Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis.

, , , and 1992. Cloning and partial nucleotide sequence of Schistosoma japonicum paramyosin: a potential vaccine candidate against schistosomiasis. International Journal for Parasitology 22: 1187–1191. Paramyosin from the blood fluke, Schistosoma mansoni, has shown promise as a vaccine candidate for schistosomiasis mansoni. Here we report the cloning and partial nucleotide sequence of a cDNA encoding paramyosin from the related human parasite, Schistosoma japonicum. Affinity purified antibodies to this clone recognized a S. japonicum antigen of molecular weight 97 kDa, equivalent to the reported size of S. mansoni paramyosin. Alignment of the cDNA sequence with that of S. mansoni paramyosin revealed 90% identity. Comparison of the predicted amino acid sequences revealed 95% identity. Although these two parasites differ in many characteristics, the substantial homology demonstrated here between S. mansoni and S. japonicum paramyosin could have important implications for the development of a S. japonicum vaccine.     

Expression of a Bacillus subtilis pectate lyase gene in Pichia pastoris

The methylotrophic yeast Pichia pastoris is an attractive heterologous protein expression host, mainly for genes from higher eukaryotes. However, no successful examples for the expression of bacterial gene encoding pectate lyase in P. pastoris have been reported. The present study reports for the first time the cloning and functional expression of the bacterial Bacillus subtilis gene encoding alkaline pectate lyase in P. pastoris. A molecular weight of 43,644 Da was calculated from the deduced amino acid sequence. A pectate lyase activity as high as 100 U/ml was attained in the fermentation broth of P. pastoris GS 115, which was about 10 times higher than when the gene is expressed in Escherichia coli. The recombinant pectate lyase was purified to homogeneity and maximal activity of the enzyme was observed at 65 °C, and pH 9.4. The recombinant enzyme showed a wider pH and thermal stability spectrum than the purified pectate lyase from B. subtilis WSHB04-02. Pectate lyase activity slightly increased in the presence of Mg²⁺ (ion) but decreased in the presence of other metal ions. Analysis of polygalacturonic acid degradation products by electrospray ionization-mass spectrometry revealed that the degradation products were unsaturated trigalacturonic acid and unsaturated bigalacturonic acid, which confirms that the enzyme catalyzes a trans-elimination reaction.     

幽门螺杆菌多价表位疫苗CWAE的表达及其免疫学性质的研究 *

目的 利用生物信息学软件评价幽门螺杆菌(Helicobacter pylori)多价表位疫苗CWAE的抗原结构,经原核表达获得高纯度CWAE蛋白,进而鉴定多价表位疫苗CWAE的免疫学性质。方法 通过生物信息学软件分析H. pylori多价表位疫苗CWAE的抗原结构;用人工合成的H. pylori多价表位肽融合基因WAE替换重组质粒pET28a-CUE中的UE基因,构建重组质粒pET28a-CWAE。然后,将pET28a-CWAE转入大肠杆菌BL21(DE3)中,经IPTG诱导表达,并通过Ni-NTP镍离子亲和层析纯化抗原蛋白CWAE;利用GM1-ELISA鉴定CWAE中CTB组分的黏膜免疫佐剂活性。最后,通过ELISA和小鼠脾脏淋巴细胞增殖实验检测CWAE激发BALB/c小鼠产生抗H. pylori抗体体液免疫和淋巴细胞免疫应答的能力。结果 通过生物信息学软件证实H. pylori多价表位疫苗CWAE具有科学合理的结构;重组表达质粒pET28a-CWAE经PCR、双酶切和基因测序鉴定,融合基因CWAE与设计序列完全一致;重组基因工程菌株pET28a-CWAE/BL21(DE3)经IPTG诱导表达,抗原蛋白CWAE主要以包涵体形式存在,经Ni-NTP镍离子亲和层析纯化,纯度约达93.2%;GM1-ELISA实验证实,CWAE中CTB组分依旧保持有较好的黏膜佐剂活性;ELISA结果证实CWAE能够激发BALB/c小鼠产生H. pylori特异性抗体,而小鼠脾脏淋巴细胞增殖实验进一步证实CWAE能够激发针对H. pylori多种致病因子的淋巴细胞免疫反应。结论 H. pylori多价表位疫苗CWAE具有科学合理的抗原结构,经原核表达可获得高纯度抗原蛋白,能够激发BALB/c小鼠产生H. pylori特异性抗体体液免疫和淋巴细胞免疫应答。为研发防治H. pylori感染的多价表位疫苗奠定实验基础。     

Heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1) using versatile autonomously replicating vector for a wide range of host

A versatile plasmid shuttle vector system pKDU7 was constructed, which is useful for the heterologous gene expression in a wide range of Kluyveromyces and Saccharomyces strains. This cloning vector was constructed using the 1.6-μm circular plasmid pKD1 of Kluyveromyces drosophilarum, the URA3 gene of K. marxianus as well as the pUC19 sequences. The stability of vector in transformants strongly depends on the integrity of the functionally important elements of pKD1. It was shown by comparison of three recombinant vectors, which possessed the pKD1 sequence inserted in different ways. The efficient transformation and stability maintenance of the vector constructed in various strains of Kluyveromyces and Saccharomyces was shown by the expression of the EPG1 gene of the Kluyveromyces marxianus encoding pectin-degrading endopolygalacturonase.     

Cloning and characterization of the cpyA gene encoding intracellular carboxypeptidase from Aspergillus nidulans.

Carboxypeptidase Y (CPY) has been used as a maker enzyme for investigations on intracellular transport of vacuolar proteins and on vacuolar biogenesis in Saccharomyces cerevisiae. We describe the cloning and characterization of the CPY homologue encoding gene (cpyA) from the filamentous fungus Aspergillus nidulans. The cpyA gene has one intron and encodes a protein with 552 amino acids containing a putative signal sequence and pro-sequence. The predicted CpyA protein is highly similar in sequence with carboxypeptidases from several yeast species and contains a catalytic triad (Asp-His-Ser) like that of serine carboxypeptidase. The cpyA disruptant cells showed reduced levels of intracellular carboxypeptidase. These results suggest that the cpyA gene encodes a vacuolar carboxypeptidase in A. nidulans.     

Cloning and preliminary characterization of a novel cuticular antigen from the filarial parasite Dirofilaria immitis

We have described here the cloning and partial characterization of a cDNA encoding a cuticular antigen of Dirofilaria immitis. A 48-h third-stage larval D. immitis cDNA library was immunoscreened with sera raised in mice against third-stage larval cuticles (mouse anti-L3 cuticle antisera). A strongly immunoreactive clone (L3MC4) was isolated. Sequence analysis of L3MC4 showed that it was a partial length cDNA. The missing 5′ end of the clone was amplified by PCR from D. immitis adult female first-strand cDNA using the nematode 22-base splice leader sequence and a L3MC4-specific antisense primer. The composite cDNA sequence comprised 616 bases (nDiL3MC4) encoding a full-length protein of 146 amino acids (DiL3MC4). GenBank analysis showed that DiL3MC4 shared some homology to an unknown C. elegans gene product (31%) at the amino acid level. However, there were no related filarial expressed sequence tags in the current GenBank™ database. Antibodies to recombinant DiL3MC4 (rDiL3MC4) identified a 19-kDa native antigen in the adults and in the L3 and L4 larval stages of D. immitis. In addition, the antibodies bound to the cortical layers of the L3 cuticle, as revealed by immuno-gold electron microscopy. The native protein was not detected in larval and adult excretory–secretory products. Immunoblot analysis showed that serum from a rabbit that was repeatedly injected with a small number of D. immitis third stage larvae reacted with rDiL3MC4. Thus, DiL3MC4 is a novel cuticular antigen of a filarial parasite.     

