墨吉明对虾(Fenneropenaeus merguiensis)CrustinⅡ型基因的克隆与原核重组表达

Crustin是一类具有广谱抗菌活性的且富含半胱氨酸的小分子抗菌肽,广泛分布于甲壳动物中。为研究CrustinⅡ基因在墨吉明对虾中的免疫功能,本研究采用了RT-PCR技术获得了墨吉明对虾CrustinⅡ基因的ORF序列,共390 bp,编码一个129个氨基酸残基的多肽,且具有典型的Ⅱ型Crustin基因结构:N-末端的信号肽、C-末端的WAP(whey acidic protein)结构域、Gly和Cys富集区;运用实时荧光定量PCR技术检测了CrustinⅡ基因在墨吉明对虾不同组织中的表达模式,数据分析表明,CrustinⅡ在血细胞中高量表达,而在肠道、鳃、肝胰腺、尾部肌肉、胃、眼柄和心脏组织中的表达量非常低,且具有显著性差异;同时本研究运用原核重组表达技术成功诱导出CrustinⅡ蛋白。本研究为进一步探究Crustin在对虾的先天性免疫功能和防御机制提供理论依据。     

三疣梭子蟹PtCrustin2成熟肽在毕赤酵母中的表达

Crustin主要分布于甲壳动物中,是一种富含半胱氨酸的小分子抗菌肽,在甲壳动物的先天免疫系统中发挥重要作用。根据crustin的一级结构特征可以将其分为不同的类型,本文以三疣梭子蟹(Portunus trituberculatus)的PtCrustin2成熟肽为研究对象,通过构建毕赤酵母表达系统,以期实现PtCrustin2成熟肽在毕赤酵母中的重组DNA表达。首先,从其鳃中提取总RNA,通过RT-PCR得到编码PtCrustin2成熟肽的cDNA(m Pt Crustin2),并在其5'和3'端分别引入EcoRⅠ和NotⅠ限制性内切酶位点;然后将此片段与表达载体p PIC9K连接,构建重组表达载体pPIC9K-m Pt Crustin2;电转入毕赤酵母GS115细胞后,以不同浓度的G418筛选到高拷贝酵母转化子,经0.5%甲醇诱导表达和固化金属离子亲和层析(IMAC)分离,获得了纯化的重组体mPt Crustin2,Tricine-SDS-PAGE分析显示其分子量约10.5 k Da;抑菌实验证明重组体mPtCrustin2对金黄色葡萄球菌和铜绿假单胞菌具有一定的抑菌效果。本研究首次实现了三疣梭子蟹Pt Crustin2成熟肽在毕赤酵母中的重组DNA表达,为进一步研究提供参考。     

抗菌肽RIKL的分子设计及生物学活性分析

天然抗菌肽具有较强的杀菌能力,但高生物相容性抗菌肽的构建一直阻碍着该领域的发展.为了提高抗菌肽的选择特异性,通过分子动力学分析探讨了抗菌肽的结构特性,并检测其生物学活性.首先以(RXKY)2(YRY)2 (X代表Ile,Y代表Leu)为模板设计新型抗菌肽分子RIKL.通过圆二色谱(circular dichroism,...     

复合抗菌肽PL在毕赤酵母中的分泌表达及其活性研究

为了获得抗菌活性较强的抗菌肽,将几种抗菌肽串联起来在毕赤酵母中表达,并比较其与单独抗菌肽的抑菌活性。以GenBank中的Protegrin-1(PG-1)、ScorpionDefensin(SD)、Metalnikowin-2A和SheepMyeloidAntibacterialPeptide(SMAP-29)(序列号分别为AAB27599,AAAB27538、P80409和P49928)成熟肽段作为模板序列,根据巴斯德毕赤氏酵母(P.pastoris)偏好密码子,设计并人工合成复合抗菌肽pl基因,同时用SOE法获得ScorpionDefensin的基因,分别克隆到pPICZαA载体中,转化P.pastoris受体菌X-33,在醇氧化酶(AOX)启动子调控下,复合抗菌肽PL及SD均获得表达。体外抑菌试验检测复合抗菌肽PL与单独的蝎子防御素SD的热稳定性、酸稳定性、最低抑菌浓度等,结果显示复合抗菌肽PL及SD具有很强的热酸稳定性,而针对不同的细菌,复合抗菌肽则表现出了强于单独的SD的活性,特别是对大肠杆菌。上述结果说明了该复合抗菌肽具有很好的开发前景。     

