大黄鱼(Pseudosciaena crocea)Cathepsin L基因cDNA片段的克隆与稳定性研究

采用RACE-PCR和RT-PCR方法获得大黄鱼组织蛋白酶L(Cathepsin L)基因的cDNA中间片段,片段长度为773bp。BLAST分析显示,大黄鱼Cathepsin L基因cDNA片段与鱼的同源性最高达到95%,与斑马鱼同源性为80%。实时定量分析了大黄鱼Cathepsin L基因在宰杀当天和冷藏期间的mRNA水平,结果表明,大黄鱼0℃条件下冷藏0、5、10、15、20d肌肉中mRNA都具有相对稳定性,但是含量水平有差异并且是呈现下降趋势,宰杀当天的大黄鱼肌肉中Cathepsin L基因mRNA含量水平最高,与冷藏5、10、15、20d的Cathepsin L基因mRNA水平差异性极显著(P<0.01),冷藏5d的Cathepsin L基因mRNA水平与15、20d的水平也达到了极显著水平(P<0.01)。同时结果表明,在不同储藏时间Cathepsin L基因的mRNA含量水平与肌肉TPA和pH等也表现了高度的相关性,表明Cathepsin L基因的mRNA含量可以作为评价大黄鱼冷藏期间肉品质指标的可行性和准确性。

小麦颖果与根部Cathepsin B-like蛋白的初步研究

利用免疫组织化学技术观察了小麦(Triticum aestivum L.)灌浆时期颖果内以及苗期根部原生韧皮部Cathepsin B-like蛋白的分布以及活性变化。结果表明,在开花后1~9 d的小麦颖果内,Cathepsin Blike蛋白主要分布在内果皮细胞以及灌浆池两侧的维管束中;在开花后12~24 d的颖果中,糊粉层内的Cathepsin B-like蛋白含量逐渐增加,两侧维管束细胞中的Cathepsin B-like蛋白消失,颖果背部内果皮细胞的Cathepsin B-like蛋白含量减少并在细胞边缘出现聚集现象;在小麦苗期根部的原生韧皮部中,随着韧皮部筛分子的成熟,筛分子内的Cathepsin B-like蛋白含量逐渐下降直至消失。

cathepsin L及其抑制剂cystatin C在急性脱髓鞘动物模型中的表达及意义

在饲料中掺入cuprizone饲育小鼠6周,制备急性脱髓鞘动物模型。进而利用髓鞘荧光染色和原位杂交方法,检测动物胼胝体内髓鞘的脱失以及cathepsin L及其抑制剂cystatin C的表达情况。结果发现,与正常动物比较,cuprizone饲育6周后小鼠胼胝体内髓鞘脱失严重,胼胝体内cathepsin L和cystatin C的表达均显著上调。结果提示,髓鞘损伤后cathepsin L高表达可能有助于髓鞘残骸的清理,其抑制剂cystatin C的表达增强可将蛋白水解程度控制在一定范围内,这对髓鞘再生具有积极的意义。

华支睾吸虫组织蛋白酶Cathepsin L1样基因全长序列的克隆和生物信息学分析

目的预测华支睾吸虫全长cDNA质粒文库中cs002d09号EST序列的结构和功能特征及编码蛋白的应用前景。方法利用NCBI和ExPaSy等生物信息学网站在线分析工具和Vector NTIsuite及PcGene等软件包，分析华支睾吸虫全长cDNA质粒文库中cs002d09号EST序列及其编码蛋白的理化性质、结构与功能特征。结果BLASTx分析该ESF序列是组织蛋白酶Cathepsin L1的同源基因，全序列长为1458bp，包含完整的编码区80～1191，编码371个氨基酸，前18个氨基酸是分泌信号肽，蛋白的理化性质较稳定。该蛋白有2个空间构象相对独立功能域：N端的抑制功能域和C端的活性功能域，抑制功能域含有3个线性B细胞抗原表位，同源性较低；C端活性功能域同源性较高，Cys177，His318和Asn338组成催化中心，位于底物结合裂缝的底部。结论华支睾吸虫Cathepsin Ll基因与多个物种的Cathepsin L1基因同源，编码蛋白可能是重要的虫体分泌排泄抗原成分，在华支睾吸虫病的免疫诊断和疫苗研究方面有较好的应用前景。

