Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1)

Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system.     

Expression and functional characterization of a giant Type I fatty acid synthase (CpFAS1) gene from Cryptosporidium parvum

A 25-kb CpFAS1 gene from Cryptosporidium parvum has been engineered and expressed as five individual maltose-binding protein (MBP)-fusion proteins: an N-terminal loading unit, three fatty acyl elongation modules, and a C-terminal reductase. Enzymatic activities of all domains (except the reductase) were individually assayed as recombinant proteins. The preferred substrate for the fatty acyl ligase (AL) domain in the loading unit was palmitic acid (C16:0). However, a competition assay suggests that the AL domain could also utilize other fatty acids as substrates (i.e., C12:0-C24:0), albeit with reduced activity. Among the three elongation modules, enzymatic activities were detected for ketoacyl synthase (KS), acyl transferase (AT), dehydrase (DH), enoyl reductase (ER), and ketoacyl reductase (KR) domains, which suggests that these modules were involved in the elongation of a saturated fatty acyl chain that would be C6 longer (e.g., C22:0) than the precursor (e.g., C16:0). In addition, the KS activity could be specifically inhibited by cerulenin (IC(50) approximately 1.5 microM), reinforcing the notion that CpFAS1 could be exploited as potential drug target. Since C. parvum lacks other fatty acid synthases, these observations imply that this parasite may not be capable of synthesizing fatty acids de novo.     

重组SARS冠状病毒棘突蛋白S1区基因片段的克隆、表达及免疫原性研究

目的构建SARS冠状病毒(SARS-CoV)棘突(spike)蛋白(S蛋白)S1区基因(40～2001bp)分段表达载体,并研究其免疫原性。方法利用PCR技术扩增S1区基因,将其定向插入pQE30质粒,在pQE30质粒上将S1切成3段(40～751、746～1344、746～2001bp),均插入pQE30质粒,构建质粒在大肠埃希菌M15中表达,表达产物经亲和层析纯化及WesternBlot和ELISA鉴定。结果构建的3种重组质粒(pQE30/S1a、pQE30/S1b、pQE30/S1c)均高效表达,重组蛋白相对分子质量分别为26700、22500和46000,表达量分别占菌体总蛋白质的35%、35%和30%。3种重组蛋白经Ni亲和层析树脂得到成功纯化。经Western-Blot及ELISA证实,重组蛋白片段S1c(746～2001bp)可以被SARS患者血清所识别。结论成功构建了SARS-CoVS蛋白S1区基因分段表达载体,重组蛋白S1c具有较强的免疫原性,它的获得为下一步疫苗的研制奠定了基础。     

Identification of a novel piscine Cryptosporidium genotype and Cryptosporidium parvum in cultured rainbow trout (Oncorhynchus mykiss)

This study reports for the first time the presence and molecular characterization of Cryptosporidium in farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792). A total of 360 fish, with no apparent clinical signs of disease, were collected and classified into groups according to their size. Cryptosporidium oocysts were detected by immunofluorescence microscopy in 33 specimens (9.2%), which were located in pyloric caeca samples (42.4%), intestinal scrapings (39.4%), or at both locations (18.2%). In the smallest (youngest) fish group, a higher percentage of positive samples were detected in the pyloric caeca relative to the intestinal location (58.8 vs. 17.6%; P = 0.01), including a cluster with more than 10 oocysts observed in the pyloric caeca of one specimen. PCR amplification and sequencing of fragments of SSU-rDNA and hsp70 genes identified a novel Cryptosporidium piscine genotype (genotype 9) in two specimens and Cryptosporidium parvum in seven fish, including the specimen in which the oocyst cluster was observed. Moreover, Cryptosporidium oocysts were detected in farm water samples (41.7 and 16.7% from influent and effluent, respectively). Although Giardia was not found in gastrointestinal samples, Giardia cysts were observed in 50.0 and 33.3% of the influent and effluent water samples, respectively. The results support the existence of natural infections by C. parvum in freshwater cultured fish, suggesting that the rainbow trout could shed infectious oocysts in aquatic environments and it may be a potential source of human infection when this edible fish is handled.

