Development of DNA-Designed Avian IgY Antibodies for Detection of Mycobacterium avium subsp. paratuberculosis Heat Shock Protein 70 (Hsp70) and Anti-Hsp70 Antibodies in the Serum of Normal Cattle

Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (MAPHsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAPHsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1-MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1-MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAPHsp70, and in a sandwich ELISA to assess the prevalence of anti-MAPHsp70 antibodies in cattle serum. Western blotting results indicate the expression of rMAP hsp70 in COS-7 cells and sandwich ELISA could detect anti-MAPHsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-MAPHsp70 could demonstrate the effective production of anti-MAPHsp70 IgY antibodies. Monospecific anti-MAPHsp70 antibody generated in chickens is useful for detection of MAPHsp70 peptide in cell culture and MAP lysate.     

Co-administration of Interleukin-2 Enhances Cellular and Humoral Immune Responses to HIV Vaccine DNA Prime／MVA Boost Regime

Interleukine-2 (IL-2) is a growth factor for antigen-stimulated T lymphocytes and is responsible for T-cell clonal expansion after antigen recognition. It has been demonstrated that DNA vaccine-elicited immune responses in mice could be augmented substantially by using either an IL-2 protein or a plasmid expressing IL-2. Twenty mice, divided into four experimental groups, were immunized with: (1) sham plasmid; (2) HIV-1 DNA vaccine alone; (3) HIV-1 DNA vaccine and IL-2 protein; or (4) HIV-1 DNA vaccine and IL-2 plasmid, separately. All the groups were immunized 3 times at a 2-week interval. Fourteen days after the last DNA vaccine injection, recombinant MVA was injected into all the mice except those in group 1. ELISA and ELISPOT were employed to investigate the effect of IL-2 on DNA vaccine immune responses. The obtained results strongly indicate that the efficacy of HIV vaccine can be enhanced by co-administration of a plasmid encoding IL-2.     

An Exopolysaccharide from Cultivated <Emphasis Type="Italic">Cordyceps sinensis</Emphasis> and its Effects on Cytokine Expressions of Immunocytes

The exopolysaccharide (EPS) is a polysaccharide from cultivated Cordyceps sinensis, which possesses immunomodulatory and antitumor effects, was purified by DEAE-32 cellulose and Sephadex G-200 gel. The preliminary characters of EPS were analyzed by IR and GC, and the molecular weight was estimated by gel filtration. The effect of EPS on proliferation ability of lymphocytes from ICR mice was assayed by MTT method. The mRNA and protein expression levels of several cytokines in spleen and thymus cells were detected by RT-PCR and ELISA. The results showed that EPS consists of mannose, glucose, and galactose in a ratio of 23:1:2.6. Its molecular weight is about 1.04 × 10⁵. EPS elevated proliferation ability of spleen lymphocytes only at 100 μg/ml after 48 h treatment. Tumor necrosis factor alpha (TNF-α), interferon-α (IFN-γ), and interleukin-2 (IL-2) mRNA levels in splenocytes and thymocytes were increased after EPS treatment for 2, 4, 8, or 20 h. EPS also significantly elevated splenic TNF-α and IFN-γ protein expressions at 100 μg/ml and increased thymic TNF-α and IFN-γ protein levels at 50 and 100 μg/ml. These data indicated that EPS may stimulate cytokine expressions of immunocytes.     

Expression, refolding, and characterization of GFE peptide-fused human interferon-α2a in Escherichia coli

Interferon-α2a (IFN-α2a) has been used for the treatment of various viral infections and cancers for many years. However some untolerable side effects have limited its application in some aspects. To evaluate whether or not an oligopeptide containing GFE motif can home human IFN-α2a to specific tissues, a fusion gene was constructed by fusing the coding sequence of GFE peptide (CGFECVRQCPERC), which was screened from phage display peptide library, to the 3′ end of human IFN-α2a gene by recombinant DNA technique. Fusion protein rhIFN-α2a-GFE was expressed in Escherichia coli as inclusion bodies using a T7 RNA polymerase expression system, pET-22b, refolded through dialysis and purified to homogeneity to >95% of purity by affinity chromatography. Characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting demonstrated the authenticity of the fusion protein. Purified rhIFN-α2a-GFE was found to be functionally active in terms of its antiviral activity for about 2.5×10⁸ IU/mg in vitro. Yields of the purified fusion protein were about 200 mg/L of culture medium. Tissue distribution assay in mouse showed that at 30 min IFN-α2a could be enriched sevenfold higher in lung in the targeted IFN group of mice than in the standard IFN group of mice, and last for a long time. At 1 h, IFN-α2a in the targeted IFN group was still 4.02-fold higher than that in the standard group. This confirmed that GFE peptide has the ability to selectively deliver its fusion partner IFN-α2a to lungs. The results also showed that the IFN-α2a-GFE could be specifically enriched in kidney and liver. Its distribution in kidney was concordant with the finding of GFE receptor, MDP, in kidney. However, the IFN-α2a-GFE in liver may imply some significance in pharmacology and toxicology.     

Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application

Adenosine deaminase (ADA) is currently used as a diagnostic marker for tuberculous pleuritis. Although ADA has been suggested as a potential marker for several types of cancer, the importance of each of ADA isoforms as well as their levels and enzymatic activities in tumors need to be further investigated. Herein we developed avian immunoglobulin Y highly specific to human ADA via hens immunization with calf adenosine deaminase. The obtained antibodies were used for the development of a sensitive double-egg yolk immunoglobulin (IgY) sandwich ELISA assay with an ADA detection limit of 0.5 ng/ml and a linearity range of up to 10 ng/ml. Specific, affinity-purified IgYs were able to recognize human recombinant ADA and ADA present in human cancer cell lines. In addition, antigen-specific IgY antibodies were able to inhibit catalytic activity of calf ADA with an IC₅₀ value of 47.48 nM. We showed that generated IgY antibodies may be useful for ADA detection, thus acting as a diagnostic agent in immunoenzymatic assays.     

Production of antibodies from chicken egg yolk to phenyl- N -methylcarbamate insecticides for analytical purposes

With the aim of developing polyclonal antibodies binding as many phenyl-N-methylcarbamate insecticides (PNMCs) as possible, IgY antibodies were produced in laying hens. Two haptens (3-(2,6-dimethyl-4-(methylcarbamoyl)phenylaminocarbonyl)propanoic acid and 4-((2,6-dimethyl-4-(methylcarbamoyl)phenylaminocarbonyl)-3,3-dimethyl)butanoic acid) were synthesized preserving the major structural features of PNMCs, by a novel synthetic pathway. These haptens differed only in the spacer arm incorporated. Immunizing antigen and coating antigen were prepared by coupling the first hapten with bovine serum albumin and the second with thyroglobulin, from porcine thyroid glands, respectively. The titre and maturation increase in the developed antibodies, in the egg yolk, were assessed by non-competitive ELISA. Avidity and cross-reactivity of the antibodies with selected pesticides were estimated by means of competitive ELISA. The produced IgYs exhibited a high binding capacity to carbaryl, trimethacarb, metolcarb, aminocarb, and promecarb. These antibodies can be used for immunosorbent preparation for analytical purposes.     

Passive Immunization of Crayfish (<Emphasis Type="Italic">Procambius clarkiaii</Emphasis>) with Chicken Egg Yolk Immunoglobulin (IgY) Against White Spot Syndrome Virus (WSSV)

White spot syndrome virus (WSSV) is a major cause of mortality in shrimp lacking a true adaptive immune response. In this study, high activity egg yolk immunoglobulin (IgY) against WSSV for passive immunization of crustaceans was already prepared as crude and purified product, while an indirect enzyme-linked immunosorbent assay test was used for quality control of IgY activity. The effectiveness of IgY of intramuscular injection, oral administration, and immersion was investigated in crayfish (Procambius clarkiaii) against WSSV. The result showed that the groups treated with IgY from inactivated WSSV and DNA vaccine were, respectively, 20% and 80% mortality, which were significant difference in survival rates (P < 0.05) from the positive control groups. The groups in diet added 10% egg yolk powder and 1% IgY power showed 53.3% and 67.7% mortality, respectively, and the immersion showed 46.7% mortality, which have significantly different compared to the positive groups (P < 0.05). These results indicated passive immunization of specific IgY antibodies through intramuscular injection, oral administration, and immersion have effective to protect crayfish against WSSV. It is noteworthy that IgY as feed additive and immersion solution is useful and feasible methods in practical work. Thus, our results suggest that the passive immunization of crayfish with IgY against WSSV will have potential development to prevent and control WSSV in practical culture.     

