河蚌C-反应蛋白的分离纯化及其性质研究

用合成的CDPC-Sepharose-6B亲和层析吸附剂一步提纯河蚌C-反应蛋白(CRP)。纯化的CRP在SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦电泳鉴定时均显示为一条区带。其等电点(pI)为5.2,分子量约为310,000道尔顿,由六个亚基组成,亚基分子量约为51,000道尔顿。在280nm处的消光系数E_(1cm)~(1mg)/ml=1.2。河蚌CRP与C_多糖(CPS)发生的沉淀反应是钙依赖性的。本文对河蚌CRP的氨基酸组成及其它性质也作了研究。     

河蚌C反应蛋白一级结构分析

经亲和层析纯化的河蚌 C反应蛋白 ( CRP)具有 SDS- PAGE纯度 ,用经改进的双偶联 Ed-man方法测得其 N端残基为谷氨酸 ,而不是高等动物 (人与家兔 ) C反应蛋白 N端的焦谷氨酸 .河蚌 C反应蛋白 N端的一级结构由固相 Edman方法测得 ,依次为 H2 N- E- T- A- Y- S- C- I- T- A- V- ;C端的一级结构由羧肽酶 A降解法测得 ,依次为 - L/V- S- S- T- Y- COOH,也不同于人和家兔的 C反应蛋白 .在河蚌 CRP的胰蛋白酶酶解肽段中 ,其 N端及 C端的结构也得到了证实 .河蚌 C反应蛋白经 V8蛋白内切酶酶解 ,溴化氰裂解 ,肽段经 HPLC反相柱分离 ,共得到 35个肽段 ,所有肽段的氨基酸序列均由气相氨基酸自动分析仪测得 .结合河蚌 C反应蛋白的胰蛋白酶酶解肽段的分析结果 ,其一级结构已初步拼接完成 .在其一级结构中发现有类似于其它 CRP的 Ca2 + 结合部位和磷酸胆碱结合部位 .河蚌 C反应蛋白的分子结构中存在微观不均一性 .从已知河蚌 C反应蛋白的分子特点 ,包括分子量 ,糖基化比例 ,一级结构不均一等特点 ,可以推测它与高等动物的免疫蛋白有许多相关之处 .对于河蚌 C反应蛋白分子结构的分析 ,将有助于免疫系统蛋白的发生 ,进化等方面的研究     

鳑鲏鱼类产卵时对河蚌鳃不同位置的选择

鰟鮍是鲤科中的一群小型鱼类,在繁殖季节,鱼类产卵于河蚌的鳃腔内,鰟鮍鱼卵在河蚌鳃上的分布有一定的规律性。以前的研究主要是关于不同的鰟鮍鱼类对河蚌不同物种的选择,而对鱼类产卵时在河蚌鳃的上具体位置研究很少。本研究分为野外和室内两部分,野外现场解剖16种河蚌并统计观察鱼卵在鳃上的位置;室内选择六种代表性的淡水河蚌鱼尾楔蚌(Cuneopsispisciculus)、圆顶珠蚌(Unioclouglasiae)、失衡丽蚌(Lamprotulatortuosa)、洞穴丽蚌(L·caveata)、背角无齿蚌(Anodontawoodianawoodiana)和射线裂脊蚌(Schistodesmuslampreyanus),在分别养殖6d和3d的条件下,观察高体(Rhodeusocellatus)卵寄生在河蚌鳃上具体位置。结果表明,不同种类的在繁殖时有不同的适应策略。在野外条件下鉴定出3种鱼类,短须(Acheilognathusbarbatulus)、越南(A·tonkinensis)和广西副近似种(P·cf·meridianus),它们用其较长的产卵管将鱼卵寄生在河蚌内鳃的前部和中部,而室内属鱼类产卵管短,对河蚌鳃的前、中、后部位置选择没有显著性的差异,但是其仔鱼所具有的发达翼状突起可以将其稳固地寄生在河蚌内鳃。本研究结果表明,不同的类群为了给其仔鱼提供最大的生存机会而采用不同的策略。这些结果与鰟鮍和河蚌之间的协同进化关系是一致的[动物学报52(2):272-278,2006]。     

