k Da,Eg G1Y162重组蛋白能与细粒棘球蚴感染40天后犬的血清发生反应;与包虫病患者血清也有阳性反应。结论：成功克隆Eg G1Y162抗原基因,序列对比分析显示Eg G1Y162的c DNA与em Y162的c DNA具有很高的相似性,基因的差异性主要存在于内含子区域,Eg G1Y162抗原基因是一种新的基因。Eg G1Y162在成虫中的表达量最多。成功诱导表达出Eg G1Y162重组蛋白,并且Eg G1Y162重组蛋白具有很好的抗原性。

Objective：To clone and identify Echinococcus granulosus eg G1Y162 gene and analysis the protein expression,adaptive evolution and identification.of antigenicity.Methods：According to the gene sequence of emy162,a pair of primers was designed from Echinococcus granulosus protoscolices,cystic wall students send layer,adults and eggs of four developmental stage respectively,extract the genomic DNA and total RNA,mRNA was reverse transcribed into c DNA,amplified eg G1Y162 gene via PCR with genomic DNA and c DNA.Construct the recombinant plasmid of PUCm-T/Eg G1Y162 and identified by PCR,enzyme digestion and sequencing to determine its correctness.The characteristics of Eg G1Y162 gene were analyzed by DNAman software and MEGA4 software,and construct the phylogenetic tree of Eg G1Y162 nucleic acid to further explore its homology.Fluorescence quantitative PCR detection eg G1Y162 gene at four different developmental stages including Echinococcus granulosus protoscolices,cystic wall students expression layer,adults and eggs.The Eg G1Y162 gene was cloned into prokaryotic expression plasmid PET-41 a by using the directed cloning technology,and the positive clones were identified by the restriction analysis and PCR identification,and the sequence was determined by sequencing.IPTG was initially induced and expressed by Eg G1Y162-GST recombinant proteins and identified by SDS-PAGE and Western blot assay.Results：Eg G1Y162 gene were cloned from both two different developmental stages of Echinococcus granulosus,from total DNA clone obtained length for1 680 bp fragment,from a c DNA clone obtained length for 459 bp fragment.The similarity of the Eg G1Y162 gene was 91%,while the similarity of em Y162 gene c DNA and Eg G1Y162 was 95%.Further analysis showed that the sequence of Eg G1Y162 gene was composed of 3 exons and 2 introns,the exon regions were 1 ～ 70,1064 ～1380and 1577 ～ 1648.The amino acids of 1 ～ 16 amino acids in the hydrophobic side constitute the Eg G1Y162 signal peptide sequence,and 35 ～ 115 amino acids form a large fibronectin splicing FN3133 ～ 152 amino acids which constitute the carboxyl terminal transmembrane region.The result of DNA sequencing showed that the length of Eg G1Y162 gene was 360 bp and 120 amino acids were encoded..By fluorescence quantitative PCR detection found eg G1Y162 in adults,germinal layer,protoscolex and the egg stage had different degrees of expression.But the majority eg G1Y162 expression in the adult stage,at most a relative value is 19.526 difference was statistically significant（P 0.01）,secondly germinal layer,5.122,again in protoscolex stage,relative value 5.083,in the egg stage at least for 1.6588.The prokaryotic expression plasmid of PET-41 a/Eg G1Y162 was induced by SDS-PAGE,and the IPTG assay showed that the recombinant protein of Eg G1Y162-GST was successfully expressed and expressed in the relative molecular weight of 44 KDa.Western blot analysis showed positive blot bands,the molecular weight of 44 kda,eg G1Y162 recombinant protein can with Echinococcus granulosus infection after 40 days of canine sera react;serum from patients with hydatid disease also have positive reaction.Conclusion：Successfully cloned eg G1Y162 antigen gene,sequence comparison analysis showed high similarity with c DNA of the eg G1Y162 c DNA and emy162,differences in gene mainly exists in the intron region,the eg G1Y162 antigen gene is a new gene.The expression of Eg G1Y162 in adult was the most.The recombinant protein was successfully induced,and the recombinant protein of Eg G1Y162 has significant antigenicity.