中国明对虾溶菌酶基因克隆、重组表达与性质分析

溶菌酶是机体先天免疫系统中一个重要的效应分子, 参与机体多种免疫反应, 在溶菌过程中形成一个水解体系, 破坏和消除侵入体内的病原, 从而实现机体的免疫防御。从中国明对虾中克隆得到了溶菌酶基因(称为FcLyz基因), 该基因全长709 bp, 其完整的阅读框为477 bp, 编码158个氨基酸, 前18个氨基酸(-1~-18)为信号肽, 成熟肽由140个氨基酸组成(1-140aa), 其分子量为16.2 kD。经SMART分析,该基因具有1个溶菌酶1(LYZ1)结构域(19-130aa)。半定量RT-PCR分析结果表明溶菌酶虽在多种组织中有较低水平的组成性表达, 但在细菌诱导的血细胞、心脏、肝胰腺和鳃等多种组织中表达上调。将中国明对虾溶菌酶基因的成熟肽亚克隆进原核表达载体pET-30a (+)中, 转化大肠杆菌BL21(DE3), 再进行诱导表达和亲和纯化, 得到了纯化的重组溶菌酶, 并进行了抑菌活性检测。结果表明, 重组对虾溶菌酶对革兰氏阳性菌的抑菌能力较强, 最小抑菌浓度达到3.43 mmol/L, 但对革兰氏阴性菌抑制作用较小。上述结果表明, 该溶菌酶作为一种重要的免疫效应分子, 参与了对虾的免疫防御反应。     

日本对虾c型溶菌酶的高效重组表达及产物分析

从日本对虾(Marsupenaeus japonicus)血液中提取总RNA,根据GenBank已登录的该cDNA序列(AB080238),通过RT-PCR技术扩增出日本对虾溶菌酶(MjLys)成熟肽基因。该基因完整的开放阅读框为477 bp,编码158个氨基酸(aa),前18 aa为信号肽,成熟肽由140 aa组成,分子量为16.4 kD,理论等电点(pI)为8.80。经分析表明,该基因含有一个完整的c型溶菌酶结构域(1-130 aa),包括c型溶菌酶特有的两个活性中心Glu33和Asp50,以及8个保守结构Cys残基。将MjLys成熟肽基因亚克隆至原核表达载体pET-32a(+),在大肠杆菌细胞BL21(DE3)pLysS中诱导发酵,实现了重组MjLys蛋白的高效表达,并测定了该重组蛋白对几种细菌的抑菌活性。结果表明,重组日本对虾溶菌酶对革兰氏阳性菌金黄色葡萄球菌和溶壁微球菌均有显著的溶菌活性。     

斑节对虾溶菌酶基因克隆及序列分析

参考对虾溶菌酶基因和类溶菌酶基因及其他多种生物的溶菌酶基因序列 ,设计并合成引物。运用RT PCR技术 ,从斑节对虾血细胞总RNA中扩增获得特异性片段。所获片段回收纯化后克隆到pGEM TEasyVector系统的T载体上。重组子的序列分析表明 ,所克隆的斑节对虾溶菌酶基因片段长 6 5 8bp ,包括溶菌酶基因开放阅读框 (ORF)4 77bp和 3′端非编码区的 181bp。 4 77bpORF共编码 15 8个氨基酸 ,包括溶菌酶成熟肽 14 0个氨基酸残基和信号肽 18个氨基酸残基。斑节对虾溶菌酶成熟肽推测分子量为 16 ,32kd ,等电点为 8 78。与南美白对虾溶菌酶基因的碱基序列及推测氨基酸序列比较 ,同源性分别为 89 5 %和 93 0 % ;与日本对虾类溶菌酶基因的同源性分别为 84 0 %和91 0 %。进一步的序列分析表明 ,斑节对虾溶菌酶氨基酸序列与多种类群生物的c 型溶菌酶氨基酸序列具有较高的同源性 ,并具有与c 型溶菌酶相同的活性位点氨基酸残基Glu51和Asp68,且与活性位点相邻的序列高度保守。斑节对虾溶菌酶氨基酸序列还具有与c 型溶菌酶相同的结构氨基酸——— 8个半胱氨酸残基。因而可认为所克隆的斑节对虾溶菌酶基因属c 型溶菌酶基因。     