Neuronal models to study amyloid precursor protein expression and processing in vitro

Most organisms obtain their fatty acids through their diet or by de novo synthesis, but human blood flukes belonging to the genus Schistosoma lack the oxygen-dependent pathways required for the synthesis of sterols and fatty acids so they are entirely dependent on their hosts for these and other complex lipids. Fatty acid binding proteins (FABPs) of the FABP/P2/CRABP/CRBP family of beta-barrel cytosolic lipid binding proteins (cLBP) appear to be particularly important to schistosomes in the uptake, transport and compartmentalisation of host-derived fatty acids and may provide important targets for immuno- and chemotherapy. Here we describe the isolation of a set of cDNAs prepared from the Asiatic schistosome, Schistosoma japonicum, which encode two groups of cLBPs based on sequence homology and unique cDNA restriction sites. Representative clones from the two groups, one encoding a complete Sj-FABP (F10), and the other encoding a deletion mutant (F25) were characterised at the nucleic acid level by Southern and Northern hybridisation analysis, and at the protein level by immunoblotting. The presence and size of introns in the genes encoding F10 and F25 were determined and, because of the interest in the Schistosoma mansoni FABP homologue (Sm14) as a putative vaccine candidate, the immunogenicity and protective efficacy of the two proteins were also evaluated. A particularly interesting finding was the degree of Sj-FABP amino acid sequence polymorphism found to occur within the S. japonicum worm population, which appears to be greater than that described from cLBPs from vertebrates or, indeed, any other group of organisms investigated to date.     

甘油脱水酶再激活酶的克隆表达及活性鉴定

运用PCR技术从克雷伯氏菌的基因组中分别扩增得到了编码甘油脱水酶再激活酶α、β两个亚基的基因gdrA、gdrB。将gdrA、gdrB克隆至pMD-18T载体上,构建克隆载体pMD-gdrAB。经测序正确后,将gdrAB亚克隆至表达载体pET-28a( )上构建表达质粒pET-28gdrAB。利用双抗生素筛选法,将pET-28gdrAB与连有甘油脱水酶基因的表达载体pET-32gldABC在大肠杆菌菌株BL21(DE3)中共表达,鉴定了甘油脱水酶再激活酶的活性。     

Efficacy of Leishmania donovani ribosomal P1 gene as DNA vaccine in experimental visceral leishmaniasis

The acidic ribosomal proteins of the protozoan parasites have been described as prominent antigens during human disease. We present here data showing the molecular cloning and protective efficacy of P1 gene of Leishmaniadonovani as DNA vaccine. The PCR amplified complete ORF cloned in either pQE or pVAX vector was used either as peptide or DNA vaccine against experimentally induced visceral leishmaniasis in hamsters. The recombinant protein rLdP1 was given along with Freund’s adjuvant and the plasmid DNA vaccine, pVAX-P1 was used alone either as single dose or double dose (prime and boost) in different groups of hamsters which were subsequently challenged with a virulent dose of 1 × 10⁷L.donovani (MHOM/IN/DD8/1968 strain) promastigotes by intra-cardiac route. While the recombinant protein rLdP1 or DNA vaccine pVAX-P1 in single dose format were not found to be protective, DNA vaccine in a prime-boost mode was able to induce protection with reduced mortality, a significant (75.68%) decrease in splenic parasite burden and increased expression of Th1 type cytokines in immunized hamsters. Histopathology of livers and spleens from these animals showed formation of mature granulomas with compact arrangement of lymphocytes and histiocytes, indicating its protective potential as vaccine candidate.     

鲈鱼IgM重链基因cDNA的克隆及其原核表达

采用同源克隆和RACE技术扩增到鲈鱼 (Lateolabrax japonicus) 免疫球蛋白M (Immunoglobulin M,IgM) 重链 (Heavy chain,H) 基因全长cDNA序列。鲈鱼IgM cDNA全长为1 901 bp,开放阅读框包含1 749 bp,编码582个氨基酸。根据鲈鱼IgM和其他硬骨鱼免疫球蛋白重链恒定区的氨基酸序列构建的系统发育树表明IgM、IgZ和IgD分别聚为一枝,其中IgM与IgZ分支的进化关系较近,而与IgD分支的进化关系较远。RT-PCR检测IgM在鲈鱼各组织器官的表达情况,其中在头肾及脾脏中表达量最高,心脏、肌肉及脑中几乎不表达。利用已获得的鲈鱼IgM cDNA序列,构建原核表达载体pQE30-IgM,并在M15大肠杆菌中成功诱导表达了分子量为63kD的重组蛋白His-IgM,Western-blotting显示鲈鱼IgM重组蛋白能与鼠源抗6×His的单克隆抗体特异性结合,说明已经获得了基因工程表达IgM重链蛋白。     

