Human Granulocyte Colony-Stimulating Factor (hG-CSF) Expression in Plastids of Lactuca sativa

Background: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. Methods: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. Results: hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. Conclusions: This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.Key Words: Plastid, Human granulocyte colony-stimulating factor (hG-CSF), Biolistics, Gene targeting     

Functional Recombinants Designed from a Fetuin/Asialofetuin-Specific Marine Algal Lectin,Rhodobindin

Plant lectins have attracted much attention for biomedical applications including targeted drug delivery system and therapy against tumors and microbial infections. The main problem of using lectins as a biomedical tool is a batch-to-batch variation in isoforms content. The production of lectins using recombination tools has the advantage of obtaining high amounts of proteins with more precise properties, but there are only a handful of functional recombinant lectins presently available. A fetuin/asialo-fetuin specific lectin, Rhodobindin, has unique tandem repeats structure which makes it useful in exploiting for recombinant lectin. We developed three functional recombinant lectins using E. coli expression system: one from full cDNA sequence and two from fragmentary sequences of Rhodobindin. Hemagglutinating activity and solubility of the recombinant lectins were highest at OD 0.7 cell concentration at 20 °C. The optimized process developed in this study was suitable for the quality-controlled production of high amounts of soluble recombinant lectins.     

Expression of biologically active measles virus hemagglutinin glycoprotein by a recombinant baculovirus

In this study, one of the measles virus membrane proteins, named hemagglutinin (H) which has a key role in tropism, receptor binding, hemagglutinating activity and also induction of protective immunity against viral infection, was expressed by the baculovirus expression system using specific plasmid (pDONR221) to produce entry clone. Measles Virus (AIK-C strain) genome was extracted from infected Vero cells. H gene was amplified by specific primers during RT-PCR reaction and inserted into the specific plasmid (pDONR221) using BP recombination reaction. Recombinant baculovirus harboring H gene was consequently constructed by LR reaction. Insect cells (Sf9) were infected with recombinant baculovirus. In order to increase viral titer, recombinant baculoviruses were passaged four times in Sf9 cells. Synthesis of H protein was verified by SDS-PAGE, western-blot and indirect immunoflourescene using goat polyclonal antibody against Measles Virus. The results showed that H protein was partially glycosylated, but it appeared to be active in hemagglutination assay.     

Designing E1 deleted adenoviral vector by homologous recombination

BACKGROUND: Adenoviruses are used extensively to deliver genes into mammalian cells, particularly where there is a requirement for high-level expression of transgene products in cultured cells, or for use as recombinant viral vaccines or in gene therapy. In spite of their usefulness, the construction of adenoviral vectors (AdV) is a cumbersome and lengthy process that is not readily amenable to the generation of large collection of clones. METHODS: In this project, to delete E1 gene in adenovirus, an adenoviral plasmid containing lateral sites of E1 region of adenovirus was made and recombination in the 293A cells between the homologous region of this linearized plasmid and the adenovirus genome resulted in the formation of the complete adenoviral recombinant. RESULTS: This recombination resulted in loss of E1 region and we constructed a recombinant adenovirus type 5 vector that E1 gene was deleted by homologous recombination. CONCLUSION: Homologous recombination is more easy and fast technique in the production of AdV.     

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.     

Effects of Peptone Supplementation in Different Culture Media on Growth,Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles

Background:

The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood.

Methods:

In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes.

Results:

In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium.

Conclusion:

The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.Key Words: CHO cells, Culture media, Peptones, Recombinant proteins     

Cloning and expression of coxsakievirus B3 viral protein-1 in E. coli

BACKGROUND: Viral protein-1 (VP1) is a major capsid protein of Coxsakievirus B3 (CVB3) that plays an important role in directing viruses towards permissive cells and acts as a main antigenic site of the virus in eliciting of host immune response, hence it seems VP1 can be considered as a vaccine candidate against CVB3 infection. In this study, cDNA of VP1 was prepared, cloned into pET expression vector and the recombinant protein (VP1) was over expressed in E. coli. METHODS: The viruses were grown in suspension cultures of Vero cells with an input virus multiplicity of 10-50 plaque-forming units/cell. After observing complete cytopathic effect, the total RNA (cells and virus) was prepared for RT-PCR and by using specific primers, VP1 cDNA was amplified and ligated into pET vectors (32 a and 28 a). The recombinant vector was transferred into competent E. coli (BL-21) and after selection of proper colony, which carried correct cDNA within the vector; cells were cultured and induced with isopropyl B-D-thiogalactopyranoside, in order to express protein (VP1). The cultures were tested for presence of VP1 by SDS-PAGE and Western-Blotting analysis. RESULTS: Molecular techniques such as PCR which showed exact defined size of the VP1 (819 bp), restriction digestion and finally immunoblot analysis of over expressed protein; all confirmed the correct cloning and expression of VP1 in this research. CONCLUSION: In this research, full length of VP1 as major capsid protein of CVB3 was over expressed in E. coli which, can be used for further studies, including neutralizing antibody production against CVB3.     

