额种子蛋白体靶向表达口服重组胰岛素原载体构建及转化水稻

应用水稻种子生物反应器开发口服重组胰岛素原具有重要应用前景。通过分子设计保证重组胰岛素原在人体肠道内的自主加工成熟,根据水稻密码子偏爱性人工合成了霍乱毒素β亚基和人胰岛素原的融合基因(cholera toxin B subunit fused with human proinsulin,CTBIN),并在C末端添加内质网滞留信号KDEL。通过PCR技术从粳稻品种日本晴全基因组中克隆谷蛋白启动子及其信号肽序列pGluB1sig(GluB1 promoter and its signal peptide)用于驱动融合基因CTBIN的表达,插入载体pCAMBIA1302,构建了水稻种子蛋白体靶向表达口服重组胰岛素原的载体pCAMBIA1302-pGluB1sig-CTBIN-Nos。采用农杆菌介导法转化日本晴,获得了46株转基因水稻植株,Western杂交检测到CTB-人胰岛素原融合蛋白在水稻种子中表达。     

Immunogenicity of a Fusion Protein Comprising Coli Surface Antigen 3 and Labile B Subunit of Enterotoxigenic Escherichia coli

Background: Enterotoxigenic Escherichia coli (ETEC) strains are the major causes of diarrheal disease in humans and animals. Colonization factors and enterotoxins are the major virulence factors in ETEC pathogenesis. For the broad-spectrum protection against ETEC, one could focus on colonization factors and non-toxic heat labile as a vaccine candidate. Methods: A fusion protein is composed of a major fimbrial subunit of coli surface antigen 3, and the heat-labile B subunit (LTB) was constructed as a chimeric immunogen. For optimum level expression of protein, the gene was synthesized with codon bias of E. coli. Also, recombinant protein was expressed in E. coli BL21DE3. ELISA and Western tests were carried out for determination of antigen and specificity of antibody raised against recombinant protein in animals. The anti-toxicity and anti-adherence properties of the immune sera against ETEC were also evaluated. Results: Immunological analyses showed the production of high titer of specific antibody in immunized mice. The built-in LTB retains native toxin properties which were approved by GM₁ binding assay. Pre-treatment of the ETEC cells with anti-sera significantly decreased their adhesion to Caco-2 cells. Conclusion: The results indicated the efficacy of the recombinant chimeric protein as an effective immunogen inducing strong humoral response. The designated chimer would be an interesting prototype for a vaccine and worthy of further investigation. Key Words: Recombinant vaccine, Enterotoxigenic Escherichia coli (ETEC), cstH, eltB     

Expression and Purification of Neurotoxin-Associated Protein HA-33/A from Clostridium botulinum and Evaluation of Its Antigenicity

Background: Botulinum neurotoxin (BoNT) complexes consist of neurotoxin and neurotoxin-associated proteins. Hemagglutinin-33 (HA-33) is a member of BoNT type A (BoNT/A) complex. Considering the protective role of HA-33 in preservation of BoNT/A in gastrointestinal harsh conditions and also its adjuvant role, recombinant production of this protein is favorable. Thus in this study, HA-33 was expressed and purified, and subsequently its antigenicity in mice was studied. Methods: Initially, ha-33 gene sequence of Clostridium botulinum serotype A was adopted from GenBank. The gene sequence was optimized and synthesized in pET28a (+) vector. E. coli BL21 (DE3) strain was transformed by the recombinant vector and the expression of HA-33 was optimized at 37°C and 5 h induction time. Results: The recombinant protein was purified by nickel nitrilotriacetic acid agarose affinity chromatography and confirmed by immunoblotting. Enzyme Linked Immunoassay showed a high titer antibody production in mice. Conclusion: The results indicated a highly expressed and purified recombinant protein, which is able to evoke high antibody titers in mice. Key Words: Botulinum neurotoxin, Expression, Purification     

A Molecular Survey of Campylobacter jejuni and Campylobacter Coli Virulence and Diversity

