家蝇GNBP3基因克隆及感染白色念珠菌后的表达

发布时间：2018-09-11 08:34

【摘要】：对家蝇GNBP3基因进行克隆及生物信息学分析,并对该基因在感染白色念珠菌Candida albicans后的表达情况进行研究。从构建的家蝇Musca domestica幼虫c DNA质粒文库中筛选到GNBP3基因,克隆并运用生物信息学方法对该基因及其编码蛋白进行预测和分析。白色念珠菌注射感染家蝇幼虫并收集标本,逆转录,通过荧光定量PCR检测感染样本中GNBP3基因的表达情况,结果进行统计学分析。研究结果表明,GNBP3基因ORF全长1473 bp,编码490个氨基酸,理论分子量为55.8 k Da,等电点为6.85;感染后不同时间点,感染组与对照组比较,在3 h、12 h、36 h、48 h GNBP3 mRNA的表达明显增高,两组比较有显著性差异(P0.05),而24 h表达差异性最小;感染后不同组织中感染组与对照组比较,3 h和12 h时,在体壁、血淋巴、脂肪体中的表达量升高,具有显著性差异(P0.05);24 h时,在血淋巴中的表达量较对照组高,而在唾液腺和脂肪体中的表达量呈下降趋势,均具有显著性差异(P0.05);48 h,在血淋巴和脂肪体中表达量较对照组高,在中肠的表达量较对照组低,均具有显著性差异(P0.01)。成功克隆GNBP3基因且在家蝇感染白色念珠菌后GNBP3的表达水平随时间的推移呈现先升高后降低再升高的趋势,在各组织中以在免疫组织(血淋巴、脂肪体)中的表达显著增高,推测其在真菌识别过程中发挥潜在作用。
[Abstract]:The GNBP3 gene of Musca domestica was cloned and analyzed by bioinformatics, and the expression of the gene was studied after infection with Candida albicans of Candida albicans. The GNBP3 gene was screened from the constructed plasmid library of Musca domestica larva c DNA of housefly, and the gene and its encoded protein were predicted and analyzed by bioinformatics. Candida albicans were injected into housefly larvae and samples were collected. The expression of GNBP3 gene in infected samples was detected by fluorescence quantitative PCR. The results were statistically analyzed. The results showed that the ORF of GNBP3 gene encoded 490amino acids with a theoretical molecular weight of 55.8k Da, isoelectric point of 6.85.The expression of GNBP3 mRNA in the infected group was significantly higher than that in the control group at 3 h, 12 h, 36 h and 48 h after infection. There was significant difference between the two groups (P0.05), but the expression difference at 24 h was the least, and the expression level in the body wall, hemolymph and fat body increased at 3 h and 12 h after infection (P0.05), and there was significant difference at 24 h after infection (P0.05). The expression level in hemolymph was higher than that in control group, but the expression level in salivary gland and fat body showed a decreasing trend (P0.05) for 48 h. The expression level in hemolymph and fat body was higher than that in control group, and the expression level in midgut was lower than that in control group. There was significant difference between the two groups (P0.01). GNBP3 gene was cloned successfully and the expression level of GNBP3 increased first and then decreased and then increased after infection with Candida albicans in housefly, and increased significantly in immune tissues (hemolymph, fat body). It is speculated that it plays a potential role in the process of fungi recognition.
【作者单位】：贵州医科大学基础医学院;毕节市第一人民医院;贵州省疾病预防控制中心;贵州博康生物工程有限公司;
【基金】：国家自然科学基金(81360254) 贵州省科技厅省校合作项目(黔科合LH字[2014]7076号) 贵州省卫生计生委科学技术项目(gzwjkj2014-2-100) 贵州省科学技术基金(黔科合J字[2013]2042号)
【分类号】：Q78;Q963

【参考文献】