一类蛙源非典型结构抗菌肽cDNA的克隆以及成熟肽的预测

抗菌肽是两栖类非特异性免疫的重要组成部分,具有广谱的抗菌、抗病毒、抗肿瘤、抗寄生虫等活性,而且不易产生耐药性.抗菌肽的功能发挥与其特有的α螺旋结构密切相关,但是一些抗菌肽在水溶液中呈无规则卷曲,在类膜溶液中才转变成α螺旋,这类抗菌肽往往表现出极强的抗菌肽活性或细胞毒性,在药物开发中提供更多的利用潜能.本文从东北林蛙(Rana dybowskii)皮肤组织中,通过RT-PCR技术,克隆了一类无规则卷曲的抗菌肽,属于chensirin-2家族.19条不同的抗菌肽的cDNA序列共编码3种长度为14个氨基酸残基的成熟肽,分子量在1450.78-1460.82之间,理论等电点在9.53-9.70之间,3种抗菌肽都有两亲性和阳离子性,二级结构呈现无规卷曲.这些理化性质预示着这3种抗菌肽可能具有特殊的药用价值.     

昆虫抗菌肽对肿瘤细胞的抑制作用研究进展

昆虫抗菌肽(antimicrobial peptide,AMPs)是一类昆虫先天免疫系统中十分重要的效应因子,它分子量小、热稳定,并且具有广谱抗菌性,能够抑制、杀死多种细菌、真菌.近几年来,由于昆虫抗菌肽具有抗肿瘤的活性且其不具有抗原性而受到研究者的广泛关注.昆虫种类多、分布广,因此昆虫抗菌肽具有很高的开发潜力和实际应用价值.但是,目前对于昆虫抗菌肽抗肿瘤能力的研究尚不够深入,对其作用机制还没有一套准确并且系统的理论.现在普遍认为昆虫抗菌肽的抗肿瘤机制与其抗菌机制类似,可以分为破坏细胞膜机制以及非破坏细胞膜机制,并且同一种昆虫抗菌肽可以通过多种方式来抑制甚至杀死肿瘤细胞,但是对正常真核细胞无明显的毒副作用.相较于传统化疗药物的无差别杀伤,昆虫抗菌肽在肿瘤治疗领域有着巨大潜力.本文简要综述了昆虫抗菌肽对肿瘤细胞的抑制作用及其作用机制,并对其开发潜力和实际应用价值进行了展望,以期为今后的研究提供理论支持.     

昆虫抗菌肽对肿瘤细胞的抑制作用研究进展

昆虫抗菌肽(antimicrobial peptide,AMPs)是一类昆虫先天免疫系统中十分重要的效应因子,它分子量小、热稳定,并且具有广谱抗菌性,能够抑制、杀死多种细菌、真菌.近几年来,由于昆虫抗菌肽具有抗肿瘤的活性且其不具有抗原性而受到研究者的广泛关注.昆虫种类多、分布广,因此昆虫抗菌肽具有很高的开发潜力和实际应用价值.但是,目前对于昆虫抗菌肽抗肿瘤能力的研究尚不够深入,对其作用机制还没有一套准确并且系统的理论.现在普遍认为昆虫抗菌肽的抗肿瘤机制与其抗菌机制类似,可以分为破坏细胞膜机制以及非破坏细胞膜机制,并且同一种昆虫抗菌肽可以通过多种方式来抑制甚至杀死肿瘤细胞,但是对正常真核细胞无明显的毒副作用.相较于传统化疗药物的无差别杀伤,昆虫抗菌肽在肿瘤治疗领域有着巨大潜力.本文简要综述了昆虫抗菌肽对肿瘤细胞的抑制作用及其作用机制,并对其开发潜力和实际应用价值进行了展望,以期为今后的研究提供理论支持.     