Background: The aim of this study was to determine the prevalence of virulence-associated genes and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) analysis of Campylobacter spp. isolated from children with diarrhea in Iran. Methods: A total of 200 stool specimens were obtained from children under 5 years during July 2012 to July 2013. Detection of C. jejuni and C. coli was performed by standard biochemical and molecular methods. The presence of virulence-associated genes and genetic diversity of isolates was examined using PCR and ERIC-PCR analyses. Results: A total of 12 (6%) Campylobacter spp. were isolated from patients including 10 (4.5%) C. jejuni and 2 (1.5%) C.coli. The flaA, cadF and ciaB genes were present in 100% of isolates, while no plasmid of virB11 gene was present in their genome. The prevalence of invasion-associated marker was 100% among C. coli and was not detected in C. jejuni isolates. The distribution of both pldA and the genes associated with cytolethal distending toxin (CDT) was 58.3% in C. jejuni isolates. Seven distinct ERIC-PCR profiles were distinguished in three clusters using ERIC-PCR analysis. Genotyping analysis showed a relative correlation with geographic location of patients and virulence gene content of isolates. Conclusion: To our knowledge, this is the first molecular survey of Campylobacter spp. in Iran concerning genotyping and virulence gene content of both C. jejuni and C. coli. ERIC-PCR revealed appropriate discriminatory power for clustering C. jejuni isolates with identical virulence gene content. However, more studies are needed to clearly understand the pathogenesis properties of specific genotypes. Key Words: Campylobacter jejuni, Campylobacter coli, Ddiarrhea, Virulence factors     

西农538 LMW-GS基因的克隆、原核表达及功能鉴定

为了探索普通小麦品种西农538的LMW-GS对面粉加工品质的影响,根据NCBI中已公布的LMW-GS基因序列,设计了一对特异性引物,从西农538基因组DNA中克隆出LMW-GS基因后,对其进行原核表达及掺粉试验。序列分析表明,克隆得到的LMW-GS基因序列(GenBank登录号为KX452081)有单一完整的开放阅读框,编码区长915bp,无内含子。同源性比对及进化树分析发现,该基因属于Glu-D3、Type V(Group 10)、m型、C组LMW-GS基因。SDS-PAGE和Western blot分析表明,该基因原核表达成功。微量掺粉试验表明,诱导表达的蛋白对小麦面粉加工品质有负效应。     

First Report of a Peroxiredoxin Homologue in Jellyfish: Molecular Cloning,Expression and Functional Characterization of CcPrx4 from Cyanea capillata

We first identified and characterized a novel peroxiredoxin (Prx), designated as CcPrx4, from the cDNA library of the tentacle of the jellyfish Cyanea capillata. The full-length cDNA sequence of CcPrx4 consisted of 884 nucleotides with an open reading frame encoding a mature protein of 247 amino acids. It showed a significant homology to peroxiredoxin 4 (Prx4) with the highly conserved F-motif (⁹³FTFVCPTEI¹⁰¹), hydrophobic region (²¹⁷VCPAGW²²²), ¹⁴⁰GGLG¹⁴³ and ²³⁹YF²⁴⁰, indicating that it should be a new member of the Prx4 family. The deduced CcPrx4 protein had a calculated molecular mass of 27.2 kDa and an estimated isoelectric point of 6.3. Quantitative real-time PCR analysis showed that CcPrx4 mRNA could be detected in all the jellyfish tissues analyzed. CcPrx4 protein was cloned into the expression vector, pET-24a, and expressed in Escherichia coli Rosetta (DE3) pLysS. Recombinant CcPrx4 protein was purified by HisTrap High Performance chelating column chromatography and analyzed for its biological function. The results showed that the purified recombinant CcPrx4 protein manifested the ability to reduce hydrogen peroxide and protect supercoiled DNA from oxidative damage, suggesting that CcPrx4 protein may play an important role in protecting jellyfish from oxidative damage.     