利用大肠杆菌在菌体外分泌生产人C反应性蛋白(CRP)

酵母工业长浜生物科学研究所利用大肠杆菌确立了在菌体外分泌生产在体内发生急性炎症和组织损伤时能在血清中迅速增加的一种蛋白——人C反应性蛋白(CRP)技术。从94年开始商业生产,抗CRP抗血清可用于急性感染疾病的诊断。目前,该公司一年可供应1000升左右抗人CRP的兔抗血清(及抗体)。作为诊断用容积需要系统化。该公司目前引进含CRP的血清和腹水,高纯度地纯化CRP,将其给兔免疫,制造抗血清。由于生产重组人CRP,省去了引进原料需花时间高纯度地纯化CRP的作业。不仅获得了抗体,还能确保作为CRP测定药盒的标准物质所必需的高品质CRP。     

人C反应蛋白在不同原核表达载体及菌株中的表达及免疫反应性分析

构建人C反应蛋白(CRP)原核表达载体,并分别将其转化进入E.coli BL21(DE3)和Rosetta gami 2(DE3)p Lys S中,诱导CRP重组蛋白的表达;主要运用了基因工程,亲和层析及透析复性等方法。成功构建了CRP原核表达载体及菌株。经IPTG诱导后,得到了不同的重组CRP蛋白。结果表明,相同表达载体在不同的表达菌株中的表达情况有一定差别,分别在大肠杆菌及Rosetta gami2(DE3)p Lys S中表达并得到了重组CRP的包涵体,对复性后的CRP重组蛋白进行Westen blotting及ELISA检测,结果表明原核表达的重组CRP蛋白的免疫反应性很低,CRP蛋白发挥免疫活性需要以五聚体形式存在。     

SP-A、CRP和PCT在新生儿肺炎中的变化及诊断意义

为了考察肺泡表面活性物质蛋白A (SP-A)、血清C反应蛋白(CRP)、降钙素原(PCT)在新生儿肺炎中的变化及诊断意义,本研究检测了80例肺炎新生儿和40例健康新生儿血清SP-A、CRP和PCT水平。然后建立了肺炎克雷伯菌诱导的肺炎小鼠模型,每天按照200 mg/kg的剂量对小鼠腹腔注射SP-A蛋白溶液,共注射3 d。研究显示,肺炎新生儿血清中SP-A水平显著低于健康新生儿,而CRP和PCT水平均显著高于健康新生儿(p0.05)。肺炎新生儿血清SP-A与CRP和PCT水平显著负相关(p0.05),CRP和PCT水平显著正相关(p0.05)。SP-A、CRP和PCT联合诊断的敏感度、特异性和曲线下面积依次为0.864、0.829和0.845,均高于单独指标诊断。SP-A处理可显著减轻肺炎小鼠肺组织的病理改变,并显著升高肺组织和血清中的SP-A水平,降低血清CRP和PCT水平。本研究表明,肺炎新生儿血清SP-A水平明显降低,而CRP和PCT水平明显升高。SP-A、CRP和PCT联合检测对新生儿肺炎的诊断价值高于单独指标诊断。通过腹腔注射SP-A蛋白溶液可显著抑制小鼠肺组织病变,并降低血清CRP和PCT水平,从而抑制炎症反应。     