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     

Potato Breeding and Seed Production System Development in Russia

First, an extensive literature review was performed with respect to Potato virus Y (PVY) resistance sources and their further utilization in a breeding programme. On the basis of that review we present a scheme of backcrossing and new cultivar creation on the basis of five detected sources of PVY resistance and one source of Potato virus X resistance. Some cultivar pedigrees are presented reflecting the differences in the breeding strategies. Moreover, results of investigations on some polygenic traits such as field resistance against late blight and starch content are presented. For these purposes progenies were screened for suitable recombinant genotypes which were used in further crossings. Also the results of investigations on resistance to the potato golden nematode and on the selection of cultivars suitable for processing are briefly analysed. We also describe a programme of parallel evaluation of identical hybrid populations in different soils and climatic zones. The development of seed potato production systems facilitated the conditions to improve the quality of potato seed material, to increase potato production and to allow Russia to participate in the international potato market. Systems of virus detection, norms and methods of laboratory tests as well as requirements for quality and tolerance levels of different seed classes (generations) were unified and harmonized with European systems.     

大豆PM2蛋白(LEA3)表达可提高酵母重组子的耐盐性

大豆PM2蛋白属LEA3(late embryogenesis abundant group3)蛋白.本实验以含大豆PM2基因的重组载体pET28a/PM2为模板,PCR法构建酵母表达载体pYES2/PM2,并转化酵母细胞得到重组菌INV/PM2.SDS-PAGE电泳结果表明,INV/PM2重组菌可被诱导表达分子量约为50 kDa的蛋白条带,与目的蛋白的理论分子量(50 kDa)接近.利用液相色谱-电喷雾质谱对酵母重组子表达的重组蛋白进行分析鉴定.分别测定未转基因重组菌INV/pYES2和转PM2基因的重组菌烈V/PM2在无胁迫、和含1.8 mol/LNaCl、2 mol/L山梨糖培养基中的生长曲线.结果表明大豆PM2蛋白的表达对酵母正常生长没有影响.在含高盐的培养基里,转PM2基因酵母INV/PM2的延滞期短于对照菌.这表明PM2蛋白的表达可以提高酵母细胞的耐盐能力.但是在含山梨糖的高渗培养基里,两种菌的生长情况无明显差异.     

重组人血清白蛋白在紫花苜蓿中的转基因表达

为获得高产重组人血清白蛋白的转基因紫花苜蓿"游客"(Medicago sativa L. cv.‘Eureka’)株系用于规模化生产rHSA,构建了rHSA植物表达载体。以紫花苜蓿子叶愈伤组织为受体,通过农杆菌介导法进行遗传转化,筛选出潮霉素抗性植株,提取抗性再生紫花苜蓿植株基因组DNA做PCR鉴定,结果表明重组人血清白蛋白基因片段已整合到紫花苜蓿基因组中;提取转基因植株总蛋白,Western blotting检测结果表明rHSA在转基因植株中成功表达。此结果表明转基因紫花苜蓿可稳定表达rHSA。      

海南PRSV-CP与rhIFNα-2b双价载体构建新策略

为了获得抗病毒能力较强的海南番木瓜转基因新品系，采用海南番木瓜环斑花叶病毒外壳蛋(Papaya Ring Spot Virus-Capsid Protein of Hainan，HPRSV-CP)和重组人源干扰索α-2b(recombinant human interferon α-2b，rhIFNα-2b)为目的基因，利用pBIl21、pCAMBIA2300载体及相关技术(如技术基因内部位点突变套叠PCR)，实施双价基因表达载体的构建策略。本研究成果可为其他多价基因表达载体的构建提供技术依据。     

Linkage analysis and integrated software GAPL for pure-line populations derived from four-way and eight-way crosses

Pure lines derived from multiple parents provide abundant variation for genetic study.However,efficient genetic analysis methods and user-friendly software are still lacking.In this study, we developed linkage analysis methods and integrated analysis software for pure-line populations derived from four-way and eight-way crosses.First, polymorphic markers are classified into different categories according to the number of identifiable alleles in the inbred parents.Expected genotypic probability is then derived for each pair of complete markers, and based on them a maximum likelihood estimate(MLE) of recombination frequency is calculated.An EM algorithm is proposed for calculating recombination frequencies in scenarios that at least one marker is incomplete.A linkage map can thus be constructed using estimated recombination frequencies.We describe a software package called GAPL for recombination frequency estimation and linkage map construction in multi-parental pure-line populations.Both simulation studies and results from a reported four-way cross recombinant inbred line population demonstrate that the proposed method and software can build more accurate linkage maps in shorter times than other published software packages.The GAPL software is freely available from www.isbreeding.net and can also be used for QTL mapping in multi-parental populations.     

Marine Origin Collagens and Its Potential Applications

Collagens are the most abundant high molecular weight proteins in both invertebrate and vertebrate organisms, including mammals, and possess mainly a structural role, existing different types according with their specific organization in distinct tissues. From this, they have been elected as one of the key biological materials in tissue regeneration approaches. Also, industry is constantly searching for new natural sources of collagen and upgraded methodologies for their production. The most common sources are from bovine and porcine origin, but other ways are making their route, such as recombinant production, but also extraction from marine organisms like fish. Different organisms have been proposed and explored for collagen extraction, allowing the sustainable production of different types of collagens, with properties depending on the kind of organism (and their natural environment) and extraction methodology. Such variety of collagen properties has been further investigated in different ways to render a wide range of applications. The present review aims to shed some light on the contribution of marine collagens for the scientific and technological development of this sector, stressing the opportunities and challenges that they are and most probably will be facing to assume a role as an alternative source for industrial exploitation.     