太平洋牡蛎i型溶菌酶基因的克隆与重组高效表达

在双壳类软体动物牡蛎体内,溶菌酶(Lysozyme)在实现宿主免疫防御,破坏和消除侵入体内的病原中发挥着重要的作用.根据GenBank已有的太平洋牡蛎溶菌酶的全长cDNA序列(GenBank:AB179775),通过RT-PCR技术,从太平洋牡蛎(Crassostrea gigas)中克隆得到溶菌酶(简称为CgLys)基因的开放阅读框(open reading frame,ORF)序列.生物信息软件分析表明,其ORF为414 bp,编码137个氨基酸(aa),前20个aa为信号肽,成熟肽由117个aa组成,其分子量为13.2 kD.通过构建分子系统发育树对其同源性进行分析比较,初步推断该CgLys属于i型溶菌酶.将该CgLys基因的成熟肽亚克隆进原核表达载体pET-32a(+)中,构建重组质粒pET32a(+)-CgLys,再转化至大肠杆菌BL21(DE3)pLysS.该基因工程菌经IPTG诱导发酵后,成功高效地表达了重组CgLys蛋白,其分子质量约为18 kD.该重组CgLya蛋白主要存在于细菌裂解液的上清液中,即以可溶性蛋白形式存在.上述结果将显著简化后续的蛋白纯化过程,为今后扩大规模生产牡蛎溶菌酶提供参考.     

小菜蛾溶菌酶Pxlys的基因克隆及其 重组蛋白的抗菌活性分析

【目的】本研究旨在通过研究小菜蛾Plutella xylostella溶菌酶的功能,进一步认识小菜蛾的免疫防御机理,为小菜蛾的生物防治提供新的思路。【方法】利用RACE技术克隆小菜蛾溶菌酶基因。构建原核表达载体pET-29a-Pxlys,利用原核表达系统表达并用镍柱亲和层析纯化重组蛋白Pxlys。利用牛津杯法检测重组蛋白Pxlys对停滞棒杆菌Corynebacterium stationis、藤黄微球菌Micrococcus luteus、金黄色葡萄球菌Staphyloccocus aureus、大肠杆菌Escherichia coli、志贺氏菌Shigella sp.、沙门氏菌Salmonella sp.和苏云金芽胞杆菌Bacillus thuringiensis的抑菌活性,并利用扫描电子显微镜观察重组蛋白Pxlys对停滞棒杆菌和大肠杆菌的溶菌特征。【结果】克隆获得开放阅读框长423 bp的小菜蛾溶菌酶基因Pxlys(GenBank登录号： MN702780)序列,它编码140个氨基酸,相对分子质量为15.79 kD。抑菌试验表明,重组蛋白Pxlys不仅对革兰氏阳性细菌的停滞棒杆菌、藤黄微球菌和金黄色葡萄球菌有较强的抑菌活性(抑菌圈直径分别为20.0±1.1, 19.0±0.5和16.5±0.5 mm),而且对革兰氏阴性细菌大肠杆菌、志贺氏菌和沙门氏菌也有抑菌活性(抑菌圈直径分别为16.3±0.5, 15.0±0.5和14.0±1.1 mm),重组蛋白Pxlys对革兰氏阳性细菌比对革兰氏阴性细菌表现出更强的抑菌活性。另外,重组蛋白Pxlys还表现出对苏云金芽胞杆菌的抑菌活性。扫描电子显微镜下,经重组蛋白Pxlys处理过的停滞棒杆菌和大肠杆菌的溶菌特征不同。【结论】Pxlys具有广谱的抗微生物活性,其对革兰氏阳性细菌和革兰氏阴性细菌的抑菌机理可能存在不同。研究结果为深入研究小菜蛾免疫防御系统提供基础。     

两种沼虾溶菌酶基因ORF的克隆和罗氏沼虾溶菌酶基因的组织表达(英文)