家蚕卵黄原蛋白及其受体基因

家蚕小卵突变体 (Smalleggmutant ,sm) ,其卵体积仅及正常卵的 2 / 3,不能受精而致死。因其卵母细胞不能正常吸收卵黄原蛋白 (Vg) ,人们认为其原因可能是卵黄原蛋白受体基因 (VgR)突变所致。本研究首先通过克隆筛选和基因组序列分析 ,获得了 2 5 6 4bp含有 ployA的家蚕卵黄原蛋白受体基因 (BmVgR)片段。将该基因片段的预测蛋白与其它物种的VgR/YPR和低密度脂蛋白受体 (LDLR)家族比较 ,发现该基因具有LDLR家族的基本结构特征。其次 ,经RT PCR检测 ,结果表明BmVgR在sm的不同时期的卵母细胞中都能正常转录。最后 ,分别对sm不同发育时期的体液和卵的总蛋白进行SDS PAGE分析 ,发现该突变体的卵母细胞不能正常摄取体液蛋白 (包括Vg)。综合分析 ,sm不能正常摄取Vg ,可能并不是VgR的功能异常导致 ,而是与滤泡细胞异常有关     

C反应蛋白与动脉粥样硬化

人类C反应蛋白 (C reactiveprotein ,CRP)是在感染和组织损伤时血浆浓度快速、急剧升高的主要的急性期蛋白。CRP可以激活补体和加强吞噬细胞的吞噬而起调理作用 ,从而清除入侵机体的病原微生物和损伤、坏死、凋亡的组织细胞 ,在机体的天然免疫过程中发挥重要的保护作用。关于CRP的研究已经有 70多年的历史 ,传统观点认为CRP是一种非特异的炎症标志物 ,但近十年的研究揭示了CRP直接参与了炎症与动脉粥样硬化等心血管疾病 ,并且是心血管疾病最强有力的预示因子与危险因子     

原肌球蛋白和副肌球蛋白晶体的电子显微镜观察

(一)自猪胃、猪膀胱平滑肌、鵝肫、鴨肫平滑肌、河蟹横紋肌和河蚌閉壳肌及斧足肌制备出原肌球蛋白的針状晶体,自河蚌的两种肌肉制备出副肌球蛋白的針状晶体,分別在电子显微鏡下观察其横紋結构与細节。(二)各种原肌球蛋白的針状晶体与河蚌的副肌球蛋白晶体相类,一般都不是具有三維規律性的真正晶体,而是具有横紋結构和整齐外形的纤維聚集物——类晶体。(三)各种原肌球蛋白类晶体的横紋周期,大都属于n×200A(n=1,2,…)数系,周期或为400A、或为200A。其具有400A周期的,有时伴有200A的細节。这些共同的特征与免原肌球蛋白真正晶体的三維网状周期相同。(四)鴨肫和鹅肫原肌球蛋白的类晶体中,除了400A,200A的横紋外,还出現800A(n=4)的周期,显示其分子长度可能至少不低于800A。(五)河蚌斧足肌和閉壳肌副肌球蛋白的类晶体,同海蚌一样,都具有周期約700A的横紋結构。条件适宜时,还可以在每一周期之間观察到4条横紋,細节間距为140A。(六)河蚌閉壳肌原肌球蛋白的类晶体中,也有接近800A的周期。(七)在高盐条件下所获得的河蚌斧足肌和閉壳肌原肌球蛋白的类晶体,与上述一般低盐条件下的类晶体不同,細节間距与副肌球蛋白的相同,为140A,而非200A。(八)根据电子显微鏡下观察到的类晶体横紋周期和細节間距,可以初步推論:一般而言,原肌球蛋白和副肌球蛋白是两类而非一类的蛋白貭。但在两种蛋白貭共存的同一肌肉,如河蚌閉壳肌或斧足肌之中,它們之間还可能存在着一定的亲属关系。     

C反应蛋白--天然防御蛋白研究进展

C反应蛋白(C-reactive protein,CRP)是急性时相蛋白中最突出的代表,在机体遭受损伤后,它在体内会急速上升,因而被认为是感染和炎症的早期指示灯。CRP是属于古老并高度保守的“pentraxin”蛋白家族,是由5个非共价联合的单体(protomers)组成。CRP具有多种生理功能,能与多种糖蛋白和磷酸酯类结合,能对补体系统进行调节,对机体具有调理作用,对细胞凋亡有一定的影响等等。CRP检测技术的发展使人们对CRP的结构和生理功能越来越了解,更认识到其在临床医学中的巨大应用价值。     

Complete cDNA sequence of SAP-like pentraxin from Limulus polyphemus: implications for pentraxin evolution

The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology.     

