团头鲂三个抗菌肽基因的鉴定与表达研究

发布时间：2018-07-03 10:30

本文选题：团头鲂 + 嗜水气单胞菌　；参考：《华中农业大学》2017年硕士论文

【摘要】：NK-LYSIN是机体自然杀伤细胞(natural killer cells,NKL)和毒性T细胞(cytotoxic T lymphocytes,CTL)分泌的小分子阳离子抗菌肽,同人类颗粒溶素granulysin是同源多肽,具有广谱的抗菌活性,对细菌、真菌、病毒、癌细胞等均有生物活性。组蛋白H2A是真核生物体细胞染色质中的一种结构蛋白,是先天性免疫的组成成分之一,其衍生的多肽片段的杀菌作用已成为目前的研究热点。团头鲂(Megalobrama amblycephala)是我国重要的淡水养殖品种,随着养殖规模的扩大,集约化程度的提高以及养殖环境的恶化,导致各类疾病频发。其中,对团头鲂养殖影响最大的疾病是由嗜水气单胞菌(Aeromonas hydrophila)感染引起的细菌性败血症,可造成鱼体各部位充血、溃疡、腹腔积水和肝脏发白等症状,从而导致鱼体大量死亡。另外,现阶段还没有很好的药物可以控制,养殖业中则主要使用抗生素提高鱼体免疫力,但抗生素会造成药物残留和细菌耐药性,严重影响水产业的发展和人类健康,新型抗菌药物的研发成为热点。本研究从团头鲂转录组中钓取到2个NK-lysin基因(nkla和nklb)和1个H2A基因,通过PCR扩增和测序验证其ORF序列,并分析序列特征。采用荧光定量PCR技术检测其在团头鲂胚胎发育不同时期以及成鱼组织和嗜水气单胞菌感染后免疫组织中的表达。构建团头鲂2个NK-lysin基因的原核表达载体,转入大肠杆菌中进行重组表达,并对其表达条件进行优化,为进一步研究NK-lysin的功能奠定了基础。本研究的主要结果如下:1.团头鲂3个抗菌肽基因(nkla、nklb和H2A)的序列分析nkla ORF全长369 bp,编码122个氨基酸,DNAstar预测其等电点为6.71,分子质量为14 kDa;nklb ORF全长411 bp,编码136个氨基酸,DNAstar预测其等电点为6.72,分子质量为15.6 kDa。多氨基酸序列比对分析显示,nkla和nklb的N端均含有信号肽,6个高度保守半胱氨酸,可形成3对分子内二硫键,以及1个SapB结构域,序列分析发现它属于鞘脂激活蛋白样蛋白家族成员。系统进化分析显示,团头鲂NK-lysin基因与斑马鱼亲缘关系最近,其次是半滑舌鳎和斑点叉尾洶。组蛋白H2A基因ORF全长387 bp,编码128个氨基酸,DNAstar预测其等电点为10.92,分子质量为16.2 kDa。H2A氨基酸序列不含跨膜结构域及信号肽,且其序列十分保守,不同物种间序列相似性极高。2.团头鲂3个抗菌肽基因在不同发育时期及成鱼各组织中的表达荧光定量PCR结果显示,nkla和nklb在胚胎发育的不同时期广泛表达。在胚胎发育的初始阶段,nkla和nklb呈低表达,而在出膜期时,nkla和nklb表达量快速上升。其中,nkla在眼球色素出现期达到最高表达,nklb在出膜后1 d时达到最高值。H2A也在胚胎发育期广泛表达,且在出膜后7 d达到最高值。在健康团头鲂各组织中,nkla在脾脏中表达量极高,为其他组织中的数百倍,而在肌肉、血中表达量极低,在肝脏、脑、肾脏、头肾、肠、鳃和心中均有明显表达;nklb则在肠中表达量相对较高,在脾脏、头肾表达相对较低,在其他组织中均表达。nkla和nklb不同的表达模式暗示着不同的NK-lysin可能存在功能上的分化。H2A在血中表达量最高,脑中其次,其他组织中表达量相对较低。3.团头鲂3个抗菌肽基因在嗜水气单胞菌感染后的表达嗜水气单胞菌感染后,团头鲂nkla和nklb两个基因在不同组织中的表达变化模式相似:在头肾中,nkla在感染后表达显著性下降,nklb则在4 h、24 h及120 h时呈显著下降水平;在肝脏中,均在4 h时上升到最高水平,而后回落;在脾脏中,则在72 h升到最高水平,120 h恢复正常;在肠中,nkla和nklb表达量在4 h时显著降低,并分别在72 h和24 h时达到最高值,120 h回到正常水平。nkla和nklb在主要免疫组织中的表达变化,暗示着它们可能在团头鲂抵抗嗜水气单胞菌侵染过程中发挥重要作用。H2A基因在肝脏和肠中的表达量均呈上升趋势,在脾脏和肾脏中,H2A的表达则是先呈下降趋势,而后显著上升。4.团头鲂nkla和nklb的原核表达成功构建了3种nkla和nklb的原核表达载体,转入到BL21(DE3)宿主菌中,获得原核表达菌株,对IPTG浓度、温度和诱导时间等各条件进行优化。在pET32a-NKLA和pET32a-NKLB重组表达体系中,融合蛋白NKLA只在沉淀中表达,NKLB则在上清和沉淀中表达,但该表达体系不稳定。而对于pET28a-NKLA和pET28a-NKLB表达体系,NKLA没有表达,NKLB在沉淀中只有少量表达。在pGEX-NKLA和pGEX-NKLB表达体系中,融合蛋白主要以包涵体形式存在,分子量分别为38.2 kDa和39.7 kDa,且该表达体系较稳定。选择诱导时间为8 h,在不同IPTG终浓度和各温度条件下,融合蛋白表达量没有明显差异,其中融合蛋白NKLA在37℃下IPTG浓度为0.05 mmol/L时,目的蛋白占菌体总蛋白比率最高,为33.8%;NKLB融合蛋白在37℃下IPTG浓度为0.02 mmol/L时,这一比率最高,为48.8%。
[Abstract]:NK-LYSIN is a small molecular cationic antibacterial peptide secreted by the natural killer cells (NKL) and toxic T cells (cytotoxic T lymphocytes, CTL). It is a homologous polypeptide with the human granular soluble peptide granulysin, with a broad spectrum of antibacterial activity and bioactivity to the bacteria, fungi, virus and cancer cells. Histone H2A is a eukaryotic generation. A structural protein in the chromatin of body cells is one of the components of innate immunity. The bactericidal effect of the polypeptide fragment derived from it has become a hot spot of research. Megalobrama amblycephala is an important freshwater breed in China. With the expansion of the culture regulation, the enhancement of the degree of intensive culture and the culture environment. The worsening of the disease