分别提取罗氏沼虾和日本沼虾血细胞总RNA,RT-PCR扩增获得特异性cDNA片段,纯化后克隆到T载体上。序列测定表明所克隆的两种沼虾溶菌酶基因的开放阅读框(ORF)为477bp,共编码158个氨基酸,包括溶菌酶成熟肽140个氨基酸残基和信号肽18个氨基酸残基。同源性分析表明,罗氏沼虾和日本沼虾溶菌酶基因的碱基序列及推测氨基酸序列高度同源,分别为99.4%和98.1%。两种沼虾溶菌酶基因的碱基序列和推测氨基酸序列与Gen-Bank上其他对虾溶菌酶的同源性达83.0%和80.0%以上。两种沼虾溶菌酶都具有c-型溶菌酶典型的两个酶活性位点(Glu51)和(Asp68),以及8个保守结构氨基酸残基Cys,且在101、106和107位上缺少Asp,因而推测本实验所克隆的两种沼虾溶菌酶基因属c-型溶菌酶基因的非钙结合亚型。以PCR法制备罗氏沼虾溶菌酶基因的生物素标记探针,斑点杂交检测感染弧菌后溶菌酶基因mRNA在各组织中的转录水平,结果表明受感染6h后在眼、肌肉、鳃、肝胰腺、肠管中的表达量均有升高,其中在肝胰腺中的表达量最高,约为对照组的560%。在不同感染时间里,肝胰腺中该基因表达量有较大的变化:感染后3h表达量最低,24h后表达量升至最高,大约为对照组的430%,48h时的表达量又有所下降,但仍明显高于对照组(约为330%)。受弧菌感染后罗氏沼虾溶菌酶基因转录的上调证明溶菌酶基因在非特异性免疫中的直接作用,同时表明肝胰腺可能在沼虾的免疫防御过程起重要作用。       

家蝇抗真菌肽-溶菌酶基因的克隆、表达及序列分析

旨在对家蝇抗真菌肽MAF-1-溶菌酶(LZM)基因进行生物信息学分析,并进行融合基因MAF-1-LMZ的克隆和表达分析。从Gen Bank获得家蝇抗真菌肽MAF-1和溶菌酶LZM的编码序列,分析和预测这两种蛋白质的结构和功能。PCR扩增融合蛋白质抗真菌肽-溶菌酶的基因MAF-1-LMZ,将其克隆到原核表达载体p ET-28a中,重组质粒p ET-28a-MAF-1-LMZ在大肠杆菌Origmi B/DE3中经用IPTG诱导表达,表达产物MAF-1-LMZ通过SDS-PAGE电泳进行鉴定,采用小试管法倍比稀释法进行活性验证。结果显示,融合蛋白质MAF-1-LMZ序列的ORF为969 bp,编码322个氨基酸残基,理论分子量为35 468.6 Da,等电点为8.31,在大肠杆菌Origmi B/DE3中得到成功表达。其纯化后的目的蛋白具有抗真菌活性。     

人赖氨酸乙酰基转移酶7结构域蛋白的原核表达及纯化

目的:克隆人赖氨酸乙酰基转移酶7(KAT7)的2个功能结构域基因,获得其原核表达产物,并纯化蛋白。方法:采用PCR技术从人乳腺文库中扩增人KAT7基因的2个功能结构域(1-330aa)和(331-611aa)编码片段,将其克隆到p ET28a载体中,在大肠杆菌Rossate中表达后,对原核表达产物进行纯化,以SDS-PAGE和Western印迹鉴定表达与纯化产物。结果:从人乳腺文库中分别扩增获得约990和840 bp的DNA片段,并克隆至p ET28a载体,测序结果表明与目的序列完全一致;在大肠杆菌Rossate中诱导表达出相对分子质量分别约为42 000和36 000的目的蛋白,经纯化后获得了纯度较高的重组蛋白KAT7(1-330aa)和(331-611aa)。结论:获得了重组蛋白KAT7(1-330aa)和(331-611aa),为后续研究KAT7与肿瘤调控奠定了实验基础。     