Certain biochemical characteristics of an insulin-like substance from the bivalve mollusk Anodonta cygnea

An insulin-like substance (ILS) was isolated from the visceral organs of the bivalve mollusc Anodonta cygnea by chromatography on a sulfocationite CU-23 and purified by reverse phase liquid chromatography. ILS was shown to be made up to several fractions with Mr ranging from 9 to 20 kDa which have identical amino acid composition but different hydrophobicity and N-terminal amino acids. It was supposed that the heterogeneity of ILS fractions is due to its genetical or posttranslational polymorphism. ILS has a low (0.02%) affinity for the mammalian insulin receptor and a low immune affinity for mammalian insulin and possesses a mitogenic activity which is commensurate with that of the epidermal growth factor. The data obtained suggest that Anodonta cygnea ILS represents a separate branch of a relatively ancient family of insulin-like hormones and growth factors responsible for metabolism and proliferation of invertebrate tissues.     

嗜水气单胞菌感染对背角无齿蚌5种免疫相关酶活力的影响

本研究以背角无齿蚌为材料,利用嗜水气单胞菌为诱导刺激物,对背角无齿蚌进行注射感染,在注射后3、6、12 、24、48h分别取血淋巴、肝胰腺,测定其超氧化物歧化酶（SOD）、酚氧化酶 (PO)、酸性磷酸酶 (ACP)、碱性磷酸酶 (AKP) 及过氧化氢酶 (CAT) 的活力,并研究各项酶活力的变化规律。结果表明：除3h肝胰腺外,实验组的SOD活力均不同程度地高于对照组;血清实验组的PO活力开始显著高于对照组,然后降低,而肝胰腺实验组的PO活力持续高于对照组;另外,与对照组相比,实验组中血淋巴与肝胰腺的ACP、AKP和CAT活力在不同的时间段虽然有所增强,但两者之间无显著差异。因此,认为SOD、PO活性可以作为背角无齿蚌免疫抗病功能指标参数,而ACP、AKP及CAT活性能否作为该参数还有待于进一步研究。     

The amino-acid sequence of multiple lectins of the acorn barnacle Megabalanus rosa and its homology with animal lectins

The amino-acid sequence of a lectin isolated from the coelomic fluid of the acorn barnacle Megabalanus rosa has been determined. The lectin (Mr 140,000) is a multimeric protein whose subunit consists of 173 amino acids and one carbohydrate chain attached to Asn-39. The amino-acid sequence was determined by the manual sequencing of peptides derived from the protein by digestion with Staphylococcus aureus V8 proteinase, lysine endopeptidase and chymotrypsin, as well as fragments produced by cleavage with cyanogen bromide. The amino-acid sequence of the lectin was compared with the sequence of one (Mr 64,000) of the multiple lectins of M. rosa. They are distinct molecules in spite of a significant homology in their amino-acid sequences. The amino-acid sequence includes some regions homologous to those in other invertebrate lectins, such as sea urchin and flesh fly lectins, and vertebrate lectins. This is the first report to show the amino-acid sequence of multiple lectins isolated from an invertebrate.     

背角无齿蚌对浮游藻类的滤食选择性与滤水率研究

以滇池及其附近水体中分布的背角无齿蚌为研究对象,分析其滤水速率及相关的影响因素,研究其食物组成和滤食浮游藻类的选择性,并初步估算其自然种群的滤水能力和控制浮游藻类的潜力.结果表明,背角无齿蚌的滤水率有一定的日变化,傍晚时较高;滤水率与水体中的悬浮物质含量密切相关,随着悬浮物质浓度增加而减少;滤水率与蚌的个体大小有关,体重增加,滤水率下降.对比水体中与蚌消化道内浮游藻类所占的百分比,发现其对浮游藻类的滤食没有显著的选择性.自然水体中背角无齿蚌的滤水能力很低.     