Controlled expression of cholera toxin B subunit from Vibrio cholerae in Escherichia coli

The ctxB gene, the causative agent of cholera epidemic was successfully cloned from V. cholerae in E. coli. The insertion of the gene was confirmed by PCR as well as restriction digestion analyses. The sequencing results for the gene confirmed that the insert was in the correct orientation and in-frame with the P(BAD) promoter and it showed that the gene was 99% homologous to the published ctxB sequence. The CTB protein was successfully expressed in E. coli using the pBAD/His vector system. The expected protein of approximately 14 kDa was detected by SDS-PAGE and Western blot. The use of pBAD/His vector to express the cholera toxin gene in E. coli would facilitate future study of toxin gene products.     

HARDY基因的克隆、原核表达及其植物表达载体的构建

为了给转HARDY(HRD)基因小麦研究奠定基础,利用PCR技术从拟南芥中克隆HRD基因,进行生物信息学分析,预测其编码的蛋白质结构与功能,构建原核表达载体pET28a(+)-HRD,转化大肠杆菌BL21(DE3),IPTG诱导蛋白的表达,同时构建pBin-HRD植物表达载体。测序分析结果表明,克隆的HRD与NCBI发布的拟南芥核苷酸序列的同源性为99%,其开放阅读框长555bp,可编码184个氨基酸,具有许多重要功能位点;成功构建了原核表达载体pET28a(+)-HRD和植物表达载体pBin-HRD,诱导表达出大小约为23.3kD的蛋白,与理论值相近。     

Recombinant Production of a Novel Fusion Protein: Listeriolysin O Fragment Fused to S1 Subunit of Pertussis Toxin

Background:Some resources have suggested that genetically inactivated PTs bear a more protective effect than chemically inactivated products. This study aimed to produce new version of PT, by cloning an inactive PTS1 in a fusion form with N-terminal half of the LLO pore-forming toxin. Methods:Deposited pdb structure file of the PT was used to model an extra disulfide bond. Codon-optimized ORF of the PTS1 was used to make recombinant constructs of PTS1 and LLO-PTS1 in the pPSG-IBA35 vector. The recombinant PTS1 and LLO-PTS1 proteins were expressed in BL21 DE3 and SHuffle T7 strains of E. coli and purified by affinity chromatography. Cytotoxic effects of the recombinant proteins were examined in the MCF-7 cell line. Results:The purity of the products proved to be more than 85%, and the efficiency of the disulfide bond formation in SHuffle T7 strain was higher than BL21 DE3 strain. No cytotoxicity of the recombinant proteins was observed in MCF-7 cells. Soluble recombinant PTS1 and LLO-PTS1 proteins were produced in SHuffle T7 strain of E. coli with high efficiency of disulfide bonds formation. Conclusion:The LLO-PTS1 with corrected disulfide bonds was successfully expressed in E. coli SHuffle T7 strain. Due to the safety for human cells, this chimeric molecule can be an option to prevent pertussis disease if its immunostimulatory effects would be confirmed in the future. Key Words: Adjuvant, Cloning, Fusion protein, Pertussis toxin     

Antibiotic Resistance Pattern of Different Escherichia coli Phylogenetic Groups Isolated from Human Urinary Tract Infection and Avian Colibacillosis

Background : The emergence and propagation of different phylogenetic groups of antimicrobial-resistant E. coli have become a worldwide health concern in human and veterinary medicine. Therefore, the evaluation of the phylogenetic distribution of antibiotic-resistant E. coli is important for therapeutic and economic purposes. The aims of this study were to determine phylogenetic groups and patterns of antibiotic resistance of E. coli strains isolated from human urinary tract infection and avian colibacillosis. Methods: A total of 50 E. coli isolates (25 from human urinary tract infection and 25 from avian colibacillosis) were characterized by culture and assigned as different phylogenetic groups (A, B1, B2, and D) by triplex PCR assay. Kirby-Bauer disk diffusion method was used to assess the susceptibility of all isolates to ten antibiotics. Results: Results showed that the majority of the human and poultry isolates belonged to phylogenetic groups A and B2 and phylogenetic group B1 of the avian pathogenic strain isolates were the most drug-resistant isolates. Most of the isolates were resistant to at least five antibiotics, and multiple drug resistance was observed in 98% of E. coli isolates. A high degree of resistance was seen against penicillin and erythromycin. Conclusion: According to the results of this study, multidrug-resistance among isolates and high relation between phylogenetic groups and resistance in both human and poultry isolates were observed. Key Words: Escherichia coli, Avian colibacillosis, Phylogenetic grouping     

