本文選題：團(tuán)頭魴 + 嗜水氣單胞菌�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：NK-LYSIN是機(jī)體自然殺傷細(xì)胞(natural killer cells,NKL)和毒性T細(xì)胞(cytotoxic T lymphocytes,CTL)分泌的小分子陽離子抗菌肽,同人類顆粒溶素granulysin是同源多肽,具有廣譜的抗菌活性,對細(xì)菌、真菌、病毒、癌細(xì)胞等均有生物活性。組蛋白H2A是真核生物體細(xì)胞染色質(zhì)中的一種結(jié)構(gòu)蛋白,是先天性免疫的組成成分之一,其衍生的多肽片段的殺菌作用已成為目前的研究熱點(diǎn)。團(tuán)頭魴(Megalobrama amblycephala)是我國重要的淡水養(yǎng)殖品種,隨著養(yǎng)殖規(guī)模的擴(kuò)大,集約化程度的提高以及養(yǎng)殖環(huán)境的惡化,導(dǎo)致各類疾病頻發(fā)。其中,對團(tuán)頭魴養(yǎng)殖影響最大的疾病是由嗜水氣單胞菌(Aeromonas hydrophila)感染引起的細(xì)菌性敗血癥,可造成魚體各部位充血、潰瘍、腹腔積水和肝臟發(fā)白等癥狀,從而導(dǎo)致魚體大量死亡。另外,現(xiàn)階段還沒有很好的藥物可以控制,養(yǎng)殖業(yè)中則主要使用抗生素提高魚體免疫力,但抗生素會造成藥物殘留和細(xì)菌耐藥性,嚴(yán)重影響水產(chǎn)業(yè)的發(fā)展和人類健康,新型抗菌藥物的研發(fā)成為熱點(diǎn)。本研究從團(tuán)頭魴轉(zhuǎn)錄組中釣取到2個NK-lysin基因(nkla和nklb)和1個H2A基因,通過PCR擴(kuò)增和測序驗(yàn)證其ORF序列,并分析序列特征。采用熒光定量PCR技術(shù)檢測其在團(tuán)頭魴胚胎發(fā)育不同時期以及成魚組織和嗜水氣單胞菌感染后免疫組織中的表達(dá)。構(gòu)建團(tuán)頭魴2個NK-lysin基因的原核表達(dá)載體,轉(zhuǎn)入大腸桿菌中進(jìn)行重組表達(dá),并對其表達(dá)條件進(jìn)行優(yōu)化,為進(jìn)一步研究NK-lysin的功能奠定了基礎(chǔ)。本研究的主要結(jié)果如下:1.團(tuán)頭魴3個抗菌肽基因(nkla、nklb和H2A)的序列分析nkla ORF全長369 bp,編碼122個氨基酸,DNAstar預(yù)測其等電點(diǎn)為6.71,分子質(zhì)量為14 kDa;nklb ORF全長411 bp,編碼136個氨基酸,DNAstar預(yù)測其等電點(diǎn)為6.72,分子質(zhì)量為15.6 kDa。多氨基酸序列比對分析顯示,nkla和nklb的N端均含有信號肽,6個高度保守半胱氨酸,可形成3對分子內(nèi)二硫鍵,以及1個SapB結(jié)構(gòu)域,序列分析發(fā)現(xiàn)它屬于鞘脂激活蛋白樣蛋白家族成員。系統(tǒng)進(jìn)化分析顯示,團(tuán)頭魴NK-lysin基因與斑馬魚親緣關(guān)系最近,其次是半滑舌鰨和斑點(diǎn)叉尾洶。組蛋白H2A基因ORF全長387 bp,編碼128個氨基酸,DNAstar預(yù)測其等電點(diǎn)為10.92,分子質(zhì)量為16.2 kDa。H2A氨基酸序列不含跨膜結(jié)構(gòu)域及信號肽,且其序列十分保守,不同物種間序列相似性極高。2.團(tuán)頭魴3個抗菌肽基因在不同發(fā)育時期及成魚各組織中的表達(dá)熒光定量PCR結(jié)果顯示,nkla和nklb在胚胎發(fā)育的不同時期廣泛表達(dá)。在胚胎發(fā)育的初始階段,nkla和nklb呈低表達(dá),而在出膜期時,nkla和nklb表達(dá)量快速上升。其中,nkla在眼球色素出現(xiàn)期達(dá)到最高表達(dá),nklb在出膜后1 d時達(dá)到最高值。H2A也在胚胎發(fā)育期廣泛表達(dá),且在出膜后7 d達(dá)到最高值。在健康團(tuán)頭魴各組織中,nkla在脾臟中表達(dá)量極高,為其他組織中的數(shù)百倍,而在肌肉、血中表達(dá)量極低,在肝臟、腦、腎臟、頭腎、腸、鰓和心中均有明顯表達(dá);nklb則在腸中表達(dá)量相對較高,在脾臟、頭腎表達(dá)相對較低,在其他組織中均表達(dá)。nkla和nklb不同的表達(dá)模式暗示著不同的NK-lysin可能存在功能上的分化。H2A在血中表達(dá)量最高,腦中其次,其他組織中表達(dá)量相對較低。3.團(tuán)頭魴3個抗菌肽基因在嗜水氣單胞菌感染后的表達(dá)嗜水氣單胞菌感染后,團(tuán)頭魴nkla和nklb兩個基因在不同組織中的表達(dá)變化模式相似:在頭腎中,nkla在感染后表達(dá)顯著性下降,nklb則在4 h、24 h及120 h時呈顯著下降水平;在肝臟中,均在4 h時上升到最高水平,而后回落;在脾臟中,則在72 h升到最高水平,120 h恢復(fù)正常;在腸中,nkla和nklb表達(dá)量在4 h時顯著降低,并分別在72 h和24 h時達(dá)到最高值,120 h回到正常水平。nkla和nklb在主要免疫組織中的表達(dá)變化,暗示著它們可能在團(tuán)頭魴抵抗嗜水氣單胞菌侵染過程中發(fā)揮重要作用。H2A基因在肝臟和腸中的表達(dá)量均呈上升趨勢,在脾臟和腎臟中,H2A的表達(dá)則是先呈下降趨勢,而后顯著上升。4.團(tuán)頭魴nkla和nklb的原核表達(dá)成功構(gòu)建了3種nkla和nklb的原核表達(dá)載體,轉(zhuǎn)入到BL21(DE3)宿主菌中,獲得原核表達(dá)菌株,對IPTG濃度、溫度和誘導(dǎo)時間等各條件進(jìn)行優(yōu)化。在pET32a-NKLA和pET32a-NKLB重組表達(dá)體系中,融合蛋白NKLA只在沉淀中表達(dá),NKLB則在上清和沉淀中表達(dá),但該表達(dá)體系不穩(wěn)定。而對于pET28a-NKLA和pET28a-NKLB表達(dá)體系,NKLA沒有表達(dá),NKLB在沉淀中只有少量表達(dá)。在pGEX-NKLA和pGEX-NKLB表達(dá)體系中,融合蛋白主要以包涵體形式存在,分子量分別為38.2 kDa和39.7 kDa,且該表達(dá)體系較穩(wěn)定。選擇誘導(dǎo)時間為8 h,在不同IPTG終濃度和各溫度條件下,融合蛋白表達(dá)量沒有明顯差異,其中融合蛋白NKLA在37℃下IPTG濃度為0.05 mmol/L時,目的蛋白占菌體總蛋白比率最高,為33.8%;NKLB融合蛋白在37℃下IPTG濃度為0.02 mmol/L時,這一比率最高,為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%..
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S943

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