捻转血矛线虫脂肪酶基因表达与生物学特性分析

摘要

To study the biological character of Lipase from Haemonchus contortus,the lipase gene of H.contortus preserved in our laboratory was cloned by PCR,and then expressed in procaryotic expression system.The expression vector was constructed to obtain the fusion protein.The antigenicity of the protein was analyzed by Western blot.The Lipase activity of the fusion protein was detected by esterase reaction.The location of the protein was tested by the indirect immunofluorescence.The transcription of lipase in different stages of H.contortus was detected by real-time quantification PCR.The results showed that the gene fragment encoding Lipase (without signal peptide region) sized as 864 bp.The recombinant protein weighted as 50 kD was fusion expressed.The fusion protein had highest lipase enzyme activity under 37 ℃ and pH7.Hc-Lipase was localized at outer and inter membrane as well as the intestinal tracts of adult worms.The expression of Hc-Lipase was higher in the male than other stages.It is indicated that Lipase from H.contortus could play important roles in worm development and modulation of the host immunity.Therefore,it is expected that Hc-Lipase could be a new target in prevention and therapy of Haemonchosis.%选取捻转血矛线虫脂肪酶(Lipase)进行基因克隆、表达并对其生物学特性进行探究.PCR克隆捻转血矛线虫lipase基因,构建表达载体,获得融合蛋白质,采用Western blot检测该蛋白质的抗原特性;酯类酶解反应检测该融合蛋白质脂肪酶活性;实时定量PCR检测该基因在捻转血矛线虫不同发育阶段的转录情况;间接免疫荧光试验检验该抗原蛋白质在虫体组织的定位情况.结果表明,去除lipase基因信号肽序列后,基因片段长864 bp,融合蛋白质大小50 ku;该融合蛋白质在37℃左右、pH为7的条件下酶活力最高;雄性成虫lipase转录量相对虫体其他阶段最高;该抗原蛋白质主要表达于虫体表皮和肠腔.Lipase具有较好的抗原性,可能与捻转血矛线虫发育和宿主免疫调节有关,深入研究捻转血矛线虫Lipase的特性对寻找新的免疫保护性抗原和药物靶标具有意义.