混合抗体的亲和色谱分离策略

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。     

Functional Expression of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Protective Antigen in <Emphasis Type="Italic">E. coli</Emphasis>

The protective antigen (PA) of Bacillus anthracis is a potent immunogen and an important candidate vaccine. In addition, it is used in monitoring systems like enzyme-linked immunosorbent assay to assess antibodies against PA in immunized subjects. The low level of PA production in B. anthracis and the difficulty of separating it from other bacterial components have made the researchers do different studies with the aim of producing recombinant PA (rPA). In this study, to produce rPA as a recombinant protein vaccine, the partial sequence of protective antigen of B. anthracis, amino acids 175–764, as a potent immunogenic target was inserted in pET21b(+). This is a prokaryotic plasmid that carries an N-terminal T7.tag sequence. The integrity of constructed plasmid was confirmed using restriction enzyme mapping. rPA was expressed after induction with isopropyl β-d-1-thiogalactopyranoside in Escherichia coli BL21. Purification of rPA was done with an affinity system using anti T7.tag antibody. Electrophoresis and Western blotting confirmed the specificity of the expressed protein. BALB/c mice were immunized with obtained PA protein and evaluation of specific immunoglobulin G antibodies against PA in sera using Western blotting method and showed that rPA is immunogenic. The challenge of immunized mice with virulent strain of B. anthracis showed that rPA is functional to protect against pathogenic strain.     

Expression and Characterization of HIV-1 Envelope Glycoprotein in Pichia Pastoris

To obtain a sufficient amount of glycoprotein for further studying the structure and function of HIV-1 envelope glycoprotein, amplified and modified HIV-1 envelope glycoprotein gene which recombined subtypes（850 amino acids） from Guangxi in China was inserted into Pichia pastoris expression vector pPICZαB; then the recombinant plasmid was transported into the yeast cells to induce the expression of Env protein with methanol. The results of SDS-PAGE and Western blot indicate that the envelope glycoprotein could be expressed in Pichia pastoris with productions of a 120000 glycoprotein and a 41000 glycoprotein, which showed satisfactory immunogenicity by indirect ELISA.     

Direct antigen capture by soluble scFv antibodies

For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag.     

Application of recombinant phage display antibody system in study of Codakia orbicularis gill proteins

We used the recombinant phage display antibody system (RPAS) to obtain chimeric single-chain fragment variable (ScFv) antibodies to gill proteins of the white clam Codakia orbicularis (Linné, 1758). After three rounds of selection on immunotubes loaded with total gill protein extract, recombinant phages exhibiting antibodies to gill proteins were isolated and tested by enzyme-linked immunosorbent assay (ELISA). Clones exhibiting a high affinity for the mollusk proteins were selected for production of soluble ScFv antibodies, which were purified for subsequent analysis. ScFv antibodies exhibited a reaction specific for a protein whose molecular mass was about 15,000 Daltons and that was detected by the antigen capture technique followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting.     

Application of fast solvent extraction processes to studies of exotic nuclides

The nucleus²³Na has been investigated by studying the primary γ-rays emitted from 53 keV neutron capture in it using a high resolution and high efficiency (100%) HPGe detector and NaI(T1) detector for anti-Compton. 24 primary γ-rays were placed in the²⁴Na, in which 3 primary γ-rays were new ones from a (n, γ) reaction, and reported for the first time. In order to obtain an exact energy calibration within 7 MeV,⁵⁶Fe(n,γ)⁵⁷Fe reaction was used at thermal neutron energy. Intensity calibration was obtained from the²⁷Al(p,γ)²⁸Si reaction atE _p=2046 keV. The neutron binding energy of²⁴Na was determined to be 6959.75 keV.     

Immunoassay for the detection of lead ions in environmental water samples

A rapid, simple, and reliable competitive immunoassay was developed for measurement of lead ions Pb(II) in environmental samples. Avian antibodies were produced against Pb(II). Since lead ions are too small to elicit an immune response, the metal was coupled to protein carrier Bovine serum albumin (BSA) using a bifunctional chelator 1-(4-isothiocyanobenzyl) ethylenediamine N,N,N′,N′-tetra acetic acid (ITCBE). Poultry birds (layers) were immunised with this Pb(II)–ITCBE–BSA immunoconjugate and the avian antibodies (IgY) isolated from egg yolk recognised Pb(II)-ITCBE complexes as capture reagent and a Pb(II)–ITCBE conjugate of Alkaline phosphatase as an enzyme label. Antibody reaction was optimised for different concentrations of antigen and antibody dilutions. Cross reactivity with other metals were below 1% in competitive ELISA. The IC₅₀ value of this avian antibody was 0.19?µg?mL^?1. The detection range and the detection limit were 0.02–1000?µg?mL^?1and 0.2?µg?mL^?1, respectively.     