Cathepsin D及Fas ligand在苦参碱诱导急性T淋巴母细胞白血病JM细胞株凋亡中的表达研究

为了检测苦参碱诱导JM细胞发生凋亡过程中Cathepsin D、Fas-L的表达情况,应用光镜及电镜观察加药及未加药组细胞形态学变化。免疫细胞化学染色观察Cathepsin D在细胞内的表达及定位。半定量PCR检测Cathepsin D mRNA在转录水平的变化并用Western Blot检测Cathepsin D、Fas-L蛋白表达。结果显示0.6mg/ml加药组细胞培养72h,出现凋亡形态学改变。免疫细胞化学染色CathepsinD阳性信号主要位于呈凋亡形态学改变的胞浆和胞核内。其阳性细胞率在处理组明显高于对照组,处理组Cathepsin D的mRNA转录上调。处理组前体Cathepsin D(52kD)的条带亮度较对照组强,而切割后产物(32kD)亮度较对照组弱;Fas-L在处理组表达上调。提示:苦参碱诱导JM细胞的凋亡伴随Cathepsin D及Fas-L的表达改变。

喉癌患者外周血Cathepsin B、L及Stefin A水平检测的意义

目的探讨喉癌患者外周血组织蛋白酶B(Cathepsin B)、组织蛋白酶L(Cathepsin L)及内源性抑制剂(Stefin A)表达水平的变化与临床诊断和预后的关系。方法收集我院确诊的喉癌患者及正常对照者血清,酶联免疫吸附测定法(Elisa)检测60例喉癌患者及30位正常对照者血清中Cathepsin B、L及Stefin A水平。结果喉癌患者血清中CathepsinB、L水平显著高于正常对照者(P<0.01),二者表达水平呈显著正相关(r=0.434 1,P<0.05);Stefin A水平显著低于正常对照者(P<0.01),但与Cathepsin B、L无显著相关性。结论喉癌患者血清中存在高水平的Cathepsin B、L及低水平的Stefin A,提示检测血清中Cathepsin B、L及Stefin A含量可协助诊断及判断预后。

曼氏迭宫绦虫cathepsin L样蛋白酶的生物信息学分析

目的通过生物信息学预测曼氏迭宫绦虫cathepsin L样蛋白酶(Smcathepsin L样蛋白酶)的生物学特征及其潜在功能和结构,为下一步Smcathepsin L样蛋白酶参与寄生虫和宿主相互作用过程研究提供依据。方法通过NCBI的ORF finder工具对Smcathepsin L样蛋白酶的开放阅读框(ORF)进行分析,利用Ex PASy网站进行蛋白的物理化学参数、信号肽、跨膜螺旋和潜在分子生物学功能的预测,通过NCBI/BLAST对蛋白保守功能域进行预测。从NCBI网站获取不同物种的cathepsin L样蛋白酶序列,并利用Vector NTI suit 8.0和Tree View软件进行分析。利用SWISS-MODEL网站和SPDBV4.10软件分析Smcathepsin L样蛋白酶的三维空间结构。结果 Smcathepsin L样蛋白酶是一个全长基因,编码336个氨基酸。蛋白由2个典型的cathepsin L样蛋白酶结构域组成,序列当中有信号肽,是一个稳定的可溶性蛋白分子。三维空间立体结构分析结果显示Smcathepsin L样蛋白酶是一个保守的蛋白。Smcathepsin L样蛋白酶编码氨基酸序列与细粒棘球绦虫、链状带绦虫、多房棘球绦虫和人的cathepsin L样蛋白酶基因的同源性分别是50%、50%、49%和46%。分子进化分析显示Smcathepsin L样蛋白酶与日本血吸虫、多房棘球绦虫和链状带绦虫亲源性更近,而与其他物种,例如原虫、线虫和哺乳动物亲源性较远。结论 Smcathepsin L样蛋白酶可能是一个潜在的分泌蛋白,该蛋白能在胞外发挥作用,即在寄生虫的消化、转移、入侵及宿主-寄生虫相互作用中起着重要的作用,是一个具有潜在研究价值的多功能分子。