Molecular cloning of human cardiac troponin I using polymerase chain reaction

We have used the polymerase chain reaction (PCR) to synthesise a cDNA encoding part of human cardiac troponin I. Amplification was achieved using fully degenerate sets of oligonucleotides corresponding to conserved regions of amino acid sequence identified in other troponin I isoforms. The cloned PCR fragment was subsequently used to isolate full-length cDNAs from a cardiac cDNA library. We describe the approach, as a general cloning strategy starting from limited amino-acid sequence data and report the cloning, and complete amino acid sequence of human cardiac troponin I. Analysis of human development using these clones demonstrates early expression of this gene in the heart.     

Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80/CD86-CD28 costimulatory pathway

The development of recombinant techniques for rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, or inclusion of self-assembly motifs, novel molecules such as bispecific Abs can be generated that possess the subset of functional properties designed to fit the intended application. We describe the engineering of Ab fragments produced in bacteria for blocking the CD28-CD80/CD86 costimulatory interaction in order to induce tolerance against transplanted organs. We designed single-chain Fv antibodies, monospecific and bispecific diabodies, and a bispecific tetravalent antibody (BiTAb) molecule directed against the CD80 and/or CD86 costimulatory molecules. These recombinant Ab molecules were expressed in Escherichia coli, followed by purification and evaluation for specific interaction with their respective antigen in an enzyme-linked immunosorbent assay (ELISA). A specific sandwich ELISA confirmed the bispecificity of the bispecific diabodies and the BiTAb.     

Identification and characterization of a cDNA encoding ribosomal protein S12 from Xenopus laevis

The complete nucleotide (nt) sequence of the cDNA clone XL-S12, encoding a Xenopus laevis (XI) homologue of the mammalian ribosomal protein S12, has been determined. The sequence predicts a XI S12 protein of 132 amino acids (aa) with a molecular mass of 14.7 kDa. XI S12 shares 95 and 97% aa sequence identity with the human and murine S12 proteins, respectively. Analysis of nt substitution patterns and rates indicates that S12 is a very highly constrained protein, evolving at an estimated rate of only 0.03 x 10˜9 non-synonymous (protein-altering) substitutions per site per year.     

Expression,purification, and characterization of minimized chicken riboflavin carrier protein from a synthetic gene in Escherichia coli

Minimized proteins have long been used to elicit immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. Here we describe molecular cloning and production of a minimized chicken riboflavin carrier protein (mini-RCP) sequence that harbours all the four neutralizing epitopes but lacks the sequences that otherwise elicit undesirable antibodies. The gene encoding mini-RCP is engineered by contiguous alignment of nucleotide sequences coding for selected epitopes of chicken RCP separated by leucyl alanine residues. The gene has been constructed from eight oligonucleotides by employing overlapping PCR strategy and expressed in Escherichia coli, using the T7 promoter system. The recombinant protein could be purified to homogeneity by a single step Ni2+ affinity chromatography. Western blot experiments using epitope specific antisera confirm that the corresponding linear amino acid sequences are available for immunorecognition in the engineered protein. This methodology enables continuous production and purification in bulk amounts of the minimized RCP as a source of candidate immunocontraceptive vaccine in mammals.     

Construction of a set Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli

We describe here the construction of a 10-Gateway-based vector set applicable for high-throughput cloning and for expressing recombinant proteins in Escherichia coli. Plasmids bear elements required to produce recombinant proteins under control of the T7 promoter and encode different N-terminal partners. Since the vector set is derived from a unique backbone, a consistent comparison of the impact of fusion partner(s) on protein expression and solubility is easily amenable. Finally, a sequence encoding a six-histidine tag has been inserted to be in frame with the cloned open reading frame either at its C terminus or at the N terminus, giving the flexibility of choosing the six-histidine tag location for further purification. To test the applicability of our vector set, expression and solubility profile and six-histidine tag accessibility have been demonstrated for two Bacillus subtilis signaling proteins' encoding genes (SBGP codes E0508 and E0511).