     

First surveillance and molecular identification of the Cryptosporidium skunk genotype and Cryptosporidium parvum in wild raccoons (Procyon lotor) in Osaka,Japan

Parasitology Research - Recent research suggests that raccoons (Procyon lotor) can transmit several important pathogens affecting humans, including protozoans. In Japan, the number of wild raccoons...     

Overexpression,purification, and characterization of full-length and mutant caldesmons using a baculovirus expression system

Summary Three recombinant chicken gizzard caldesmon (CaD) baculovirus vectors that contained the full-length CaD codon sequence (Pv1CaD), the full-length CaD codon sequence and a six-histidine tag at the 5-end (pBlueBacHisCaD), or the full-length CaD codon sequence and an extra six-histidine codon sequence at the 3-end (PvlHisCaD) were constructed. Spodoptera frugiperda (Sf9) cells transfected with these constructs overexpressed full-length CaD, yielding 2, 20, and 50 g per 10⁶ cells for pBlueBacHisCaD, PvlHisCaD, and PvlCaD, respectively. Time course assays for the expressed proteins demonstrated that the optimum harvest time was 36 h postinfection. Immunofluorescence microscopy revealed PvlCaD localized on the plasma membrane of Sf9 cells at 24 h postinfection and distributed throughout the cytoplasm at 36–48 h postinfection. Analysis of the purified recombinant full-length CaD revealed most of the characteristics of the authentic CaD, including (a) an electrophoretic mobility corresponding to 125 kDa, (b) heat stability, (c) binding to actin, tropomyosin-actin, myosin, and calmodulin, (d) ability to inhibit actin-activated ATP hydrolysis by smooth muscle myosin, and (e) ability of Ca²⁺-calmodulin to reverse the inhibition. A CaD mutant with a deletion of 159 amino acids from the carboxyl terminus of the full-length CaD was also expressed at high levels in Sf9 cells. However, this mutant showed a decreased ability to bind to actin, tropomyosin-actin, and calmodulin, whereas the myosin binding was unaffected; actin-activated ATP hydrolysis by smooth muscle myosin was not inhibited by this mutant.     

A recombinant human immunodeficiency virus type-1 capsid protein (rp24): its expression, purification and physico-chemical characterization

An expression system has been established in Escherichia coli to facilitate the preparation of the HIV-1 capsid protein in amounts sufficient for structural analysis. A plasmid vector pTCA5, containing the gene for the recombinant HIV-1 capsid protein rp24 under the control of the λ-P_R-promoter, was constructed which gave an expression product that spanned 234 amino acid residues. It differs at the N-terminus from the authentic sequence in that the residues Pro-Ile- are replaced by Met-Asn-Ser-Ala-Met-. Recombinant p24 was produced, as inclusion bodies in E. coli LE392 containing pTCA5, at a level of approximately 15% of the total cellular protein. After dissolution of the inclusion bodies in the acidic urea system, the protein was easily reconstituted in a soluble state by dialysis. The yield of reconstituted and purified protein was 12 mg per liter in rich medium. Recombinant rp24 consists of about 40% -helix and 10% β-sheet from circular dichroism measurements and the two cysteine residues, within the rp24 sequence, are bridged by a disulfide bond.     

鼠源性α-1,3-半乳糖基转移酶催化结构域的表达及纯化

目的：表达和纯化鼠源性的α-1,3-半乳糖基转移酶的催化结构域（α1,3 GTcd）以及为在肿瘤细胞表面生成α-gal表位提供了可行性手段.方法：利用pET-15b载体以可溶性形式表达鼠源性α1,3 GTcd.利用高效液相色谱阴离子交换柱检测酶的活性.结果：①成功地构建了带有His-tag的α1,3GTcd重组质粒.②可溶性高效表达与纯化了带有His-tag的α1,3GTcd.③利用HPLC阴离子交换柱检测α1,3GTcd的活性.结论：可溶性地表达了α1,3 GTcd.     