溶酶体蛋白酶cathepsin L在阿尔茨海默病模型大鼠海马组织中的表达及意义

目的观察阿尔茨海默病(AD)大鼠海马组织中溶酶体蛋白酶cathepsin L的表达。方法采用Aβ-淀粉样肽(Aβ)大鼠海马注射制作AD动物模型。用免疫荧光染色和Western印迹法检测大鼠海马组织cathepsin L的表达。结果 Aβ海马内灌注造成Aβ沉积的AD模型在行为学和病理改变上一定程度地模拟了AD。AD大鼠海马区cathepsin L蛋白表达与正常组大鼠相比显著升高,cathepsin L蛋白荧光检测发现AD大鼠海马区cathepsin L阳性神经元数量明显增加。结论在AD大鼠海马组织中cathepsin L表达升高。

日本血吸虫(中国大陆株)Cathepsin L基因的克隆、表达及其免疫保护功能的研究

目的开展日本血吸虫组织蛋白酶L(SjCL)功能研究,为研发血吸虫病抗病疫苗提供基础。方法应用RT-PCR技术分离、克隆日本血吸虫中国大陆株组织蛋白酶L保守功能域编码基因,并在大肠杆菌系统中表达,应用免疫亲和层析法纯化组织蛋白酶L重组抗原,用该基因纯化重组表达产物进行小鼠免疫保护实验。结果获得了672 bp的血吸虫组织蛋白酶L保守功能域cDNA序列,成功构建了原核表达载体pET28a(+)-SjCL,并在大肠杆菌中进行了表达,经免疫亲和层析获得了高纯度的SjCL融合蛋白,以该纯化物免疫小鼠,结果实验组减虫率为36.04%,肝组织减卵率为34%,粪卵减少率达49%,与对照组进行方差分析,差异均显著。结论获得日本血吸虫组织蛋白酶L保守功能域cDNA克隆,其原核表达产物免疫小鼠对抗血吸虫感染具有一定的保护性。

Plasma cathepsin L:A prognostic marker for pancreatic cancer

AIM:To assess the prognostic significance of cathepsin L,a cysteine protease that degrades the peri-tumoral tissue,in patients with pancreatic cancer.METHODS:Plasma samples from 127 pancreatic cancer patients were analyzed for cathepsin L levels by ELISA.Out of these patients,25 underwent surgeryand their paraffin-embedded tissue was analyzed for cathepsin L expression by immunohistochemistry.Survival of patients and clinicopathological parameters was correlated with cathepsin L expression in plasma and tissue using appropriate statistical analysis.RESULTS:The mean(±SD)cathepsin L in plasma samples of pancreatic cancer patients was 5.98±2.5ng/m L that was significantly higher compared to the levels in healthy controls(3.83±0.45)or chronic pancreatitis patients(3.97±1.06).Using ROC curve,a cut-off level of 5.0 ng/m L was decided for survival analysis.Elevated plasma levels of cathepsin L were found to be associated with poor prognosis(P=0.01)in multivariate analysis.The plasma levels of the protease decreased after surgery.Though no significant correlation was seen between plasma and tissue expression of this protease,a trend did emerge that high cathepsin L expression in tissue correlated with its high levels in plasma.CONCLUSION:Cathepsin L levels in plasma of pancreatic cancer patients may be used as a potential prognostic marker for the disease.