leads to the frequent occurrence of various diseases. Among them, bacterial septicemia caused by Aeromonas hydrophila (Aeromonas hydrophila) infection, which is the most influential disease of bream bream, can cause hyperemia, ulcers, peritoneal effusion and liver whitening in all parts of the fish body, resulting in a large number of death of the fish. Drugs can be controlled, the main use of antibiotics in the breeding industry to improve the immunity of the fish body, but antibiotics will cause drug residues and bacterial resistance, seriously affecting the development of the aquatic industry and human health. The development of new antibiotics has become a hot spot. 2 NK-lysin genes (nkla and nklb) and 1 H were caught from the blunt snout bream transcriptional group. The 2A gene was amplified and sequenced by PCR to verify its ORF sequence and analyze the sequence characteristics. Fluorescence quantitative PCR was used to detect its expression in the different period of embryonic development of bream blunt snout and the expression of the immuno tissues after the infection of the adult fish tissue and Aeromonas hydrophila. 2 NK-lysin based prokaryotic expression vectors were constructed and transferred into the Escherichia coli. The main results of this study are as follows: 1. the sequence analysis of 3 antimicrobial peptide genes (nkla, nklb and H2A) of 1. blunt snout bream: nkla ORF full length 369 BP, 122 amino acids, DNAstar predicted that the isoelectric point is 6.71, the molecular weight is 14 kDa; nklb OR. The total length of F is 411 BP, encoding 136 amino acids, and DNAstar predicts its isoelectric point is 6.72. The molecular mass of 15.6 kDa. multi amino acid sequence alignment analysis shows that the N end of nkla and nklb contains signal peptides and 6 highly conserved cysteines, which can form 3 pairs of intramolecular two sulfur bonds and 1 SapB domains. Sequence analysis found that it belongs to sphingolipid activator. Phylogeny family members. Phylogenetic analysis showed that the NK-lysin gene was closest to zebrafish, followed by semi smooth tongue sole and spotted forktail. Histone H2A gene ORF was 387 BP, encoded 128 amino acids, DNAstar predicted its isoelectric point 10.92, and molecular mass of 16.2 kDa.H2A amino acid sequences without transmembrane domains and Signal peptide, and its sequence is very conservative, the expression of 3 antimicrobial peptide genes of high.2. blunt snout bream with different species sequence in different developmental periods and adult fish tissues, the results of fluorescence quantitative PCR show that nkla and nklb are widely expressed in different stages of embryonic development. In the initial stage of embryo development, nkla and nklb are low expression, but in the early stage of embryo development, the expression of nkla and nklb is low. During the membrane phase, the expression of nkla and nklb increased rapidly. Among them, nkla reached the highest expression in the appearance period of the eye pigment. Nklb reached the highest value at 1 d after the film, and it was widely expressed in the embryonic