凡纳滨对虾次要过敏原肌质钙结合蛋白的质谱鉴定及免疫交叉反应

目的:质谱鉴定凡纳滨对虾相对分子质量(Mr)为21 000的次要过敏原组分肌质钙结合蛋白(SCP),分析它与其他虾、蟹等甲壳类过敏原的免疫交叉反应,以阐明SCP可作为甲壳类食物过敏原检测、检验及脱敏的标准过敏原物质。方法:运用MALDI-TOF/TOF-MS鉴定凡纳滨对虾Mr为21000的过敏原组分,利用软件BLAST、ClustalW分析甲壳类食物中该蛋白的氨基酸序列同源性,同时用纯化的21 000过敏原免疫小鼠制备其特异性多克隆抗体,通过该抗体与其他8种甲壳类食物蛋白粗提液的Western blot法结果分析该过敏原的免疫交叉反应。结果:质谱鉴定结果显示,凡纳滨对虾21 000过敏原为肌质钙结合蛋白;氨基酸序列同源性分析显示其与斑节对虾、中国对虾、克氏原螯虾、细趾小龙虾、褐虾的SCP序列一致性为81%～100%;Western blot结果显示,针对SCP的特异性多克隆抗体与凡纳滨对虾、刀额新对虾、斑节对虾、口虾蛄、罗氏沼虾、克氏原螯虾、远海梭子蟹、锈斑鲟、中华绒螯蟹粗提液在SCP对应的21 000左右处均有反应条带。结论:凡纳滨对虾Mr为21 000的次要过敏原为肌质钙结合蛋白,其氨基酸序列与多种甲壳类具有很高的同源性以及较强的免疫交叉反应。     

凡纳滨对虾主要过敏原-原肌球蛋白单克隆抗体的制备及其过敏原表位分析

目的制备凡纳滨对虾主要过敏原-原肌球蛋白的单克隆抗体(mAb),运用它们和虾过敏患者血清分析凡纳滨对虾原肌球蛋白的过敏原表位。方法纯化的凡纳滨对虾原肌球蛋白免疫Balb/c小鼠,间接ELISA和Western blot筛选并建立稳定分泌抗原肌球蛋白抗体的杂交瘤细胞株;腹水型mAbs经硫酸铵沉淀、Protein G亲和层析纯化;叠加ELISA分析mAbs的抗原结合表位;虾过敏患者血清lgE与mAbs的抑制Western blot和间接竞争ELISA分析原肌球蛋白的过敏原表位。结果共筛选出5株mAbs,它们之间叠加ELISA的叠加值均高于40%;其中B5和A5能显著抑制虾过敏患者血清IgE与原肌球蛋白的结合,抑制率分别为58.1%和48.6%,同时也能抑制66.7%和44.3%的血清IgE与虾蛋白粗提液反应。结论成功制备了5株分别结合原肌球蛋白不同抗原表位的单克隆抗体,其中B5和A5能结合其过敏原表位。     

酿酒酵母烯醇化酶单克隆抗体的制备、筛选与鉴定

<正>烯醇化酶(enolase,ENO)是糖酵解过程中必需的关键酶,能催化磷酸甘油酸向磷酸烯醇式丙酮酸转变,也能在糖异生过程中催化逆向反应。白念珠烯醇化酶由440个氨基酸组成,相对分子质量(Mr)47 000左右。据研究,其含量分别约占白念珠菌孢子相和菌丝相的0.7%和2%[1],是白念珠菌含量最为丰富的蛋白质之一。近年来,住院患者院内侵袭性真菌感染(invasive fungi infection,IFI)的问题日益     

脑烯醇化酶同工酶的研究进展

脑烯醇化酶(Enolase E、C,4,2,1,11)同工酶为脑糖元酵解途径中的关键酶。由于它在神经内分泌肿瘤,尤其是肺小细胞癌和神经母细胞瘤有异常过量的表达,可以作为有用的生物标记物应用于临床     