Anthropometric correlates of C-reactive protein among indigenous Siberians

C-reactive protein (CRP) is an inflammatory marker, which at low-level elevations is associated with increased cardiovascular risk. Although CRP has been extensively investigated in North American and European settings, few studies have measured CRP among non-Western groups. The present study used dried whole blood spot samples to examine high-sensitivity CRP concentrations among the Yakut (Sakha) of Siberia (85 females, 56 males; 18-58 years old). Our goals were: (1) to compare Yakut CRP concentrations with other populations; (2) to investigate sex differences; and (3) to explore anthropometric correlates of CRP. Results indicate that serum equivalent CRP concentrations are similar to those from industrializing nations, lower than US and European values, and greater than Japanese concentrations. Yakut men and women display similar CRP concentrations; however, CRP was significantly higher among men after adjustment for body fat, age, and smoking. Positive associations were documented between CRP and BMI, body fat, and central adiposity.     

中枢神经系统感染患儿血清和脑脊液CRP、PCT、TNF-α及MMP-9水平及其临床意义

目的:探讨中枢神经系统感染患儿血清和脑脊液C反应蛋白(CRP)、降钙素原(PCT)、肿瘤坏死因子-α(TNF-α)及基质金属蛋白酶-9(MMP-9)水平及其临床意义。方法:选择2017年1月~2018年6月期间南京市第二医院收治的中枢神经系统感染患儿93例作为研究对象,其中化脓性脑膜炎62例记为化脓性脑膜炎组,病毒性脑炎31例记为病毒性脑炎组,另选取同期于我院治疗的非中枢神经系统感染患儿40例作为对照组,比较各组血清、脑脊液CRP、PCT、TNF-α、MMP-9水平及阳性率,并计算血清和脑脊液CRP、PCT、TNF-α、MMP-9诊断中枢神经系统感染的灵敏度、特异度及准确度。结果:化脓性脑膜炎组患儿血清、脑脊液CRP、PCT、TNF-α及MMP-9水平及阳性率高于病毒性脑炎组和对照组,病毒性脑炎组患儿血清、脑脊液CRP、TNF-α及MMP-9水平及阳性率高于对照组(P<0.05),病毒性脑炎组与对照组血清、脑脊液PCT水平及阳性率比较无统计学差异(P>0.05)。血清或脑脊液CRP+PCT+TNF-α+MMP-9联合检验对中枢神经系统感染具有一定的诊断价值。结论:中枢神经系统感染患儿血清、脑脊液CRP、TNF-α、PCT及MMP-9水平明显升高,其中化脓性脑膜炎患儿血清、脑脊液PCT水平高于病毒性脑炎患儿,血清或脑脊液CRP、PCT、TNF-α及MMP-9联合检验对儿童中枢神经系统感染的鉴别诊断具有较高的价值。     

Increased parasite abundance associated with reproductive maturity of the clam Anodonta piscinalis.

Several studies on vertebrates have demonstrated that reproductive activities may increase the parasite load, but this has not been shown in invertebrate hosts. We studied abundance of a potentially harmful gill parasite, the ergasilid copepod Paraergasilus rylovi, from the freshwater bivalve host Anodonta piscinalis in relation to reproductive maturity of the host in the field. Prevalence of this previously unstudied parasite varied from 90 to 100%, and the mean parasite abundance from 16.3 to 28.8 among 3 study populations. Abundance of P. rylovi increased with host size. In the maturating age groups (3-5 yr) the length-adjusted mean parasite abundance among mature, reproducing female clams that brooded glochidia larvae was 2 times higher than in nonreproducing females, the observed pattern being consistent among the 3 study lakes. Alternative, mutually nonexclusive explanations may be found for the result. For example, changes in clam behavior or filtration activity accompanying maturation can increase host exposure to parasites, or reproduction may decrease energy available to host immunologic defense. However, the present result indicates that maturation, and reproduction, is associated with increased parasite abundance in A. piscinalis, an invertebrate host.