美洲大蠊Per a7基因的克隆、表达及免疫学鉴定

根据原肌球蛋白基因序列设计引物,以我国南方地区美洲大蠊Periplaneta Americana RNA为模板,用RT-PCR方法扩增出852 bp的全长编码片段,经序列分析发现该基因与NCBI公布的Per a7有3个核苷酸发生改变。将目的片段克隆到pET24a(+)表达载体中,在大肠杆菌BL21 Star获得表达,融合蛋白的分子量约为33 kD。利用蟑螂过敏性患者血清对表达产物进行Western blotting检测,出现明显的识别条带,说明表达产物具有IgE结合活性。     

美洲大蠊变应原Pera2的生物信息学分析

目的:了解美洲大蠊变应原Per a 2的分子生物学特征。方法:从Genbank中获得Per a 2的核酸序列,用ExPaSy、EBI和NCBI网站的在线软件推导出编码氨基酸序列及其理化性质、空间结构、功能位点,并在Blastp比对后选择不同物种同源序列计算相似率、构建分子进化树。结果:Pera2由351个氨基酸细戍,分子量为38119Da、等电点为4.90、分子式为C1217H1825N285O297S18,为细胞外疏水性蛋白、属于肽酶a家族,信号肽位于1～20氨基酸处,三个跨膜螺旋区域依次位于1～19aa、51～78aa、282～300aa处;二级结构由α-螺旋(9.4%)、β延伸(28.49%)、随机线圈(62.11%)组成;美洲大蠊和德国小蠊相似率为55%、与马德拉蜚蠊相似率为51%,三者在Pera2与不同物种的同源序列构建的分子进化树中聚成一簇。结论:通过对Per a 2的生物信息学分析获得了该变应原分子特征,为进一步研究奠定基础。     

美洲大蠊主要变应原蛋白的质谱鉴定与分析

为了建立美洲大蠊Periplaneta americana变应原蛋白的质谱鉴定方法,我们将美洲大蠊粗浸液通过DEAE-52离子交换层析、Sephacryl S-200凝胶过滤层析等分离步骤得到纯化的74 kD蛋白,对纯化前后的该74 kD蛋白分别进行SDS-PAGE及凝胶内胰酶酶切,再经液相色谱-电喷雾-串联质谱（HPLC-ESI-MS/MS）在线联机分析,所得质谱数据进入网站（http://www.matrixscience.com）进行Mascot检索比对。通过对两者质谱鉴定结果的比较来评估美洲大蠊天然主要变应原蛋白的纯化效果。结果表明,纯化蛋白经HPLC-ESI-MS/MS鉴定是美洲大蠊主要变应原蛋白;离子交换层析等纯化步骤可以去除同一分子量的杂蛋白(如卵黄原蛋白),从而获得较好的鉴定结果。我们首次成功地运用质谱建立起变应原蛋白的新鉴定方法。     

美洲大蠊变应原Per a 2的生物信息学分析

目的：了解美洲大蠊变应原Per a 2的分子生物学特征。方法：从Genbank中获得Per a 2的核酸序列,用ExPaSy、EBI和NCBI网站的在线软件推导出编码氨基酸序列及其理化性质、空间结构、功能位点,并在Blastp比对后选择不同物种同源序列计算井刖双率、构建分子进化树。结果：Per a 2由351个氨基酸细戍,分子量为38119Da、等电点为490、分子式为C1217H1825N285O297S18,为细胞外疏水性蛋白、属于肽酶a家族,信号肽位于1～20氨基酸处,三个跨膜螺旋区域依次位于1～19aa、51～78aa、282～300aa处;二级结构由α-螺旋（9．4％）、β延伸（28．49％）、随机线圈（62．11％）组成;美洲大蠊和德国小蠊相似率为55％、与马德拉蜚蠊相似率为51％,三者在Per a 2与不同物种的同源序列构建的分子进化树中聚成一簇。结论：通过对Per a 2的生物信息学分析获得了该变应原分子特征．为进一步研究奠定基础。     

美洲大蠊胸腺素THY3的表达模式分析

美洲大蠊Periplaneta americana胸腺素基因具有THY1、THY2和THY3三个不同剪接体,其中THY3结构域最多。本研究使用实时荧光定量PCR技术比较分析了THY3在美洲大蠊不同性别、不同发育阶段及不同组织中的表达差异,以及大肠杆菌Escherichia coli诱导对美洲大蠊血淋巴和脂肪体中THY3表达的影响。结果表明:THY3在成虫期的表达量显著高于其他虫期,雌虫表达量显著高于雄虫,脂肪体表达量显著高于血淋巴、头部、肌肉、体壁组织。雌性成虫体腔注射大肠杆菌3 h后血淋巴中THY3表达量显著增高,而在脂肪体中12 h后才检测到THY3表达明显上调,研究结果为进一步研究美洲大蠊胸腺素的功能打下了基础。     