Characterization of a recombinant canine coronavirus with a distinct receptor-binding (S1) domain

Canine alphacoronaviruses (CCoV) exist in two serotypes, type I and II, both of which can cause severe gastroenteritis. Here, we characterize a canine alphacoronavirus, designated CCoV-A76, first isolated in 1976. Serological studies show that CCoV-A76 is distinct from other CCoVs, such as the prototype CCoV-1-71. Efficient replication of CCoV-A76 is restricted to canine cell lines, in contrast to the prototypical type II strain CCoV-1-71 that more efficiently replicates in feline cells. CCoV-A76 can use canine aminopeptidase N (cAPN) receptor for infection of cells, but was unable to use feline APN (fAPN). In contrast, CCoV-1-71 can utilize both. Genomic analysis shows that CCoV-A76 possesses a distinct spike, which is the result of a recombination between type I and type II CCoV, that occurred between the N- and C-terminal domains (NTD and C-domain) of the S1 subunit. These data suggest that CCoV-A76 represents a recombinant coronavirus form, with distinct host cell tropism.     

Isolation and characterization of a novel HS1 SH3 domain binding protein, HS1BP3

We have isolated a novel gene, HS1BP3, which encodes an HS1 binding protein. Analysis of HS1BP3 cDNA indicates several potentially important segments, including a PX domain, a leucine zipper, immunoreceptor tyrosine-based inhibitory motif-like motifs and proline-rich regions. HS1BP3 associates with HS1 proteins in vivo as confirmed by immunoprecipitation in B and T cell lines. HS1BP3 preferentially associates with the HS1 SH3 domains rather than with other SH3 molecules, suggesting a role of HS1BP3 as an HS1 signaling mediator. Overexpression of mutant HS1BP3 protein in T cell lines results in decreased IL-2 production. Our data suggest a novel role for HS1BP3 in lymphocyte activation.     

戊型肝炎病毒Ⅰ型ORF3蛋白的表达、纯化及抗原性分析

目的　表达戊型肝炎病毒 (HEV)Ⅰ型ORF3全长基因片段 ,纯化表达产物并进行抗原性分析。方法　将Ⅰ型HEVORF3基因连接到融合表达载体pThioHisB ,IPTG诱导表达 ;Westernblot分析表达产物的抗原活性 ;用经高效液相色谱纯化的表达产物作为抗原 ,制备血清抗 HEVIgG及抗 HEVIgM诊断试剂 ,用国家参考品进行考核 ;用所得抗原检测临床血清中抗 HEV抗体 ,比较其与Genelabs试剂盒检测结果的差异。结果　含有Ⅰ型pThioHisB/ORF3质粒的菌株表达了相对分子质量(Mr)为 2 6× 10 3 的融合蛋白 ,免疫印迹表明其能与戊型肝炎患者恢复期血清发生特异性反应。国家参考品考核结果显示 ,用表达产物制备的抗HEVIgG诊断试剂阳性符合率为 10 0 % (10 / 10 ) ,阴性符合率为 96 .7% (2 9/ 30 ) ,总符合率为 97.5 % (39/ 4 0 ) ;制备的抗 HEVIgM诊断试剂阳性符合率为 83.3%(10 / 12 ) ,阴性符合率为 10 0 % (2 0 / 2 0 ) ,总符合率为 93.8% (30 / 32 )。与Genelabs公司试剂盒检测结果比较 ,抗 HEVIgG和抗 HEVIgMELISA总符合率分别为 94 .5 %和 87.0 %。结论　本实验所得全长ORF3基因工程表达产物具有良好的抗原性 ,可用于制备抗 HEV诊断试剂。     

Immunologic characterization of a recombinant American cockroach (Periplaneta americana) Per a 1 (Cr-PII) allergen

BACKGROUND: Previously, we have identified several Per a 1 (Cr-PII) allergens from a deltagt22A cDNA library of Periplaneta americana. This study aimed to sequence clone C42 and determine its molecular and antigenic properties. METHODS: The cDNA of C42 was sequenced and ligated into a bacteria expression vector, pET21. The recombinant proteins were purified by ion-exchange and affinity chromatographies. Their antigenicities were analyzed by immunoblotting, ELISA, and binding inhibition with human IgE. RESULTS: The nucleotide of the cDNA has been sequenced and the deduced amino acid which encodes a 446-amino-acid protein (50kDa) determined. The recombinant C42 protein can bind both anti-Per a 1 monoclonal antibodies and human IgE and showed a 54.4% (12/22) skin reactivity in atopic patients. Sequence homology searches revealed a high degree of identity to two other members of the Per a 1 family, C17 and C6, and the German cockroach (Blattella germanica) Bla g Bd90K allergen. Interestingly, these allergens all contain internal repeats, and the crude B. germanica extract, Per a 1, and recombinant allergens share similar antigenic determinant(s) as defined by ELISA and IgE-binding inhibition studies. In IgE-binding epitope studies, an immunopositive C42 fragment was first identified from partial protease digestion. Overlapping peptides were then generated by expression of restriction enzyme fragments in E. coli. The shortest peptide, C42-P560, identified by monoclonal antibodies and human specific IgE, can inhibit IgE binding to C42. CONCLUSIONS: An additional Per a 1 allergen has been defined at the molecular level and characterized and preliminary results showed that a potential IgE-reactive region is located within amino-acid residue 358-446 of C42, which is an internal repeat. The results defined the boundaries of the antigenic site and will facilitate further epitope-mapping studies.     