Identification and molecular characterization of Cathepsin L gene and its expression analysis during early ontogenetic development of kuruma shrimp Marsupenaeusjaponicus

Cathepsin L gene is a member of the cysteine proteinase gene group. In this study Cathepsin L gene was isolated from Kuruma shrimp Marsupenaeusjaponicus （Mj-Cathepsin L） and the full-length DNA sequence was 1 963 bp. Mj-Cathepsin L protein showed high homologies with other Cathepsin L proteins documented in vertebrates, mollusks and other crustaceans. Expression analysis of Mj-Cathepsin L gene in different tissues revealed that it was predominant in hepatopancreas. During early ontogenetic development stages Mj-Cathepsin L showed a development-regulated expression, and the Mj-Cathepsin L showed a molting stage-regulated expression during the five molting stages, inferring its role in the ontogenic development of M.japonicus. Two kinds of forms of Mj- Cathepsin L protein： pro-Cathepsin L and Cathepsin L were measured in hepatopancreas, stomach and intestine by Western Blotting.

Effect of vitamin E and human placenta cysteine peptidase inhibitor on expression of cathepsins B and L in implanted hepatoma Morris 5123 tumor model in Wistar rats

AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Norris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Norris 5123 hepatoma.METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time,tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates.RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls.The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 μg of CPI was used, i.e.7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment (P＜0.0001).CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy.

PCI手术对急性冠脉综合征患者体内CatL及hs-CRP水平的影响

目的探讨PCI手术对急性冠脉综合征患者体内CatL及hs-CRP水平的影响。方法选择2010年11月30日至2011年11月30日期间在南华大学第二附属医院心内科住院的105例急性冠脉综合征患者,以是否接受PCI手术将患者分为PCI术干预组65例和未经PCI术干预组40例,采用酶联免疫吸附试验分别于第1、7、30天采两组患者血清测定其CatL及hs-CRP水平并进行统计学分析。结果 PCI术后患者CatL水平降低(7天4.88±0.18 vs 5.56±0.15,30天4.20±0.17 vs 5.65±0.18,P<0.001),hs-CRP下降无统计学意义(7天11.10±1.85 vs11.29±1.67,30天11.35±1.23 vs 11.36±1.32,P>0.05)。结论 PCI术可能使CatL血清水平下降而与hs-CRP早期下降无关。

Cathepsin L, transmembrane peptidase/serine subfamily member 2/4, and other host proteases in COVID-19 pathogenesis – with impact on gastrointestinal tract

Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)seems to employ two routes of entrance to the host cell;via membrane fusion(with the cells expressing both angiotensin converting enzyme 2(ACE2)and transmembrane peptidase/serine subfamily member 2/4(TMPRSS2/4))or via receptor-mediated endocytosis(to the target cells expressing only ACE2).The second mode is associated with cysteine cathepsins(probably cathepsin L)involvement in the virus spike protein(S protein)proteolytic activation.Also furin might activate the virus S protein enabling it to enter cells.Gastrointestinal tract(GIT)involvement in SARS-CoV-2 infection is evident in a subset of coronavirus disease 2019(COVID-19)patients exhibiting GIT symptoms,such as diarrhea,and presenting viral-shedding in feces.Considering the abundance and co-localization of ACE2 and TMPRSS2 in the lower GIT(especially brush-border enterocytes),these two receptors seem to be mainly involved in SARS-CoV-2 invasion of the digestive tract.Additionally,in vitro studies have demonstrated the virions capability of infection and replication in the human epithelial cells lining GIT.However,also furin and cysteine cathepsins(cathepsin L)might participate in the activation of SARS-CoV-2 spike protein contributing to the virus invasiveness within GIT.Moreover,cathepsin L(due to its involvement in extracellular matrix components degradation and remodeling,the processes enhanced during SARS-CoV-2-induced inflammation)might be responsible for the dysregulation of absorption/digestion functions of GIT,thus adding to the observed in some COVID-19 patients symptoms such as diarrhea.

