大鲵β-defensin基因的生物信息学分析和表达特性研究

发布时间：2018-05-25 13:53

本文选题：大鲵 + β-defensin基因　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：β-defensin是一种结构稳定,抗菌抗病毒活性强,具备多种生物学活性的小分子多肽。本研究对来自于大鲵皮肤转录组数据库中的大鲵β-defensin基因进行了鉴定和结构分析;采用荧光实时定量PCR检测了大鲵β-defensin基因在不同组织、不同发育阶段以及细菌和病毒感染情况下的表达特性和变化;并对大鲵β-defensin基因进行了重组原核表达载体的构建和重组蛋白活性的检测。获得如下结果:1.大鲵β-defensin基因全长cDNA为771 bp,其中5’非编码区为90bp,3’非编码区为531 bp,开放阅读框150bp,共编码49个氨基酸,编码的蛋白质分子量为5.52 kD,等电点为9.23,在pH等于7的溶液中的电荷为5.87,无信号肽,为内源性抗菌肽。基于氨基酸序列的同源聚类关系表明,其和两栖动物亲缘关系最近。2.实时荧光定量PCR表明,大鲵在皮肤中表达量最高,且在幼体时即大量表达。细菌和病毒感染会显著上调大鲵β-defensin基因的表达,其中细菌感染后大鲵β-defensin基因在24小时达到峰值,而病毒感染后则在120小时达到峰值。3.构建获得的大鲵重组表达载体pET32a(+)-AdBD经IPTG诱导,获得了大鲵重组β-防御素蛋白,纯化后的重组蛋白对革兰氏阴性菌和革兰氏阳性均有杀灭效果,其中对革兰氏阴性菌中的溶藻弧菌、大肠杆菌和哈维氏菌最有效,最小抑菌浓度仅为19μg/mL。以上结果表明大鲵β-defensin基因是大鲵皮肤主要免疫效应基因,对细菌和病毒感染均有积极响应,其中对细菌感染响应迅速。同时重组原核表达的大鲵β-defensin蛋白对多种细菌均有很好的抗菌活性,是生物活性肽资源开发的潜在对象。
[Abstract]:尾 -defensin is a small polypeptide with stable structure, strong antibacterial and antiviral activity and various biological activities. In this study, the 尾 -defensin gene from the skin transcriptional database of giant salamander was identified and its structure was analyzed, and the 尾 -defensin gene in different tissues was detected by real-time fluorescence quantitative PCR. The expression characteristics and changes of 尾 -defensin gene in different stages of development and bacterial and viral infection were studied, and the recombinant prokaryotic expression vector was constructed and the activity of recombinant protein was detected. Get the following result: 1. The full-length cDNA of the 尾 -defensin gene of giant salamander is 771bp, of which the 5'noncoding region is 90bpf3'and the open reading frame is 150bp. it encodes 49 amino acids. The encoded protein has a molecular weight of 5.52 KD, a isoelectric point of 9.23 and a charge of 5.87 in a solution with pH = 7. It is an endogenous antimicrobial peptide. The homologous clustering relationship based on amino acid sequence shows that it has the closest genetic relationship with amphibians. Real-time fluorescence quantitative PCR showed that Giandrias davidianus had the highest expression in skin and was highly expressed in larvae. Bacterial and viral infection significantly up-regulated the expression of 尾 -defensin gene. The peak of 尾 -defensin gene reached a peak at 24 hours after bacterial infection, and reached a peak value at 120 hours after viral infection. The recombinant expression vector pET32a was constructed. The recombinant 尾 -defensin protein of salamander was induced by IPTG. The purified recombinant protein could kill both Gram-negative bacteria and Gram-positive bacteria. Among them, vibrio alginolyticus, Escherichia coli and Harvey's bacteria in Gram-negative bacteria were the most effective, the minimum inhibitory concentration was only 19 渭 g / mL. The results showed that the 尾 -defensin gene was the main immune effector gene in the skin of giant salamander, and had a positive response to both bacterial and viral infection, and the response to bacterial infection was rapid. At the same time, the recombinant 尾 -defensin protein of giant salamander has good antibacterial activity against a variety of bacteria, and it is a potential target for the exploitation of bioactive peptide resources.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4;S947.3

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