工业化大豆蛋白7S和11S组分分离技术研究进展

该文围绕目前工业化生产11S和7S组分的分离技术成果,结合实验室的分离技术(Guo法),比较了各种分离方法的差异.结果表明:分离提取技术利用的原理几乎均建立在"碱溶酸提"和"冷沉"作用基础之上.Wu首次成功地进行了7S和11S的工业化分离,之后的研究多是对此方法的改进和修饰.而Guo法是唯一采用中性条件抽提的工业化分离方法,在节省了原料碱的同时,避免了蛋白的变性.同时,该文提出了目前工业化生产大豆组分蛋白中存在的问题,虽然不断在加工方式上进行改进,但仍存在步骤复杂、产率低、生产成本高等方面的问题,因此作者进一步对实现工业化分离大豆组分蛋白提出了需要改进和努力的方向.     

Seaweed Protein Hydrolysates and Bioactive Peptides: Extraction,Purification, and Applications

Seaweeds are industrially exploited for obtaining pigments, polysaccharides, or phenolic compounds with application in diverse fields. Nevertheless, their rich composition in fiber, minerals, and proteins, has pointed them as a useful source of these components. Seaweed proteins are nutritionally valuable and include several specific enzymes, glycoproteins, cell wall-attached proteins, phycobiliproteins, lectins, or peptides. Extraction of seaweed proteins requires the application of disruptive methods due to the heterogeneous cell wall composition of each macroalgae group. Hence, non-protein molecules like phenolics or polysaccharides may also be co-extracted, affecting the extraction yield. Therefore, depending on the macroalgae and target protein characteristics, the sample pretreatment, extraction and purification techniques must be carefully chosen. Traditional methods like solid–liquid or enzyme-assisted extraction (SLE or EAE) have proven successful. However, alternative techniques as ultrasound- or microwave-assisted extraction (UAE or MAE) can be more efficient. To obtain protein hydrolysates, these proteins are subjected to hydrolyzation reactions, whether with proteases or physical or chemical treatments that disrupt the proteins native folding. These hydrolysates and derived peptides are accounted for bioactive properties, like antioxidant, anti-inflammatory, antimicrobial, or antihypertensive activities, which can be applied to different sectors. In this work, current methods and challenges for protein extraction and purification from seaweeds are addressed, focusing on their potential industrial applications in the food, cosmetic, and pharmaceutical industries.     

香蕉多酚氧化酶成熟蛋白的原核表达

笔者所在课题组从巴西香蕉中分离到1个PPO基因全长,并进行了原核表达,但结果未表达出目的蛋白。鉴于上述情况,笔者对香蕉PPO全蛋白进行转移肽分析,结果发现其N端含有转移肽,长度为47个氨基酸。切除转移肽并进行香蕉PPO成熟蛋白原核表达,SDS-PAGE电泳检测结果表明,在62 ku处有一条特异的蛋白条带,与预测大小相符;并且进行质谱分析,结果确定重组表达蛋白为香蕉PPO,初步说明转移肽对香蕉PPO体外表达的影响,为进一步研究香蕉PPO功能及在香蕉褐变生理和调控机制中的作用提供理论基础。     

Omega-3-Rich Oils from Marine Side Streams and Their Potential Application in Food

Rapid population growth and increasing food demand have impacts on the environment due to the generation of residues, which could be managed using sustainable solutions such as the circular economy strategy (waste generated during food processing must be kept within the food chain). Reusing discarded fish remains is part of this management strategy, since they contain high-value ingredients and bioactive compounds that can be used for the development of nutraceuticals and functional foods. Fish side streams such as the head, liver, or skin or the cephalothorax, carapace, and tail from shellfish are important sources of oils rich in omega-3. In order to resolve the disadvantages associated with conventional methods, novel extraction techniques are being optimized to improve the quality and the oxidative stability of these high-value oils. Positive effects on cardiovascular and vision health, diabetes, cancer, anti-inflammatory and neuroprotective properties, and immune system improvement are among their recognized properties. Their incorporation into different model systems could contribute to the development of functional foods, with market benefits for consumers. These products improve the nutritional needs of specific population groups in a scenario where noncommunicable diseases and pandemic crises are responsible for several deaths worldwide.     

Cloning,Expression and Characterization of Recombinant Exotoxin A-Flagellin Fusion Protein as a New Vaccine Candidate against Pseudomonas aeruginosa Infections

Background: Infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A (exo A) and killed whole cell. However, none of them are currently available clinically. Methods: In this research, recombinant exoA-flagellin (fliC) fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated. Results: Expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections. Conclusion: These results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Key Words: Pseudomonas aeruginosa, Exotoxin A (exoA), Flagellin (fliC), Vaccines