抗菌肽的设计及其应用

抗菌肽(AMP)是生物体内先天免疫系统的一个组成部分,保护机体免受致病微生物的入侵.抗菌肽具有很强的广谱抗菌活性,可抑制革兰氏阳性菌、革兰氏阴性菌、真菌和病毒的生长.为克服微生物对抗生素耐药性的问题,目前阳离子抗菌肽已被考虑作为抗生素的潜在替代品.本文将阐述抗菌肽的作用机理、选择性抗菌肽的设计及其应用.     

基于BERT与Text-CNN的抗菌肽识别方法

抗菌肽(antimicrobial peptides,AMPs)广泛存在于生命体中,是一种具有广谱抗菌活性、免疫调节功能的小分子多肽。抗菌肽不易产生耐药性,适用范围广,具有极大的临床价值,是传统抗生素的有力竞争者。识别抗菌肽是抗菌肽研究领域中的重要研究方向,湿实验法在进行大规模抗菌肽识别时存在成本高、效率低、周期长等难点,计算机辅助识别法是抗菌肽识别手段的重要补充,如何提升准确率是其中的关键问题。蛋白质序列可以被近似地看作是由氨基酸组成的语言,运用自然语言处理(natural language processing,NLP)技术可能提取到丰富的特征。本文将自然语言处理领域中的预训练模型BERT和微调结构Text-CNN结合,对蛋白质语言进行建模,提供了开源可用的抗菌肽识别工具,并与已发表的5种抗菌肽识别工具进行了比较。结果表明,优化“预训练-微调”策略带来了准确率、敏感度、特异性和马修相关系数的整体提升,为进一步研究抗菌肽识别算法提供了新思路。     

家蝇幼虫抗菌肽MDL-3分子结构的荧光特性

研究了家蝇幼虫抗菌肽MDL-3的荧光光谱和淬灭剂对内源性荧光的影响.家蝇幼虫抗菌肽MDL-3在280nm波长的激发光时,荧光光谱为Tyr残基和Trp残基共同提供,KI不能淬灭抗菌肽MDL-3的Trp残基的荧光,而Acr只能淬灭71%(f=0.71)的抗菌肽MDL-3中的Trp残基的荧光,说明Trp残基不是位于抗菌肽分子的表面,而是位于分子的内部.     

The expression of prophenoloxidase mRNA in red swamp crayfish, Procambarus clarkii, when it was challenged

The expression of the prophenoloxidase (proPO) gene was investigated in nine tissues of red swamp crayfish Procambarus clarkii, by real-time PCR after challenges by CpG oligodeoxynucleotide (ODN), Aeromonas hydrophila and white spot syndrome virus (WSSV). The results can be summarized as follows: (i) the expression level of the proPO gene in haemocytes was highest among nine studied tissues before the challenge; (ii) the expression of proPO increased in all studied tissues after stimulation by CpG ODN and WSSV, and also increased in all tissues, except the ovary, after the A. hydrophila challenge; (iii) the whole expression profiles were different, suggesting that different immune mechanisms may exist for crayfish that are resistant to WSSV and A. hydrophila, although the expression in haemocytes was similar before and after the WSSV and A. hydrophila challenges.     

