凡纳滨对虾血蓝蛋白酶解多肽的凝集与抑菌活性

以凡纳滨对虾(Litopenaeus vannamei)为研究对象, 通过分子筛层析、Tricine-SDS-PAGE、Western-blotting、凝集实验、抑菌实验和Edman N端测序等方法探索血蓝蛋白酶解多肽的凝集和抑菌活性。结果发现, 血蓝蛋白经胰蛋白酶酶解后可产生分子量约为6—70 kD的7条多肽, 该酶解混合物对副溶血弧菌(Vibrio parahaemolyticus)具有显著的凝集活性, 与未酶解的血蓝蛋白相比, 其凝集活性可提高4—16倍。在此基础之上, 进一步分离纯化该7条多肽, 发现多肽-3对副溶血弧菌表现出较强的抑菌活性, 且具有较好的浓度依赖性。在浓度为75 μg/mL时, 其抑菌率为(93.76±1.60)%, 与阴性对照组相比, 存在极显著性差异 (P<0.01)。进一步研究显示该多肽位于血蓝蛋白N端α-螺旋区域。由此推测, 凡纳滨对虾血蓝蛋白在体外经胰蛋白酶酶解后可产生具有凝集、抑菌等免疫活性的多肽, 这对研究血蓝蛋白的降解机制及其在先天免疫中的作用具有重要意义。     

5种凡纳滨对虾血蓝蛋白的糖基化修饰及功能对比分析

血蓝蛋白是近年来发现的一种多功能蛋白,但其功能多样性的分子基础尚不清楚.本研究采用亲和层析、糖含量测定、凝集素印迹等技术在凡纳滨对虾血清中发现 5 种糖含量各不相同的血蓝蛋白：HMC、HMCb^－、HMCb、HMCs^－ 和HMCs,其中HMCs^－ 糖含量最高,HMCb最低,前者约为后者的5倍.继而运用非特异性免疫学实验技术对其功能进行对比分析.结果显示,不同糖基化血蓝蛋白的免疫学活性不同, HMCs^－ 具有较强的红细胞凝集活性和酚氧化酶活性,HMCb^－和HMC分别具有较强的细菌凝集活性和溶血活性.特别是当血蓝蛋白糖基被氧化后,5种血蓝蛋白的凝集活性、溶血活性全部丧失,酚氧化酶活性下降约11～28倍.由此推测,血蓝蛋白糖基修饰多样性可能是其功能多样性的分子基础之一.     

与不同病原菌相结合的凡纳滨对虾血蓝蛋白溶血活性对比分析

以凡纳滨对虾（Litopenaeus vannamei）为研究对象,采用亲和孵育、PAGE、SDS-PAGE、Western-blotting、溶血活性测定等技术,探索与 6 种不同病原菌相结合的血蓝蛋白溶血活性的差异。结果发现,与副溶血弧菌（Vibrio parahaemolyticus）、溶藻酸弧菌（Vibrio alginolyticus）、河弧菌（Vibrio flurialis）、大肠杆菌（Escherichia coli K12）、乙型链球菌（Beta Streptococcus）和金黄色葡萄球菌（Staphylococcus aureus） 6 种不同细菌相结合的血蓝蛋白（分别命名为 HMC-VP、HMC-VA、HMC-VF、HMC-EC、HMC-BS、HMC-SA）对鸡红细胞表现出不同的溶血活性,其中HMC-VP、HMC-SA溶血活性最高（100.00%）,HMC-VA溶血活性最低（39.68%）。在此基础之上,进一步采用糖基氧化和胰蛋白酶消化等策略探索引起该6种血蓝蛋白溶血活性差异的分子基础。结果表明,该6种血蓝蛋白经糖基氧化后,溶血活性大幅度下降抑或丧失,而经胰蛋白酶水解后,溶血活性大幅度升高抑或达到100.00%。由此说明,与不同病原菌相结合的血蓝蛋白免疫学功能（溶血活性）存在显著性差异,造成该差异的原因可能与血蓝蛋白的糖基化修饰、蛋白构象的多样性有关。       

九香虫血淋巴及其纯化蛋白抑菌活性的研究

对九香虫AsporgopuschinensisDallas血淋巴及其血淋巴蛋白质分离物的抗菌活性进行了研究,抗菌活性检测指示菌为大肠杆菌Escherichiacoli和金黄色葡萄球菌Staphilocalliesacereus。测定结果表明,九香虫血淋巴及其离心上清液都具有明显的抗菌活性。用凝胶过滤法从血淋巴蛋白分离提纯获得一种小分子肽,SDS PAGE电泳为单一带,分子量约为1～1 4 4kD。该小分子蛋白对大肠杆菌和金黄色葡萄球菌都有抑菌作用,与血淋巴对2种细菌的抗菌性一致,表明其是九香虫血淋巴中具抗菌作用的主要物质之一。     