抗人脑神经元特异性烯醇化酶单克隆抗体的制备和鉴定

本文采用离子交换和凝胶过滤层析技术，分离纯化了人脑神经元特异性烯醇化酶，并以此为抗原免疫ＢＡＬＢ／ｃ小鼠，通过杂交瘤技术，获得了两株稳定分泌单克隆抗体的杂交瘤细胞株，命名为５Ｂ１和３Ａ８。两株杂交瘤细胞均分泌ＩｇＧ１型类抗体。间接ＥＬＩＳＡ实验显示：５Ｂ１与ＮＳＥ有特异性反应。与ＮＳＥ的同工酶－非神经元特异性烯醇化酶不发生交叉反应；３Ａ８与ＮＳＥ和ＮＮＥ有同等强度的阳性反应。免疫印迹实验结果表明：     

凡纳滨对虾过敏原蛋白Lit v 1.2 的原核表达与纯化

目的:诱导表达凡纳滨对虾(Litopenaeus vannamei)过敏原蛋白Lit v 1.2,并利用6-His标签获得纯化蛋白。方法:用NdeⅠ和PstⅠ双酶切实验室前期构建保存的p GEM-T-Lit v 1.2质粒,将目的基因与表达载体p ET44a连接,转入表达菌株Rosetta,通过氨苄青霉素(Amp)抗性基因筛选,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,获得Lit v 1.2蛋白。通过亲和层析纯化蛋白,SDS-PAGE凝胶电泳检测纯化效果。结果:成功构建了重组质粒p ET44a-Lit v 1.2,SDS-PAGE结果显示Lit v1.2基因在大肠杆菌Rosetta中获得高效的可溶性表达,蛋白质分子量与理论值相符,且获得的目标蛋白纯度高。结论:本研究通过原核表达及亲和层析获得高产量、高纯度的凡纳滨对虾过敏原蛋白Lit v 1.2,为进一步研究Lit v 1.2的免疫学特性奠定基础。     

神经元特异性烯醇化酶单克隆抗体的制备、鉴定及初步应用

用本科从人脑中提纯的神经元特异性烯醇化酶(NSE),免疫BALB/c小鼠,利用杂交瘤技术,成功地制作了8株分泌特异性抗NSE-McAb的杂交瘤细胞。分别用正辛酸和DEAE纤维素纯化法获得了高纯度McAb。经鉴定分属IgG1和IgG2a。用改良过碘酸钠法,对4株McAb分别标记辣根过氧化物酶,经多次方阵滴定,建立了双McAb-ELISA夹心法,初步应用于SCLC血清学检测,阳性率83％。免疫组化ABC法研究结果表明,69例APUD肿瘤免疫染色均为阳性。62例非APUD肿瘤9例呈弱阳性。与丹麦DAKD公司NSE-McAb对比,特异性及敏感性无显著差别。     

小鼠α烯醇化酶基因真核表达载体的构建与鉴定

目的构建小鼠α烯醇化酶（En01）基因的真核表达载体。方法以健康小鼠肾脏组织细胞的eDNA为模板,采用聚合酶链式反应（PCR）扩增Enol基因编码区的全部序列,克隆至真核细胞表达载体pCDNA3．1^＋中,测序鉴定目的基因用阳离子脂质体转染的方法转染中国仓鼠卵巢CHO细胞。结果PCR扩增的特异性片段长度为1304bp,以此构建重组质粒pCDNA3．1^＋-Enol,测序结果与Gen．bank中的鼠源Enol基因eDNA序列一致。转染中国仓鼠卵巢细胞后可检测到Enol蛋白的表达。结论构建的真核表达载体pCDNA3．1^＋-Enol可在真核细胞内正确表达,这为进一步的疫苗研究奠定了基础。     

Identification and characterization of an interleukin-16-like gene from pacific white shrimp Litopenaeus vannamei