Overexpression of a C-type lectin enhances bacterial resistance in red swamp crayfish Procambarus clarkii

C-type lectins play important roles in the innate immune system of crustaceans. In this study, a novel C-type lectin gene, designated as PcLec4, was obtained from the red swamp crayfish (Procambarus clarkii). Quantitative real-time polymerase chain reaction revealed that PcLec4 is mainly expressed in the crayfish hepatopancreas and intestine, and the PcLec4 mRNA expression is upregulated after challenged with the bacteria Vibrio anguillarum. PcLec4 was recombinantly expressed in Escherichia coli and anti-PcLec4 polyclonal antiserum was prepared. Binding experiments revealed that the recombinant PcLec4 binds to various bacteria and polysaccharides on the bacterial surface, which suggests that PcLec4 recognizes bacterial pathogens. Overexpression of PcLec4 in crayfish using the pIeLec4 vector was performed. The results show that the crayfish overexpressing PcLec4 eliminate injected V. anguillarum more quickly than the control, which suggests that PcLec4 elicits further immune response for removing invading bacteria. The results of the survival experiment confirmed the function of PcLec4 in resisting V. anguillarum because PcLec4 overexpression in crayfish significantly increased the crayfish survival rate. These results reveal that PcLec4 has an important role in the antibacterial immunity of crayfish, and in vivo PcLec4 overexpression might be used as a disease control strategy in aquiculture.     

鳌虾血细胞中一种新的astakine样造血因子基因的克隆及表达

甲壳动物的血细胞参与多种免疫反应,能够激活酚氧化酶原,产生抗菌肽或进行血细胞的封装、吞噬等. 本研究在克氏原螯虾血细胞中发现一种造血激素astakine,是与脊椎动物细胞因子前动力蛋白prokineticin同源的基因,命名为PcAst,该基因的开放阅读框为357 bp,编码118个氨基酸,具有保守的半胱氨酸残基. 半定量PCR结果显示,该基因只在血细胞中表达. 实时荧光定量PCR结果显示,在金黄色葡萄球菌和鳗弧菌刺激后,其mRNA表达量大幅上调. 白斑综合征病毒刺激后,该基因也呈持续上调表达趋势. 为进一步研究其功能,重组表达了PcAst蛋白,并对其蛋白水平表达模式进行了分析. 所有结果表明,该基因可能参与鳌虾血细胞的固有免疫应答反应,在抗细菌和抗病毒中起重要作用.     

White spot syndrome virus (WSSV) transmission from rotifer inoculum to crayfish

To test the possibility that shrimp pond rotifer resting eggs and hatched rotifers could transmit white spot syndrome virus (WSSV) to crayfish (Procambarus clarkii), we injected crayfish with rotifer and resting egg inocula that were WSSV-positive only by dot-blot analysis of PCR products. No crayfish became WSSV-positive after challenge with the resting egg inoculum. However, 1/15 crayfish became WSSV-positive after challenge with the rotifer inoculum. The results demonstrated that rotifers constitute a potential risk for WSSV transmission to crayfish and other cultivated crustaceans. However, the actual quantitative risk of transmission in an aquaculture setting depends on many variables that remain untested.     

A putative G protein-coupled receptor involved in innate immune defense of Procambarus clarkii against bacterial infection

The immune functions of G protein-coupled receptor (GPCR) were widely investigated in mammals. However, limited researches on immune function of GPCRs were reported in invertebrates. In the present study, the immune functions of HP1R gene, a putative GPCR identified from red swamp crayfish Procambarus clarkii were reported. Expression of HP1R gene was significant up-regulated in response to heat-killed Aeromonas hydrophila challenge. HP1R gene silencing mediated by RNA interference significantly enhanced the susceptibility of red swamp crayfish to A. hydrophila and Vibrio alginolyticus, indicating that HP1R was required for red swamp crayfish to defend against bacterial challenge. In HP1R-silenced crayfish, increased bacterial burden and decreased THC in response to bacterial challenge were observed when compared with control crayfish. No significant difference of proPO gene expression was observed between HP1R-silenced and control crayfish after challenge with heat-killed A. hydrophila. However, PO activity in response to bacterial challenge was significantly reduced in HP1R-silenced crayfish. The results collectively indicated that HP1R was an important immune molecule which was required for red swamp crayfish to defend against bacterial infection.     