Cloning and Expression of a LMW-i Glutenin Gene

Endosperm-specific low-molecular-weight (LMW) glutenins are an important component of the polymeric gluten and, as such, play a key role in end-use functionality. Reports of N-terminal amino acid sequences of LMW glutenin fractions revealed that they have either a methionine or a serine residue at the first position of the mature peptide. These subunits were therefore called LMW-m and LMW-s type glutenins. A gene that is predicted to encode a LMW glutenin subunit having an isoleucine amino acid residue at position one of the mature protein was amplified and cloned from extra strong bread wheat cultivar Glenlea (pGH3.1). The predicted N-terminal sequence of this gene is truncated as compared to the m-type and s-type. The gene still codes for the expected number of eight cysteine residues which are all located in the C-terminal region. We propose to call it LMW-i based on the same nomenclature. Analysis of 277 doubled haploid lines derived from a single cross showed perfect co-segregation of the cloned PCR fragment with a rare LMW glutenin called LMW-50. The gene was subcloned in an expression vector and the protein was expressed in E. coli. Western blot analysis using a prolamin-specific monoclonal antibody confirmed the co-migration of the cloned protein with LMW-50 from Glenlea.     

Heterologous expression and protein engineering of wheat gluten proteins

A range of systems are available for the production of recombinant wheat gluten proteins, from simple and widely used systems based on Escherichia coli to more sophisticated eukaryotic systems in yeasts or cultured insect cells. The characteristics of these systems are summarised and their advantages and disadvantages for application to wheat gluten proteins discussed. We then review the applications of heterologous expression systems to the synthesis and characterisation of wheat gluten proteins, including the production of wild type and mutant proteins for structure–function studies. We also discuss the use of heterologous expression to establish model systems including perfect repeat peptides based on motifs present in gliadins and glutenin subunits and ‘analogue glutenin proteins’ based on C hordein of barley. It is concluded that the pET series of vectors and E. coli are suitable for most applications, providing high-level expression and being rapid and easy to use.     

Yield Traits and Associated Marker Segregation in Elite Introgression Lines Derived from O. sativa × O. nivara

Introgression lines(ILs)derived from interspecific crosses are a source of new genetic variability.A total of 55 ILs derived from two crosses Swarna×O.nivara IRGC81848(population A)and Swarna×O.nivara IRGC81832(population B)were characterized for yield and yield-related traits/QTLs.Segregation of 103 simple sequence repeat(SSR)markers associated with yield-related QTLs was studied.Population A showed an average of 12.6%homozygous O.nivara alleles and population B showed 10.6%.Interestingly,three SSR markers,RM223,RM128 and RM517,showed conspicuous pattern of segregation.The distribution of parental alleles at three loci RM223,RM128 and RM517 linked to yield-related traits was unique.These markers flanked to several yield-related QTLs.RM223,flanking to qyld8.3,was heterozygous in almost all the 55 ILs except in IL10-3S and IL131S.RM128 on chromosome 1 and RM517 on chromosome 3 were mutually exclusive in 46 out of 55 ILs.These 46 ILs showed either of the marker allele RM128 or RM517 from O.nivara but not both.IL166S had both RM128and RM517 from O.nivara and the other ILs showed homozygous Swarna allele at RM517 except IL65S.Population structure assigned the 55 ILs to three sub-populations based on their genomic diversity.IL65S,IL166S,IL248S,IL7K and IL250K showed high yields in multi-location trials,and IL248S was released for cultivation as DRRDhan 40.     

Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli

α-Conotoxin LvIA is derived from Conus lividus, native to Hainan, and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. In this study, an efficient approach for the production of recombinant α-Conotoxin LvIA is described. Tandem repeats of a LvIA gene fragment were constructed and fused with a KSI gene and a His₆ tag in a Escherichia coli (E. coli) expression vector pET-31b(+). The recombinant plasmids were transformed into E. coli and were found to express well. The KSI-(LvIA)_n-His₆ fusion protein was purified by metal affinity chromatography and then cleaved with CNBr to release recombinant LvIA (rLvIA). High yields of fusion protein ranging from 100 to 500 mg/L culture were obtained. The pharmacological profile of rLvIA was determined by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing rat nAChR subtypes. The rLvIA antagonized the α3β2 nAChR subtype selectively with a nano-molar IC₅₀. The rLvIA was analgesic in a mouse hot-plate test model of pain. Overall, this study provides an effective method to synthesize α-conotoxin LvIA in an E. coli recombinant expression system, and this approach could be useful to obtain active conopeptides in large quantity and at low cost.     

Characterization of an Alginate Lyase,FlAlyA, from Flavobacterium sp. Strain UMI-01 and Its Expression in Escherichia coli

A major alginate lyase, FlAlyA, was purified from the periplasmic fraction of an alginate-assimilating bacterium, Flavobacterium sp. strain UMI-01. FlAlyA showed a single band of ~30 kDa on SDS-PAGE and exhibited the optimal temperature and pH at 55 °C and pH 7.7, respectively. Analyses for substrate preference and reaction products indicated that FlAlyA was an endolytic poly(mannuronate) lyase (EC 4.2.2.3). A gene fragment encoding the amino-acid sequence of 288 residues for FlAlyA was amplified by inverse PCR. The N-terminal region of 21 residues except for the initiation Met in the deduced sequence was predicted as the signal peptide and the following region of six residues was regarded as propeptide, while the C-terminal region of 260 residues was regarded as the polysaccharide-lyase-family-7-type catalytic domain. The entire coding region for FlAlyA was subjected to the pCold I—Escherichia coli BL21(DE3) expression system and ~eight times higher yield of recombinant FlAlyA (recFlAlyA) than that of native FlAlyA was achieved. The recFlAlyA recovered in the periplasmic fraction of E. coli had lost the signal peptide region along with the N-terminal 3 residues of propeptide region. This suggested that the signal peptide of FlAlyA could function in part in E. coli.     

Identification of KCS gene family and functional analysis of FAE-like genes from Malania oleifera

KCS(3-ketoacyl-CoA synthase)is the key enzyme catalyzing the first step of very long chain fatty acid(VLCFA)biosynthesis.Studies showed that different KCSs possessed different substrate preference.Malania oleifera are abundance of VLCFAs in its mature seeds,especially the nervonic acid,which is essential for human health.In this study,we identified and characterized 18 KCS genes in M.oleifera genome.Phylogenetic analysis showed that these KCS genes were classified into four subfamilies,including two FAE-like,six KCS-like,eight FDH-like and two CER6.We concentrated on the functional role of two FAE-like genes,Maole003085.T1 and Maole004215.T1 which encoded predicted amino acid residues of 516 and 518 in protein,respectively.Multiple sequence alignment showed that their two proteins contained the known and conserved active sites among FAE-like subfamily.Upon heterologous expression in wild type yeast(Saccharomyces cerevisiae)INVSc1,we found that Maole004215.T1 could produce four new fatty acids including C22:0/C22:1 and C24:0/C24:1,but Maole003085.T1 only produced C22:1.Besides,upon heterologous expression in mutant yeast BY4741-△elo3,we found the Maole003085.T1 could produce C24:0 and C26:0,while the Maole004215.T1 could catalyze the formation of fatty acids C24:0,C26:0 and C28:0.These results showed Maole003085.T1 and Maole004215.T1 had fatty acid elongation activity in yeast,and possessed different substrate preference in the production of different VLCFAs.Interestingly,we found Maole004215.T1 could produce nervonic acid in yeast,which provides molecular basis on the genetic improvement and genic engineering for producing nervonic acid resources by using biotechnological methods.     

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is T3SS. PcrV is an important structural protein of the T3SS. Methods:In the current investigation, a recombinant scFv mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results:Increased efficiency was achieved by EnBase® compared to LB broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC₅₀ for anti-PcrV scFv protein was determined to be approximately 7 μg/mL. Conclusion:Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+). Key Words: Fed Batch, recombinant protein, Pseudomonas aeruginosa, scFv