Molecular cloning and functional characterization of porcine nucleotide-binding oligomerization domain-1 (NOD1) recognizing minimum agonists, meso-diaminopimelic acid and meso-lanthionine

In this study, we isolated a complementary DNA encoding nucleotide-binding oligomerization domain-1 (NOD1) from Peyer's patches (Pps) of swine gut-associated lymphoid tissues (GALT). The complete open reading frame of porcine NOD1 contains 2862 bp, encoding a 953-amino acid polypeptide. The porcine NOD1 amino acid sequence is more closely related to the human sequence (83.8% identity) than the mouse counterpart (79.2% identity). To examine the subcellular expression and function of porcine NOD1, we overexpressed it in human embryonic kidney 293 cells. Immunostaining with an anti-porcine NOD1 polyclonal antibody revealed that the protein was expressed in transfectants as an intracellular membrane-bound molecule. In the transfected cells, both gamma-d-glutamyl-meso-diaminopimelic acid, and meso-diaminopimelic acid and meso-lanthionine activated nuclear factor-kappa B. Quantitative real-time PCR detected NOD1 mRNA in multiple tissues isolated from adult and newborn swine, including the esophagus, duodenum, jejunum, ileum, ileal Pps, colon, spleen, and mesenteric lymph nodes. In the newborn and adults, NOD1 was highly expressed in the esophagus and GALT, such in the ileal Pps and mesenteric lymph nodes. Furthermore, Toll-like receptor and NOD1 ligands as well as immunobiotic lactic acid bacteria enhanced the expression of NOD1 in GALT of adult and newborn swine. Our results should help clarify how the intestinal immune system is modulated by low-molecular weight peptidoglycan fragments through NOD1.     

Che a 1: recombinant expression, purification and correspondence to the natural form

BACKGROUND: Pollinosis to chenopods is one of the main causes of allergy in desertic regions and it is increasing in the South of Europe and Western USA. Che a 1 is a major allergen for chenopod-allergic subjects and belongs to the Ole-e-1-like family of proteins. METHODS: Pichia pastoris yeast has been used as expression system to produce the recombinant form of Che a 1 (rChe a 1). The allergen was isolated using a gel permeation column and reverse-phase/high-performance liquid chromatography. Molecular characterization was performed using Edman degradation, mass spectrometry and concanavalin A staining. Sera from patients allergic to chenopod pollen, as well as polyclonal and monoclonal antibodies raised against Ole e 1, were used in immunoblotting, ELISA and inhibition assays for immunological characterization of rChe a 1. RESULTS: The allergen was purified to homogeneity with a final yield of 15 mg/l of cell culture and showed a glycosylated character. N-terminal amino acid sequence of rChe a 1 and molecular mass were according to those of the protein isolated from chenopod pollen. The recombinant allergen maintained the IgG and IgE epitopes of the natural allergen deduced from the immunological assays. CONCLUSIONS: Structural and in vitro immunological properties of rChe a 1 produced in P. pastoris were equivalent to those of the natural form of the allergen and, thus, it could be used in testing patients allergic to chenopods.     

Expression, purification, characterization and clinical relevance of rAed a 1--a 68-kDa recombinant mosquito Aedes aegypti salivary allergen.