美洲大蠊microRNA的初步研究

美洲大蠊Periplaneta americana是一种世界性的卫生害虫,但其提取物有较高的药用价值。本研究对饲养的雌雄美洲大蠊成虫进行microRNA测序,分别在雄性和雌性中得到12 155 616条和9 847 263条序列。序列长度主要为18～23 nt,且在22 nt和29 nt处有2个峰值。将所得序列与数据库(NCBI、Rfam)进行比对注释,最终在雄性成虫中鉴定到57种已知的microRNA和152种潜在的新microRNA,在雌性成虫中鉴定到53种已知的microRNA和94种潜在的新microRNA。差异表达分析发现只有一种microRNA:miR-750在雌雄之间差异表达,其在雌虫中表达量显著高于雄虫。本研究首次在基因组水平研究了美洲大蠊microRNA的组成并对其功能进行了预测,为其后续研究奠定了基础。     

美洲大蠊3-磷酸甘油醛脱氢酶基因的克隆及表达

旨在通过5’-RACE获得美洲大蠊3-磷酸甘油醛脱氢酶(GAPDH)基因的全长cDNA序列,进行生物信息学分析,构建原核表达载体,诱导重组蛋白表达,为进一步研究其功能奠定基础.通过3’-RACE技术,PCR扩增获取编码美洲大蠊GAPDH蛋白的全长cDNA序列;采用生物信息学方法推导出该序列编码的氨基酸序列及其理化性质;预测信号肽、蛋白疏水性、可溶性、跨膜区结构、二级结构、三级结构,并构建系统发育树;构建原核表达载体pET28a-GAPDH,IPTG诱导重组蛋白表达,并用Histag抗体Western blotting验证.结果显示,美洲大蠊GAPDH基因,其完整阅读框含999个碱基,编码332个氨基酸.序列分析显示,该蛋白与家蚕GAPDH相似性为89％,具有GAPDH保守功能域,经IPTG诱导获得重组蛋白.     

美洲大蠊内生菌的分离鉴定及酶活性、抑菌作用测定

分离美洲大蠊内生细菌并测定菌体的淀粉、纤维素降解酶活性及抑菌特性。对虫体表面消毒,以LB和高氏一号培养基分离虫体内部细菌并进行16S rRNA基因序列分析。变色圈法测定降解酶活性,抑菌圈法测定抑菌活性。结果共分离到11株细菌,4株属于链霉菌属(Streptomyces),6株分别属于肠杆菌属(Enterbacter)、变形菌属(Proteus）、肠球菌属（Enterococcus）、沙雷氏菌属（Serratia）、芽胞杆菌属（Bacillus）和漫游球菌属（Vagococcus）。另有1株菌在NCBI中与肠杆菌的相似度最高, 为95%,可能代表着一个潜在的新种。4株链霉菌均有淀粉和纤维素降解能力。有3株菌对青枯雷尔氏菌有抑制效果。研究结果表明,美洲大蠊内生菌具有某些功能,甚至含有尚未发现的新菌种,是重要的菌种资源。     

美洲大蠊化学成分及生物活性的研究进展

美洲大蠊是源自西南民间的传统中药,目前市面已有"康复新液"、"心脉隆注射液"、"肝龙胶囊"和"消癥益肝片"四种相关成药在售。大量临床实践证明,其在抗炎消肿,保肝,组织修复,心肌保护方面有不错的疗效,但是其药理活性的物质基础和作用机制尚不明确,近年来已有不少学者致力于相关的系统研究,并且取得一些进展,从中获得了异黄酮、异香豆素等次生代谢物质,并且认为其机制是多因素,多细胞因子作用的结果。本文将上述的相关成果做了系统介绍,以期对未来美洲大蠊的研究有所帮助。     

美洲大蠊粪便粗提物的聚集活性

用不同溶剂不同方法提取美洲大蠊Periplaneta americana(L.)粪便,测定美洲大蠊各虫态的诱集活性。结果表明,采用直接浸泡提取方法,丙酮、乙醇、正己烷和二氯甲烷4种粗提物对美洲大蠊各虫态都具有明显的诱集作用,其中乙醇和丙酮粗提物的引诱效果最好,正己烷次之,二氯甲烷最弱。4种溶剂粗提物对美洲大蠊雄成虫和高龄若虫聚集活性最强,对低龄若虫聚集活性最弱。用乙醇溶剂对粪便粗提,3种提取方法均对美洲大蠊有很强的诱集效果,其中索氏抽提诱集效果最弱,直接浸泡和超声波提取效果好,且差异不显著,但直接浸泡提取效果更好。     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.     