development period, and reached the highest value in the 7 d after the film. In the healthy group, nkla was very high in the spleen, which was hundreds of times in the other tissues. The expression of nklb in the liver, brain, kidney, head kidney, intestines, gills and heart is obviously expressed in the liver, brain, kidney, head kidney, intestine, gill and heart. The expression of.Nkla and nklb in the spleen is relatively low. The expression of different expressions of.Nkla and nklb in the other tissues suggests that different NK-lysin may have functional differentiation in the blood. The expression in the brain was the highest, the brain was second, the expression of the other tissues was relatively low, and the expression of two genes in.3. and nklb was similar in different tissues after the infection of Aeromonas hydrophila after the infection of Aeromonas hydrophila. The expression of nkla in the head kidney decreased significantly in the head kidney after infection, nklb There was a significant decrease in 4 h, 24 h and 120 h; in the liver, it rose to the highest level at 4 h and then fell; in the spleen, it rose to the highest level at 72 h and 120 h returned to normal; in the intestine, the expression of nkla and nklb decreased significantly at 4 h, and reached the highest value at 72 h and 24 h, 120 h returned to normal levels The expression changes in the main immune tissues suggest that they may play an important role in the process of resistance to Aeromonas hydrophila. The expression of.H2A gene in the liver and intestines is on the rise. In the spleen and kidney, the expression of H2A is in the downward trend, and then the prokaryotic expression of nkla and nklb of.4. blunt snout bream is significantly increased. The prokaryotic expression vector of 3 kinds of nkla and nklb was successfully constructed and transferred into the host bacteria of BL21 (DE3). The prokaryotic expression strain was obtained. The conditions of IPTG concentration, temperature and induction time were optimized. In the pET32a-NKLA and pET32a-NKLB recombinant expression system, the fusion protein NKLA was expressed only in the precipitate, but NKLB was expressed in the supernatant and precipitate. The expression system is not stable, but for the expression system of pET28a-NKLA and pET28a-NKLB, NKLA is not expressed, and only a small amount of NKLB is expressed in the precipitation. In the pGEX-NKLA and pGEX-NKLB expression systems, the fusion protein mainly exists in the form of inclusion body, the molecular weight is 38.2 kDa and 39.7 kDa respectively, and the expression system is more stable. The selective induction time is 8 h, There was no significant difference in the expression of fusion protein at the final concentration of IPTG and the temperature conditions. When the concentration of fusion protein NKLA at 37 C was 0.05 mmol/L, the total protein ratio of the target protein was the highest, which was 33.8%. The ratio of NKLB fusion protein at 37 C was 0.02 mmol/L, and the ratio was the highest, which was 48.8%..
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

【参考文献】