Accurate diagnosis of mosquito allergy has been precluded by the difficulty of obtaining salivary allergens. In this study, we expressed, purified, characterized and investigated the clinical relevance of a recombinant Aedes aegypti salivary allergen, rAed a 1. Two cDNA segments were ligated together to form the full-length Aed a 1 gene. rAed a 1 was expressed using a baculovirus/insect cell system, and purified using a combination of anion-exchange and gel-filtration chromatography. The purified rAed a 1 bound to human IgE, as detected by ELISA, ELISA inhibition tests and immunoblot analyses. Epicutaneous tests with rAed a 1 and a commercial whole-body AE: aegypti extract, and AE: aegypti bite tests were performed in 48 subjects. Nine of 31 (29%) of the subjects with positive immediate bite tests also had a positive rAed a 1 immediate skin reaction and 32% had an positive immediate test to the commercial extract. Six of 33 (18%) of the subjects with positive delayed bite tests also had a positive rAed a 1 delayed skin reaction and 6% had a positive delayed test to the commercial extract. Furthermore, rAed a 1-induced flare sizes significantly correlated with mosquito bite-induced flare sizes. None of the subjects with negative bite tests had a positive skin test to rAed a 1 or to commercial extract. We conclude that the rAed a 1 has identical antigenicity and biological activity to native Aed a 1, can be used in the in vitro and in vivo diagnosis of mosquito allergy, and is more sensitive than mosquito whole-body extract for detecting delayed skin reactions.     

Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum

Giardia lamblia and Cryptosporidium parvum are recognized as the most common protozoan infections in Saudi Arabia. Microscopic examination of stool samples, either direct or concentrated, for the recovery of G. lamblia cysts and trophozoites and C. parvum oocysts is still the most commonly used for the diagnosis of both parasites. We compared the conventional parasitological techniques of iodine-stained wet mount for G. lamblia and Kinyoun's acid-fast for C. parvum against ImmunoCard STAT® Cryptosporidium/Giardia and real-time polymerase chain reaction (PCR) detecting the 18S rRNA gene of G. lamblia and conventional PCR detecting the same gene of C. parvum at a tertiary hospital in Dhahran, Saudi Arabia. Out of 148 stool samples, 19 and 12 true positives were identified for G. lamblia and C. parvum, respectively, using a composite reference standard. In this case, true positives and negatives were considered as those with at least two positive or negative results out of the three tests. Both ImmunoCard STAT! and PCR methods were more sensitive than the microscopic tests of a single stool specimen of 85.7 % (CI?=?62.6–96.2 %) and 85.7 % (CI?=?56.2–97.5 %) for G. lamblia and C. parvum, respectively. However, specificity of microscopic tests was higher than other techniques for both parasites. Although PCR seems to be most sensitive for both G. lamblia and C. parvum, its low specificity may render its superiority over other techniques. When a single stool sample is used for detection of G. lamblia and C. parvum, better results can be obtained when coupled with serological testing. Although PCR is the most sensitive method for the detection of both G. lamblia and C. parvum, its use requires attention in relation to the increased possible false positives.     

Production of recombinant antigens of Ureaplasma parvum serotypes 3 and 6 for development of a serological assay

Recombinant antigens of Ureaplasma parvum serotypes 3 and 6 were produced in order to develop a serological assay for Ureaplasma antibody detection. The genes of the multiple banded antigen (MBA) were amplified by PCR and cloned in a pTrcHis TOPO plasmid. Purified recombinant proteins were evaluated in Western blotting and enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies and human sera. Our approach was successful in the production of the recombinant MBAs (rMBAs) for serotypes 3 and 6. The antigens tested positive with serotype-specific monoclonal antibodies in Western blotting and in ELISA. Prominent reactions were detected with the rMBAs and their homologous monoclonal antibodies. Strong cross-reactions were visible in ELISA between rMBA 3 and the monoclonal antibodies from the other U. parvum serotypes. A weak cross-reaction was seen with rMBA 3 and the monoclonal antibody from serotype 4. rMBA 6 showed cross-reaction only with the monoclonal antibody from U. parvum serotype 1. Fifty-one percent of the sera obtained from culture-positive women reacted with one or both rMBAs. Only three (15%) of the sera from culture-negative women reacted with the rMBA. The positive reactions were observed only with rMBA 6. These preliminary tests showed the potential usefulness of the rMBAs produced for detecting an antibody response against Ureaplasma antigens.