家蚕30 kD特异性变应原的分析、纯化与质谱鉴定

以Coca's提取液分别提取到不同时期家蚕Bombyx mori的粗浸液,利用SDS-PAGE和Western blotting鉴定其特异性变应原,然后用DEAE-52离子交换层析及切胶纯化出30 kD的特异性变应原,再经MALDI-TOF在线联机分析,所得质谱数据进入网站搜索分析。结果显示：1～5龄家蚕均有20条左右蛋白带,其中 5龄家蚕有23条蛋白带,主带有11条（82、79、60、51、46、38、32、30、28、24和18 kD）。选用家蚕过敏患者阳性血清进行免疫印迹,1～4龄家蚕均显示出82和79 kD的特异性变应原;但只有5龄家蚕的30 kD蛋白为特异性变应原,通过离子交换层析和经切胶纯化出30 kD蛋白,再经MALDI-TOF-MS鉴定该蛋白为外膜蛋白。提示家蚕不同时期抗原成分有所变化,5龄家蚕新出现的30 kD蛋白为特异性变应原。     

Expression of recombinant chinese bovine enterokinase catalytic subunit in P. pastoris and its purification and characterization

EK (enterokinase) is a serine proteinase which consistsof a heavy chain and a light chain linked by a disulfidebond. The light chain of EK contains a chymotrypsin-likeserine proteinase domain sufficient for the normal recog-nition and cleavage of EK subst…     

Expression, purification, and immunological characterization of Cr PI

An efficient preparation of Periplaneta americana nymphae allergen, Cr PI (54 kDa) is described. It was expressed as a GST-tag fusion protein in Escherichia coli, strain BL21 (DE3). Expression of recombinant Cr PI (rCr PI), denaturation/renaturation of the inclusion bodies and the effects of protein and L-arginine concentration on inclusion body aggregation were optimized. The fusion protein was purified by affinity chromatography and size exclusion chromatography, and Cr PI fusion protein was purified to >95%. rCr PI bound strongly to IgE in the sera of individuals with cockroach allergies as shown by western blot and ELISA. Highly refolded and purified recombinant protein was obtained, providing a basis for the large-scale preparation of Cr PI allergen.     

Purification and functional characterization of lectin with phenoloxidase activity from the hemolymph of cockroach,Periplaneta americana

Lectins also identified as hemagglutinins are multivalent proteins and on account of their fine sugar‐binding specificity play an important role in immune system of invertebrates. The present study was carried out on the hemolymph lectin of cockroach, Periplaneta americana with appropriate screening and purification to understand its molecular as well as functional nature. The lectin from the hemolymph was purified using ion‐exchange chromatography. The approximate molecular weight of purified lectin was 340 kDa as determined by FPLC analysis. Rabbit erythrocytes were highly agglutinated with purified lectin from the hemolymph of P. americana. The hemagglutination activity (HA) of lectin was specifically inhibited by fucose. Glycoproteins also inhibited the HA activity of lectin. The amino acid sequences of the purified lectin revealed homology with amino acid sequences of allergen proteins from P. americana. Purified lectin showed the highest phenoloxidase activity against dopamine. The activators such as exogenous proteases and LPS from Escherichia coli and Salmonella minnesota significantly enhanced the PO activity of the purified lectin. Besides, the presence of copper and hemocyanin conserved domain in the purified lectin provided a new facet that insects belonging to the ancient clade such as cockroaches retained some traces of evolutionary resemblance in possessing lectin of ancient origin.     

Isolation and structure of corazonin, a cardioactive peptide from the American cockroach

Corazonin, a new cardioaccelerating peptide, has been isolated from the corpora cardiaca of the American cockroach, Periplaneta americana, and its structure determined to be Glp-Thr-Phe-Gln-Tyr-Ser-Arg-Gly-Trp-Thr-Asn-amide. The peptide stimulated heart beat at concentrations as low as 0.2 nM, which makes it the most potent insect cardioactive neuropeptide.