鸭疫里默氏杆菌AS87-01740基因生物信息学分析及其原核表达

发布时间：2018-04-16 03:31

本文选题：鸭疫里默氏杆菌 + AS-基因　；参考：《南方农业学报》2017年11期

【摘要】：【目的】了解鸭疫里默氏杆菌AS87-01740基因分子生物学特性,确定其编码蛋白抗原性,为鸭疫里默氏杆菌亚单位疫苗研发提供新的靶标抗原选择。【方法】采用PCR克隆鸭疫里默氏杆菌AS87-01740基因,以DNASTAR和NCBI数据库分别进行基因生物信息学分析;利用p Cold-TF原核表达载体在大肠杆菌BL21感受态细胞中进行诱导表达,并分别以SDS-PAGE和Western blotting检测融合蛋白的表达形式及其抗原特异性。【结果】从鸭疫里默氏杆菌基因组扩增获得的AS87-01740基因编码框全长618 bp,编码205个氨基酸,理论分子量约21.87 kD,理论等电点(p I)8.2,正电荷氨基酸总数17个,负电荷氨基酸总数16个。AS87-01740蛋白序列在同种属分离株中,与2型血清型分离株(11845、YM、GD和CH-2)有很高的相似性(99%),与1型血清型分离株(CH-1和CH-3)的相似性为92%;在不同种属中,与金黄杆菌(Chryseobacterium sp.)、动物溃疡伯格菌(Bergeyella zoohelcum)和脑膜炎败血伊丽莎白菌(Elizabethkingia meningosepticum)的相似性分别为61%、60%和57%。经异丙基硫代-β-D半乳糖苷(IPTG)诱导表达获得的融合蛋白分子量约77 kD,以可溶性表达形式为主。Western blotting检测结果表明,融合蛋白能与鸭疫里默氏杆菌阳性血清发生特异性免疫反应。【结论】AS87-01740基因编码蛋白在不同鸭疫里默氏杆菌分离株中具有很高的保守性,且能与其阳性血清发生特异性免疫反应,是研发鸭疫里默氏杆菌疫苗的潜在抗原。
[Abstract]:[objective] to understand the molecular biological characteristics of the AS87-01740 gene of Riemer Duck, and to determine the antigenicity of its encoded protein.[methods] PCR was used to clone the AS87-01740 gene of Riemer's disease, and the gene bioinformatics analysis was carried out by DNASTAR and NCBI database.The expression of p Cold-TF was induced in Escherichia coli BL21 receptive cells by using prokaryotic expression vector.The expression and antigen specificity of the fusion protein were detected by SDS-PAGE and Western blotting, respectively. [results] the full length of AS87-01740 gene encoding 205 amino acids was obtained by amplification from the genome of Riemer's disease.The theoretical molecular weight is about 21.87 KD, the theoretical isoelectric point is 8.2, the total number of positively charged amino acids is 17, and the total number of negatively charged amino acids is 16. AS87-01740 protein sequences are in the same species.There was a high similarity with serotype 2 isolates 11845YMN GD and CH-2, and the similarity with serotype 1 isolates CH-1 and CH-3 was 92.In different species,The similarity with Chryseobacterium sp., Bergeyella zoohelcumum and Elizabeth septicum were 61% and 57%, respectively.The molecular weight of the fusion protein induced by isopropylthiothio- 尾 -D galactoside (IPTG) was about 77kD.Western blotting analysis showed that the fusion protein was mainly expressed in soluble form.The fusion protein can react specifically with the positive serum of Riemer pestis. [conclusion] the AS87-01740 gene encoding protein is highly conserved in different isolates of Riemer's disease.It is a potential antigen for the development of Riemer's disease vaccine.
【作者单位】：广西大学动物科学技术学院;中国农业科学院上海兽医研究所;
【基金】：国家自然科学基金项目(31272591) 上海市科技兴农计划项目(2016HNG4-1) 中央级公益性科研院所科研基金项目(2017JB12)
【分类号】：S852.61

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