Assessment of the Roles of Cathepsins B, H and L in the Progression of Colorectal Cancer

Cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes, while they concurrently play an essential role in cancer progression, invasion and metastasis. The purposes of our study were to: a) compare the expression levels of cathepsins B, H and L in the supernatants of colon cancer tissues from 74 patients versus the corresponding enzymic expressions of supernatants in the adjacent normal colorectal tissues;and b) correlate our results to the grade of the malignancy by using an enzyme-linked immunosorbent assay (ELISA). The findings indicated that cathepsins B, H and L of all malignant tissues exhibited significantly higher expression levels than their corresponding controls. Furthermore, cathepsin B expression levels doubled in all tumor samples and this increase remained quite steady with tumor stage advancement, in contrast to cathepsin H expression which rose significantly as malignancy progressed. Specifically, cathepsin H concentration was higher than the corresponding control: 155% in B1 stage and 204.44% in D stage. Among the three investigated proteases, cathepsin L has shown the highest increase, which in D stage stood 261.03% higher than the corresponding control. The results at hand suggested that cysteine protease H and L expression levels could be of critical value in the diagnosis and progression of colon cancer.

Construction and transfection of sense/antisense eukaryotic expression vectors for human cathepsin L gene

Objective: To obtain sense/antisense eukaryotic expression vectors for human cathepsin L gene,and study the biological effects on human osteosarcoma cell line MG-63 after transfection. Methods:Cathepsin L gene sense/antisense eukaryotic expression vectors were constructed with recombinant technology and transfected into the human osteosarcoma cell line MG-63. The expression of cathepsin L gene mRNA was examined with RT-PCR and the expression of cathepsin L was examined with Western blot. Results:The sense/antisense recombinant eukaryotic expression vectors for cathepsin L were successfully constructed and transfected into MG-63 cell. Conclusion:Antisense cathepsin L gene can significantly inhibit the expression of cathepsin L mRNA and protein.

肝片形吸虫重组组织蛋白酶L的免疫反应性和免疫原性分析

目的分析肝片形吸虫(Fasciola hepatica)重组组织蛋白酶L(CatL)的免疫反应性,以及对SD大鼠的免疫原性。方法诱导含重组原核表达质粒pET30a-FhCatL的大肠埃希菌BL21(DE3)宿主菌表达,SDS-PAGE分析表达产物,以感染肝片形吸虫山羊血清为一抗,蛋白质印迹(Western blotting)分析其免疫反应性。20只SD大鼠随机均分为重组蛋白免疫组和佐剂对照组,免疫组大鼠皮下注射纯化的重组FhCatL,200μg/(只·次),共免疫3次,每次间隔3周;对照组用等量PBS与佐剂混合后注射。于第2次和末次免疫前,以及末次免疫后3、6和9周尾静脉采血,分离血清。利用间接ELISA法检测免疫大鼠血清IgG抗体水平,噻唑蓝比色法(MTT法)检测脾淋巴细胞增殖情况。结果经纯化获得重组FhCatL蛋白,相对分子质量(Mr)约42 000。Western blotting分析结果表明,纯化重组蛋白能被感染肝片形吸虫的山羊血清识别。重组FhCatL蛋白免疫SD大鼠后可诱导产生特异性IgG抗体,随着免疫时间的延长抗体水平逐渐升高,于末次免疫后3周抗体效价达到峰值(1∶102 400),明显高于对照组(1∶1 000)。免疫组脾淋巴细胞刺激指数为2.176±0.047,显著高于对照组(1.171±0.032)(P<0.05)。结论重组FhCatL蛋白具有较好的免疫反应性和免疫原性。

池蝶蚌组织蛋白酶L基因的组织表达及免疫应激分析

应用cDNA文库筛查及同源片段克隆拼接技术,克隆了池蝶蚌组织蛋白酶L(Hs-CtsL)cDNA基因全长序列(GenBank注册号为JN604558)。其cDNA全长1152 bp,5′-非翻译区(Untranslated Region,UTR)长1 bp,3′-UTR长149 bp包括1个多聚腺苷信号AATAAA和Poly(A)尾巴,开放阅读框(Open reading frame ORF)为1002 bp,编码333个氨基酸组成的多肽链。其分子量约37.7 kD,理论等电点为7.16,包含信号肽、前体域和成熟域。系统进化分析显示,Hs-CtsL同无脊椎动物组织蛋白酶L聚为一支,且同三角帆蚌亲缘关系最近,其次为褶纹冠蚌。组织表达分析结果显示,池蝶蚌组织蛋白酶L在肠、鳃、性腺、外套膜、斧足、闭壳肌、血细胞、肝胰腺、肾和心脏均有表达,其中血细胞中表达量最高。应激实验表明,经嗜水气单胞菌刺激后,Hs-CtsL在血细胞、鳃、肝胰腺和外套膜中的表达量显著上调。其中在肝胰腺中刺激后6h表达量到达峰值,在血细胞、鳃和外套膜中的表达模式近似,表现为一个波动变化,在4h、12h和48h被上调。结果暗示着Hs-CtsL除参与了池蝶蚌血细胞的先天性免疫防御以外,还参与了其消化腺免疫器官的免疫应答反应。