致病性鸡大肠杆菌pilA基因和外膜蛋白C基因的克隆、表达及其免疫原性

根据GenBank公布的致病性鸡大肠杆菌的Ⅰ型菌毛pilA基因和外膜蛋白C基因序列,分别设计了两对引物,并以分离的致病性鸡大肠杆菌基因组为模板,经PCR特异性扩增出pilA基因和ompC基因,基因产物大小为别为549 bp和1104 bp,与GenBank报道的参考菌株的两个基因序列的同源性为高达98.18%和97.28%.将扩增得到的两个基因分别定向克隆到原核表达载体pET-28a中,得到两个重组质粒pETpilA和pETompC.转化大肠杆菌BL21(DE3)中,得到重组菌株BL21(pETpilA)和BL21(pETompC),经IPTG诱导后,SDS-PAGE分析分别可见表达的20 kD和40.9 kD的特异条带;Western blotting结果表明,两种蛋白可与抗体发生特异性结合,说明其具有良好的免疫原性.将表达的菌毛蛋白和外膜蛋白的菌株分别制成基因工程疫苗,免疫小鼠后,具有很好的保护能力.表明这两株基因工程菌株有望作为鸡致病性大肠杆菌基因工程疫苗的候选生产菌株.     

重组人纤溶酶原K1-3蛋白的抗肿瘤活性研究

人纤溶酶原K1-3功能区是一个血管生成抑制因子。以人纤溶酶原k1-3基因在大肠杆菌中表达的重组K1-3蛋白进行鸡胚绒毛尿囊膜(chorioallantoicmembrane,CAM)血管生成抑制活性分析和小鼠B16黑色素瘤抑瘤实验,结果证实重组K1-3蛋白具有抑制毛细血管生成和抗肿瘤活性。     

杀菌/渗透性增强蛋白功能及应用前景

杀菌/通透性增强蛋白(BPI)是存在于中性粒细胞中的阳离子蛋白,约为55 kD.BPI能与革兰氏阴性菌外膜上的脂多糖结合,增加外膜对抗菌药的通透性,具有特异性杀灭革兰氏阴性菌及结合/中和内毒素的生物学功能,在革兰氏阴性菌感染的治疗方面有良好的发展前景.近年来,国内外对BPI研究颇多,并逐渐发现BPI还具有调理 作用、抗真菌、抗原虫和抑制血管生成等许多功能,被学者称为未来的“超级抗生素 ”.本文主要就BPI的结构、分布、生理功能、作用机理及临床研究方面的研究进展作一综述.     

香樟树种子中两种Ⅱ型核糖体失活蛋白凝集素活性的研究

从香樟树成熟的种子分离到两种新的Ⅱ型核糖体失活蛋白：新丰毒蛋白和辛纳毒蛋白. 两者A链的分子质量虽然相差近一倍,但B链的分子质量相同. Ⅱ型核糖体失活蛋白的A链是RNA N-糖苷酶,B链是凝集素,A链进入细胞表现毒性在很大程度上依赖于B链的糖结合活性和特异性.对辛纳毒蛋白和新丰毒蛋白B链的凝集素活性进行了测定和比较. 红细胞凝集实验显示辛纳毒蛋白和新丰毒蛋白具有相同的细胞凝集活性,半抗原抑制实验发现它们都属于半乳糖型核糖体失活蛋白,荧光光谱法显示它们的糖结合常数也相同.     

孔石莼(Ulva pertusa)凝集素的分离纯化及性质的研究

为抑制肿瘤细胞增殖和防治有关病害提供基础理论依据 ,将孔石莼 (Ulva pertusa)经磷酸盐缓冲液抽提 ,2 0 %～ 75%硫酸铵分级沉淀 ,牛甲状腺球蛋白 - Sepharose4B亲和层析 ,可以从绿藻孔石莼中纯化出孔石莼凝集素 (UPL) ,在 PAGE上显示单一蛋白染色带 ,在等电聚焦电泳上显示单一蛋白染色带 ,其 p I为 8.40 .纯化后的 UPL的最大紫外吸收峰在 2 85nm,用 Sephadex G- 2 0 0分子筛层析测得其分子量为 1 1 0 4 7.该凝集素可以凝集人的 A、B、AB、O型红细胞 ,且凝集活性相同 ,在对人 (A、B、AB、O)兔、鲤、鲫的红细胞的凝集作用中 ,兔的凝集作用最强 .该凝集素凝集兔红细胞的作用不被 D-半乳糖、D-果糖、葡萄糖、蔗糖、甘露聚糖、γ球蛋白、卵清蛋白所抑制 ,仅被牛甲状腺球蛋白抑制 ,最小抑制浓度为 6.2 0 g/L.该凝集素在 p H4.0～ 1 0 .1 4范围内均有活性 ,但在p H6.50～ 9.51范围内活性较高 ,该凝集活性在 85℃加热 1 h,活力仍未改变 ,说明具有很强的耐热性 .     

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50–200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ?OmpA and ?OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.     

SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei

In this study, we identified a variable region in the C-terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288-2503bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR-SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus-treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus-resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C-terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.     

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.     

Downregulation of Tsx and OmpW and upregulation of OmpX are required for iron homeostasis in Escherichia coli

Upregulation of outer membrane (OM) proteins was systematically investigated in response to poor iron availability in the host and natural environments, but downregulation of OM proteins was ill-defined in this response. We utilized proteomic methodologies to characterize altered OM proteins in the sarcosine-insoluble fraction of Escherichia coli K12 cultured in LB medium with iron limitation. Notably, three novel proteins, Tsx, OmpW, and OmpX, related to iron homeostasis were identified; Tsx and OmpW were downregulated, and OmpX was upregulated. These alterations were functionally validated with the use of gene overexpression and deletion methods. Of the two downregulated proteins, Tsx was more sensitive to an iron-deficient environment than OmpW. In addition, the significantly negative correlation between Tsx with OmpW was achieved when overexpressed strains were used. These findings strongly indicate that the downregulation of Tsx and OmpW and the upregulation of OmpX are required for iron homeostasis in E. coli.     

Cloning and expression of the 5'-nucleotidase gene of Vibrio parahaemolyticus in Escherichia coli and overproduction of the enzyme

The gene encoding the membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus was cloned and expressed in Escherichia coli. Cells of E. coli harboring a plasmid, pNUT5, which carries the 5'-nucleotidase gene were able to grow on ATP as the sole source of carbon, although the original cells were not. The 5'-nucleotidase activity was detected in whole cells of E. coli harboring pNUT5 and in membrane vesicles prepared from these cells. Most properties of the 5'-nucleotidase produced in E. coli, that is, its requirements for Cl- and Mg2+, substrate specificity, and inhibition by Zn2+, were similar to those observed in V. parahaemolyticus, but some alterations in properties were observed: The 5'-nucleotidase was partially inducible in V. parahaemolyticus, but its expression in E. coli was completely constitutive. The specific activity of the 5'-nucleotidase in membrane vesicles of E. coli harboring the plasmid was 30 times that observed in whole cells, whereas the specific activities in membrane vesicles and in whole cells of V. parahaemolyticus were almost the same. A new, dense band of protein with an apparent molecular mass of 63 kDa was detected when membrane proteins of E. coli harboring the plasmid were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Identification and characterization of an outer membrane protein, OmpX, in Escherichia coli that is homologous to a family of outer membrane proteins including Ail of Yersinia enterocolitica.

We previously reported that a region of the Escherichia coli chromosome at 18 min increased E sigma E activity when cloned in multicopy (J. Mecsas, P. E. Rouviere, J. W. Erickson, T. J. Donohue, and C. A. Gross, Genes Dev. 7:2618-2628, 1993). In the present report, we identify and characterize the gene responsible for the increase in E sigma E activity. This gene is in a monocistronic operon with two promoters and a rho-independent terminator. Sequence analysis of this gene indicated that it encodes an outer membrane protein which is 83% identical to OmpX in Enterobacter cloacae, leading us to name this gene ompX. There are four other proteins that are homologous to OmpX. Several of these proteins, Ail of Yersinia enterocolitica and Rck and PagC of Salmonella typhimurium, have properties that allow bacteria to adhere to mammalian cells, survive exposure to human serum, and/or survive within macrophages. We therefore characterized strains deleted for ompX for their growth phenotypes, E sigma E activity, serum resistance, and adherence to mammalian cells. No differences in growth rates, serum resistance, or adherence to mammalian cells were observed; however, E sigma E activity was dependent on expression of OmpX in certain strain backgrounds.     

Expression of human c-raf-1 oncogene proteins in E. coli

Full length and truncated versions of the human c-raf-1 cDNA were cloned into the inducible E. coli expression vector pJL6. C-raf proteins of 73 kD, 57 kD and 39 kD were produced upon induction. p73 differs from normal p73 c-raf by deletion of the two first N-terminal amino acids and their replacement by 16 amino acids encoded by the vector. The p57 and p39 represent N-terminal deletions which leave the transforming protein kinase domain intact. These proteins could be readily purified from E. coli lysates by immunoprecipitation with raf-specific antisera.     

家蝇幼虫抗菌相关蛋白/多肽的诱导及抗菌活性分析

对家蝇Musca domestica 3龄幼虫进行针刺、带菌针刺、热激和超声4种处理,并于处理后不同时间分别收集提取家蝇幼虫体内耐热总蛋白,比浊法测定其抗菌活性,经逐步回归分析确定抗菌相关蛋白/多肽。结果表明,4种处理均能诱导家蝇幼虫产生抗菌物质,其中表观分子量为22 kD的蛋白对藤黄微球菌和大肠杆菌均有抗菌作用,50 kD,13 kD,26 kD,7 kD的蛋白抗菌活性具有专一性。还发现一种37 kD的蛋白对抗菌活性有负作用,推测它可能是促进细胞生长的物质。