长毛明对虾酚氧化酶原基因cDNA克隆及组织表达分析

酚氧化酶(phenoloxidase,PO)属于对虾免疫系统的核心酶,在机体抵抗微生物感染过程中起到重要的作用。本研究利用RT-PCR和RACE技术克隆长毛明对虾(Fenneropenaeus penicillatus)酚氧化酶原(prophenoloxidase,proPO)基因(F-proPO)。cDNA全长3 003 bp,包含63 bp的5′-UTR、879 bp的3′-UTR和编码686个氨基酸的2 061 bp的开放阅读框(ORF),预测分子量为78.05 kD,理论等电点(pI)为5.79;含有3个串联式血蓝蛋白结构功能域、2个保守的Cu~(2+)结合位点和6个组氨酸残基;多重比对结果表明,F-proPO与中国明对虾proPO相似度最高(98%),与其他甲壳类动物proPO相似度为51%～93%。实时荧光定量PCR检测结果表明,该基因的表达具有组织特异性,在血淋巴中表达量最高。人工感染溶藻弧菌后,该基因在鳃、血淋巴、肠和性腺中表达水平显著上调;人工感染白斑综合征病毒后,该基因在脑、鳃、心、血淋巴和肠中表达水平显著上调。可见,长毛明对虾PO是参与机体免疫防御反应的一种重要分子。     

海刺参i型溶菌酶基因的重组表达及抑菌谱分析

将本实验室已分离到的海刺参 (Stichopus japonicus) i型溶菌酶的基因(GenBank Accession No. EF036468)亚克隆进原核表达载体pET32a(+)中, 构建重组质粒pET32a(+)-SjLys, 转化至大肠杆菌BL21(DE3)pLysS。阳性克隆子经诱导表达、亲和纯化和透析复性, 得到了酶活力为19.2 U/mg的重组SjLys (rSjLys), 并对rSjLys进行了抑菌谱测定。结果表明, rSjLys对革兰氏阳性菌和阴性菌均有抑制作用, 尤其对常见的海洋致病菌副溶血弧菌和铜绿假单胞菌具有很强的抑菌活性。更为显著的结果是, rSjLys经100oC、40 min加热处理后, 失活的rSjLys对革兰氏阳性菌和阴性菌的抑菌能力高于rSjLys的9%~25%。上述结果表明, 海刺参溶菌酶是一种具有糖苷酶活性和非酶抑菌活性的特殊的i型溶菌酶, 且具有很广的抑菌活性, 在海刺参机体先天免疫系统中是一个重要的效应分子。     

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae

C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.     

High-level Recombinant Expression and Refolding of Lysozyme from the Marine Shrimp <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>

Shrimp lysozyme is as an antibacterial enzyme that participates in the innate defense against the invasion of bacterial pathogens. In this study, the lysozyme gene from hemocytes of the shrimp Marsupenaeus japonicus was isolated and characterized. The M. japonicus lysozyme (MjLys) encodes a polypeptide of 158 amino acids (aa) that includes an 18 aa signal peptide. The gene fragment encoding the mature MjLys protein was subcloned into the expression vector pET-32a(+) and transformed into E. coli BL21(DE3)pLysS, and the protein was strongly expressed in insoluble inclusion bodies. Following extraction using urea, the denatured recombinant protein was refolded by on-column Ni²⁺ affinity chromatography or dialysis with a gradient of decreasing urea concentration. Approximately 50% of the recombinant MjLys was successfully refolded into monomeric protein using urea gradient dialysis, while 30% was salvaged using on-column refolding. Purified MjLys exhibited significant antibacterial activity against Gram-positive bacteria Micrococcus lysodeikticus and Staphylococcus aureus. This efficient over-expression and refolding method can provide the large quantities of biologically active protein required for further biochemical and structural studies and potential biotechnological applications.     

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.     