合浦珠母贝组织蛋白酶L2基因的特征与组织表达分析

为了研究合浦珠母贝(Pincatada fucata)的先天免疫调控机制,利用cDNA文库筛选和重测序技术克隆了1个合浦珠母贝组织蛋白酶L的全长cDNA序列(命名为poCL2)。poCL2cDNA全长1094bp,5′-非编码区(Untranslated region,UTR)长21bp,3′-UTR长80bp,开放阅读框(Open reading frame,ORF)为993bp,编码330个氨基酸组成的多肽链,分子量为37.1kD,理论等电点为6.9。poCL2蛋白由信号肽(Met1-Ala16)、前体域(Arg17-Asp113)和成熟域(Leu114-Val330)三部分组成,存在一个潜在的糖基化位点(Asn97),6个底物结合位点(Leu182,Met183,Ala249,Leu275,Gly278和Ser324),1个由半胱氨酸(Cys138)、组氨酸(His277)和天冬酰胺(Asn297)残基构成的催化位点和2个组织蛋白酶L签名序列(E42X3RX3WX2NX3IX3N61和G74X1NX1YX1D80)。同源性分析表明,poCL2氨基酸序列与其他物种高度保守,相似性在61.5%～73.9%之间,同源性在44.4%～59.8%之间。进化分析显示,poCL2与其他无脊椎动物的组织蛋白酶L聚为一支,和淡水螺(Radix peregra)亲缘关系最近。组织表达分析表明poCL2mRNA在消化腺、外套膜、性腺、闭壳肌、肠、血淋巴和鳃组织中均表达,外套膜表达量最高。应激实验表明,经溶藻弧菌(Vibrio alginolyticus)或脂多糖(Lipopolysaccharide,LPS)刺激后,poCL2mRNA在消化腺中的表达量显著上调,说明poCL2参与了合浦珠母贝的先天免疫应答反应,暗示其在合浦珠母贝先天免疫调控中发挥重要作用。

脊尾白虾组织蛋白酶L基因的克隆及其表达分析

根据本实验室构建的脊尾白虾(Exopalaemon carinicauda)血细胞全长cDNA文库获得的EST序列,利用RACE技术克隆获得脊尾白虾组织蛋白酶L基因的cDNA全长,命名为EcCatL基因。该序列全长1136bp,包括5'非编码区24bp,开放阅读框960bp和3'非编码区152bp,开放阅读框共编码319个氨基酸,预测相对分子量为35.30×103,理论等电点为5.27。同源性分析表明,脊尾白虾组织蛋白酶LEcCatL氨基酸序列与其它甲壳动物高度保守,与变色小长臂虾(Palaemonetes varians)及北极甜虾(Pandalus borealis)CatL的同源性分别为92%和76%。系统进化分析表明,EcCatL基因氨基酸序列与变色小长臂虾的CatL聚为一支。荧光定量PCR分析结果表明,EcCatL基因在血细胞、鳃、肝胰腺、肌肉、卵巢、肠、胃及眼柄中均有表达,其中肝胰腺中的相对表达量最高。感染鳗弧菌及WSSV后6h和12h,脊尾白虾血细胞和肝胰腺中EcCatL的表达量较对照组均极显著增加(P<0.01),且具有明显的时间差异性,表明EcCatL基因在脊尾白虾免疫反应中具有重要作用。