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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2016, Vol. 36 Issue (4): 78-87    DOI: 10.13523/j.cb.20160412
研究报告     
细粒棘球蚴EgG1Y162基因进化分析、表达及鉴定
祖力皮也·吐尔逊1, 曹春宝1, 温浩1, 丁剑冰2, 德力夏提·依米提1,2
1. 新疆重大疾病医学实验室-省部共建国家重点实验室培育基地 新疆医科大学第一附属医院 乌鲁木齐 830054;
2. 新疆医科大学科研处 乌鲁木齐 830011
Analysis of Gene Evolution, Protein Expression and Identification of Echinococcus granulosus EgG1Y162
TUERXUN Zulipiye1, CAO Chun-bao1, WEN Hao1, DING Jian-bing2, YIMITI Delixiati1,2
1. Base to Foster a State Key Lab for Major Disease Research in Xinjiang , The First Affiliated Hospital of Xinjiang Medical University, Urumuchi 830054, China;
2. College of Preclinical Medicine, Xinjiang Medical University, Urumuchi 830011, China
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摘要：

目的:克隆和鉴定细粒棘球蚴的EgG1Y162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emY162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cDNA,利用PCR的方法以基因组DNA和cDNA为模板扩增EgG1Y162基因;构建PUCm-T/EgG1Y162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EgG1Y162基因特点进行分析并构建EgG1Y162核酸序列的进化树进一步探讨其同源性。荧光定量PCR检测EgG1Y162 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EgG1Y162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EgG1Y162-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EgG1Y162基因,从总DNA克隆得到片段长度1 680bp,从cDNA克隆得到片段长度459bp。相似性比较表明,EgG1Y162基因序列与emY162相似性为91%,而EgG1Y162基因cDNA与emY162相似性为95%。进一步分析显示,EgG1Y162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EgG1Y162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EgG1Y162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EgG1Y162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EgG1Y162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/EgG1Y162原核表达质粒,经IPTG诱导后,SDS-PAGE 检测表明EgG1Y162-GST重组蛋白得到成功表达,在相对分子量为44kDa 处有表达条带。Western blot 分析显示阳性印迹条带,分子量为44kDa,EgG1Y162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EgG1Y162抗原基因,序列对比分析显示EgG1Y162的cDNA与emY162的cDNA具有很高的相似性,基因的差异性主要存在于内含子区域,EgG1Y162抗原基因是一种新的基因。EgG1Y162在成虫中的表达量最多。成功诱导表达出EgG1Y162重组蛋白,并且EgG1Y162重组蛋白具有很好的抗原性。
关键词： 原核表达;  EgG1Y162抗原;  进化树;  荧光定量PCR    
Abstract:

Objective: To clone and identify Echinococcus granulosus egG1Y162 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 cDNA,amplified egG1Y162 gene via PCR with genomic DNA and cDNA . Construct the recombinant plasmid of PUCm-T/EgG1Y162 and identified by PCR, enzyme digestion and sequencing to determine its correctness. The characteristics of EgG1Y162 gene were analyzed by DNAman software and MEGA4 software, and construct the phylogenetic tree of EgG1Y162 nucleic acid to further explore its homology. Fluorescence quantitative PCR detection egG1Y162 gene at four different developmental stages including Echinococcus granulosus protoscolices, cystic wall students expression layer, adults and eggs. The EgG1Y162 gene was cloned into prokaryotic expression plasmid PET-41a 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 EgG1Y162-GST recombinant proteins and identified by SDS-PAGE and Western blot assay. Results: EgG1Y162 gene were cloned from both two different developmental stages of Echinococcus granulosus, from total DNA clone obtained length for 1 680bp fragment , from a cDNA clone obtained length for 459bp fragment .The similarity of the EgG1Y162 gene was 91%, while the similarity of emY162 gene cDNA and EgG1Y162 was 95%. Further analysis showed that the sequence of EgG1Y162 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 EgG1Y162 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 EgG1Y162 gene was 360bp and 120 amino acids were encoded.. By fluorescence quantitative PCR detection found egG1Y162 in adults, germinal layer, protoscolex and the egg stage had different degrees of expression. But the majority egG1Y162 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-41a/EgG1Y162 was induced by SDS-PAGE, and the IPTG assay showed that the recombinant protein of EgG1Y162-GST was successfully expressed and expressed in the relative molecular weight of 44KDa. Western blot analysis showed positive blot bands, the molecular weight of 44kda, egG1Y162 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 egG1Y162 antigen gene, sequence comparison analysis showed high similarity with cDNA of the egG1Y162 cDNA and emy162, differences in gene mainly exists in the intron region, the egG1Y162 antigen gene is a new gene. The expression of EgG1Y162 in adult was the most. The recombinant protein was successfully induced, and the recombinant protein of EgG1Y162 has significant antigenicity.
Key words: EgG1Y162 antigen    Phylogenetic tree    Fluorescence quantitative PCR    Prokaryotic expression
收稿日期: 2015-09-16 出版日期: 2016-01-06
ZTFLH:  Q819  
基金资助:

国家自然科学基金(31000411,81160378)资助项目
通讯作者: 丁剑冰     E-mail: susan8s8@126.com
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引用本文:

祖力皮也·吐尔逊, 曹春宝, 温浩, 丁剑冰, 德力夏提·依米提. 细粒棘球蚴EgG1Y162基因进化分析、表达及鉴定[J]. 中国生物工程杂志, 2016, 36(4): 78-87.

TUERXUN Zulipiye, CAO Chun-bao, WEN Hao, DING Jian-bing, YIMITI Delixiati. Analysis of Gene Evolution, Protein Expression and Identification of Echinococcus granulosus EgG1Y162. China Biotechnology, 2016, 36(4): 78-87.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160412    或    https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I4/78

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