七鳃鳗g型溶菌酶的分子特征和抗菌活性

溶菌酶是先天免疫系统中对抗细菌病原体感染的一种关键蛋白．本研究从七鳃鳗中克隆g型溶菌酶基因. 其酶基因cDNA为701 bp（GenBank 序列号KP204854）,开放阅读框为555 bp,编码由184个氨基酸组成的多肽,理论分子质量为20.24 kD,等电点为5.48,含有1个半胱氨酸残基,无信号肽．实时荧光定量PCR分析表明,七鳃鳗g型溶菌酶基因在各组织中广泛表达,其中在肠中表达量最高．脂多糖（LPS）体内刺激七鳃鳗后发现,溶菌酶在口腔腺和头肾表达量显著升高．以溶壁微球菌和哈维弧菌为底物检测重组g型溶菌酶的活性时,均表现出抗菌活性,最适pH为7.5,最适温度为35℃．扫描电镜分析表明,重组酶能够使溶壁微球菌破裂．以上结果均表明,g型溶菌酶在七鳃鳗的先天免疫系统防御病菌感染中起到重要作用．     

Cloning and characterization of the tiger shrimp lysozyme

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections. A lysozyme gene isolated from tiger shrimp, Penaeus monodon, was cloned, sequenced and characterized. The cDNA consists of a signal peptide of 18 amino acids and a mature peptide of 140 amino acids. The lysozyme is presumed to be a chicken-type lysozyme for it possesses two catalytic sites and eight cysteine residues which are highly conserved across species of chicken-type lysozymes. The lysozyme cDNAs of Penaeus semisulcatus, Litopenaeus vannamei, Macrobrachium nipponense and Macrobrachium rosenbergii were also cloned. High similarities existed among shrimp and prawn lysozymes but phylogenetic relationship of shrimps and prawns based on lysozyme molecules did not quite consistent with traditional taxonomic classification. High mRNA expression was detected in hepatopancreas, haemocytes and gill of tiger shrimp. Recombinant lysozyme exhibited potent lytic activities against fish pathogens providing evidence of the involvement of lysozyme in shrimp immunity.     

克氏原螯虾一种新型甲壳素基因的鉴定和抑菌功能研究

抗菌肽是无脊椎动物非特异性免疫系统中重要的免疫因子,抗菌肽具有良好且广谱的抗菌活性,并且不易产生耐药性,有望替代传统抗生素药物发挥功能.甲壳素(Crustin)是目前甲壳类动物中研究比较广泛的一类抗菌肽家族.研究鉴定了来自克氏原螯虾(Procambarus clarkii)中的一种新型Crustin基因,命名为Pc-C...     

Identification of an antibacterial protein as L-amino acid oxidase in the skin mucus of rockfish Sebastes schlegeli

Fish skin mucus contains a variety of antimicrobial proteins and peptides that seem to play a role in self defense. We previously reported an antibacterial protein in the skin secretion of the rockfish, Sebastes schlegeli, which showed selective antibacterial activity against Gram-negative bacteria. This study aimed to isolate and structurally and functionally characterize this protein. The antibacterial protein, termed SSAP (S. schlegeli antibacterial protein), was purified to homogeneity by lectin affinity column chromatography, anion-exchange HPLC and hydroxyapatite HPLC. It was found to be a glycoprotein containing N-linked glycochains and FAD. Its molecular mass was estimated to be 120 kDa by gel filtration HPLC and 53 kDa by SDS/PAGE, suggesting that it is a homodimer. On the basis of the partial amino-acid sequence determined, a full-length cDNA of 2037 bp including an ORF of 1662 bp that encodes 554 amino-acid residues was cloned by 3' RACE, 5' RACE and RT-PCR. A blast search showed that a mature protein (496 residues) is homologous to l-amino acid oxidase (LAO) family proteins. SSAP was determined to have LAO activity by the H(2)O(2)-generation assay and substrate specificity for only l-Lys with a K(m) of 0.19 mm. It showed potent antibacterial activity against fish pathogens such as Aeromonas hydrophila, Aeromonas salmonicida and Photobacterium damselae ssp. piscicida. The antibacterial activity was completely lost on the addition of catalase, confirming that H(2)O(2) is responsible for the growth inhibition. This study identifies SSAP as a new member of the LAO family and reveals LAO involvement in the innate immunity of fish skin.