Interleukins are a group of cytokines that play essential roles in immune regulation. Almost all interleukin genes are only found in vertebrates. In this study, an interleukin-16-like gene (LvIL-16L) was identified from Pacific white shrimp, Litopenaeus vannamei. LvIL-16L was predicted to encode a precursor (pro-LvIL-16L) with 1378 amino acids, sharing similarities with predicted pro-IL-16-like proteins from insects. The C-terminus of pro-LvIL-16L protein contained two PDZ domains homologous to the mature IL-16 cytokine of vertebrates. In tissues, LvIL-16L could be processed into a ∼36 kDa mature peptide through a caspase-3 cleavage site, which was verified by in vitro site mutation analysis and in vivo RNA interference (RNAi) experiments. The LvIL-16L mRNA could be detected in all the analyzed tissues and the expression of LvIL-16L was significantly up-regulated after immune stimulation. Using RNAi strategy, the role of LvIL-16L in immune responses was initially investigated. Interestingly, knockdown of LvIL-16L could significantly increase the mortality of the Vibro parahaemolyticus infected shrimps but reduce that of the WSSV infected shrimps, suggesting that LvIL-16L could have opposite effects on the antiviral and antibacterial immune responses in shrimp. To our knowledge, this is the first study of an IL-16-like gene in invertebrates, which could help to elucidate interleukin evolution and regulatory mechanisms of shrimp immune responses.     

Evidences of abundant hemocyanin variants in shrimp Litopenaeus vannamei

Hemocyanin (HMC) is a multifunctional immune molecule present in mollusks and arthropods and functions as an important antigen non-specific immune protein. Our previous evidences demonstrated that Litopenaeus vannamei HMC might display extensive molecular diversities. In this study, bioinformatics analysis showed dozens of variant sequences of the HMC subunit with higher molecular weight from L. vannamei (LvHMC). Three variant fragments, named as LvHMCV1-3, which shared 85–99% nucleotide identity with that of the classical form of LvHMC (AJ250830.1), were cloned and characterized. Spatial expression profiles showed that LvHMCV1-3 had different tissue-specific distribution, which were affected by stimulation with six pathogenic bacteria, including Escherichia coli K12, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis, Streptococcus pyogenes and Staphylococcus aureus, with each variant fragment showing a specific stress pattern to different bacterial pathogens. Full length cDNA of LvHMCV3 was further cloned and characterized. The deduced amino acid sequence shared 92% identity with that of LvHMC, possessed a conserved structure characteristic of the HMC family and could be clustered into one branch along with other arthropod HMC in a phylogenetic tree. In addition, the recombinant protein of LvHMCV3 (rLvHMCV3) showed obvious agglutination activities against three aquaculture pathogenic bacteria including E. coli K12, V. parahaemolyticus and S.aureus at concentrations ranging from 31.25–62.5 g/mL. It also showed obvious antibacterial activity against V. parahaemolyticus at concentrations 0.02–0.5 mg/mL, and possessed the best inhibitive effects compared with those of rLvHMCV4 and rLvHMC. Co-injection of V. parahaemolyticus and rLvHMCV3 in L. vannamei showed significant decrease of the mortality rate at 24–72 h after injection. Therefore, these studies suggested that L. vannamei had abundant HMC variants, which possessed obvious resistance to pathogenic infection and might specifically target on different pathogens in shrimp.     

Localization and bacteriostasis of Vibrio introduced into the Pacific white shrimp, Litopenaeus vannamei

Although numerous mechanisms of immune defense have been described in crustaceans, the tissue distribution and fate of live bacteria introduced into the host remain unclear. In the present study, Litopenaeus vannamei were injected with a sub-lethal dose of kanamycin-resistant Vibrio campbellii expressing green fluorescent protein. Accumulation of intact bacteria was quantified by real-time PCR, while bacteriostasis was quantified as the percentage of intact bacteria that could not be recovered by selective plating. Over the 240 min examined, the lymphoid organ contained the greatest number of intact V. campbellii per gram tissue as well as the lowest percentage of culturable V. campbellii compared to other tissues, including the hemolymph. In contrast, the gills and hepatopancreas accumulated intact bacteria, but contained a significantly greater percentage of culturable bacteria than the hemolymph after 240 min. These data suggest that the lymphoid organ plays a major role in bacterial uptake and bacteriostasis in penaeid shrimp.