C-type lectin from red swamp crayfish Procambarus clarkii participates in cellular immune response

Lectins are potential immune recognition proteins. In this study, a novel C-type lectin (Pc-Lec1) is reported in freshwater crayfish Procambarus clarkii. Pc-Lec1 encodes a protein of 163 amino acids with a putative signal peptide and a single carbohydrate recognition domain. It was constitutively expressed in various tissues of a normal crayfish, especially in the hepatopancreas and gills. Expressions of Pc-Lec1 were up-regulated in the hepatopancreas and gills of crayfish challenged with Vibrio anguillarum, Staphylococcus aureus, or the white spot syndrome virus. Recombinant mature Pc-Lec1 bound bacteria and polysaccharides (peptidoglycan, lipoteichoic acid, and lipopolysaccharide) but did not agglutinate bacteria. Pc-Lec1 enhanced hemocyte encapsulation of the sepharose beads in vitro, and the blocking of beads by a polyclonal antibody inhibited encapsulation. Pc-Lec1 promoted clearance of V. anguillarum in vivo. These results suggest that Pc-Lec1 is a pattern recognition receptor and participates in cellular immune response. Pc-Lec1 performs its function as an opsonin by enhancing the encapsulation or clearance of pathogenic bacteria.     

Molecular mechanisms of the shrimp clotting system

Shrimp, like other invertebrates, relies solely on its innate immune system, to combat invading pathogens. The invertebrate immune system has ancient origins that involve cellular and humoral responses. The clotting system of the humoral immune response is the first line of defense against pathogens and also serves to prevent blood loss during injury and wound healing. Tranglutaminase and clotting protein are molecules involved in the blood clotting system of crayfish and shrimp. Studies have shown that the shrimp clotting system is linked with the activation of antimicrobial peptides, similar to that of the horseshoe crab. Unlike the horseshoe crab and crayfish blood coagulation which are well studied systems, blood clotting in shrimp remains poorly understood. Here we review the shrimp clotting system and its involvement in innate immunity.     

不同抗应激反应能力克氏原螯虾肝胰腺的代谢组分析

为探讨不同抗应激反应能力克氏原螯虾体内代谢物的差异, 研究通过运输应激和温度应激处理后, 选取抗应激反应能力强(SSR)和抗应激反应能力弱(WSR)的克氏原螯虾, 取肝胰腺, 通过液相色谱-质谱/质谱(LC-MS/MS)进行代谢组学分析。质谱共检测到10292个离子, 从中筛选、鉴定出了464个显著差异的代谢物(差异倍数>1.20 或 <0.83,P<0.05, 且 VIP>1.0), 其中与WSR相比, 在SSR中下调的代谢物227个, 上调代谢物237个。KEGG分析显示, 这些差异代谢物主要富集在氨基酸代谢通路, 包括组氨酸代谢、牛磺酸和亚牛磺酸代谢、赖氨酸降解、缬氨酸和亮氨酸及异亮氨酸生物合成、谷胱甘肽代谢等, 同时也富集到抗坏血酸和醛酸盐代谢途径、碳水化合物代谢途径(戊糖和葡萄糖醛酸相互转化)和脂肪酸代谢途径(不饱和脂肪酸生物合成)等。这些结果表明, 克氏原螯虾在应对运输和温度应激时存在广泛的代谢应答, 其中一些与抗氧化应激和增强免疫力相关的代谢物, 如γ-L-谷氨酰-L-半胱氨酸、牛磺酸和油酸等可能在抗应激反应过程中发挥重要作用。研究不仅可为动物抗逆境机制的研究提供新的思路, 而且在克氏原螯虾优良品种的培育及寻找应对克氏原螯虾应激反应的策略等方面也具有重要价值。