具有生物活性的人带3蛋白胞质片段融合蛋白的克隆、表达与纯化

用PCR法从质粒pHB3中扩增了人红细胞带3蛋白胞质片段(CDB3)基因.PCR产物经限制性内切酶切割后与多克隆位点处带有编码6个组氨酸序列的高效表达载体pET28b连接,构建为重组子pCDBHistag.重组子经酶切及序列测定后在大肠杆菌BL21(DE3)中获得高效表达,可溶性目的蛋白占菌体总蛋白的40%左右.C端带有6个连续组氨酸的带3蛋白胞质片段作为融合蛋白不仅可以降低宿主菌蛋白酶对其水解程度,而且简化了目的蛋白的纯化过程.经一步螯合Ni²⁺的亲和层析获得了电泳纯的带3蛋白胞质片段融合蛋白.活性测定结果表明,带3蛋白胞质片段融合蛋白能够抑制醛缩酶(Aldolase)活性的70%,与文献报道的人红细胞内带3蛋白胞质片段具有相同的功能.     

Regulation and role of arachidonate cascade during changes in life cycle of adipocytes

Although some eicosanoids serve as potent natural ligands to activate peroxisome proliferator-activated receptor (PPARγ), the ability of adipocytes to produce eicosanoids and regulate PPARγ remains unclear. Here, adipogenic 3T3-L1 cells were employed to determine the gene expression of isoforms of biosynthetic enzymes in the arachidonate cyclooxygenase (COX) pathway and the synthesis of prostaglandins (PGs). The expression of COX-2 was induced transiently in a biphasic manner upon the triggering of the differentiation and maturation phases while COX-1 was constitutive. The exclusive expression of lipocalin-type PGD synthase occurred and gradually increased during the maturation process along with the stable expression of PPARγ. Moreover, we confirmed the formation of PGD₂ from arachidonic acid by the mature adipocytes, suggesting conversion into PGJ₂ derivatives. Even though cytosolic and membrane-associated subtypes of PGE synthase were expressed at relatively constant levels, the ability of preadipocytes to produce PGE₂ was greater than that of mature adipocytes in the cell response. The treatment of the mature adipocytes with exogenous PGD₂, 15-deoxy-Δ^12,14-PGJ₂ and PGE₂, in the presence of aspirin, enhanced the adipogenesis. These findings imply the specific roles of prostanoids produced by the mature adipocytes in the maintenance of terminal differentiation through an autocrine control mechanism.     

Chitosan–Alginate Microcapsules for Oral Delivery of Egg Yolk Immunoglobulin (IgY): Effects of Chitosan Concentration

In our previous study, chitosan–alginate microcapsules were developed to protect egg yolk immunoglobulin (IgY) from gastric inactivation. The present study was undertaken to determine the effect of chitosan concentration (0–0.8%; w/v) on various properties of the microcapsules in order to produce the optimum chitosan–alginate microcapsules for use in the oral delivery of IgY. The properties investigated included microcapsule morphology, loading capacity for IgY (expressed as the IgY loading percentage, w/w, of microcapsules), encapsulation efficiency (EE%), in vitro gastroresistance, and IgY release. IgY loading percentage and EE% were both highest at 0.2% (w/v) chitosan, and, above this level, further increases were not observed. The stability of IgY in simulated gastric fluid (pH 1.2) was significantly improved by encapsulation in alginate microcapsules (IgY retained 43.5% of its activity) and was further improved by including chitosan at any of the chitosan concentrations assessed (IgY retained an average of 69.4% activity) although there was no difference in protection of gastric inactivation among concentrations of chitosan varying from 0.05% to 0.8% (w/v). Higher chitosan concentrations (i.e., ≥0.2%; w/v) prolonged the release of IgY from the microcapsules during simulated intestinal fluid incubation (pH 6.8). However, above the 0.2% (w/v) level, no significant differences were observed. We conclude that the optimum chitosan concentration for microencapsulation is 0.2% (w/v).     

Thermal behavior and signature patterns of human cytokine and soluble cytokine receptors investigated using dielectric thermal analysis and thermogravimetry

Cytokines are small regulatory proteins secreted mostly by cells of the immune system. Cytokines participate in anti-inflammatory and pro-inflammatory processes in the body and in responses to host exposure to pathogens. In this study, the thermal behavior of human recombinant cytokines and soluble cytokine receptors; IFNγ, TNFα, IL-1 receptor antagonist, soluble TNF-receptor types 1 and 2, and sIL-2 receptor α were analyzed by dielectric thermal analysis at 37 °C and by thermogravimetry. Measurements were performed at a frequency range of 0.1–300,000 Hz. Permittivity and loss factor measurements were used to calculate mobility of charges (tan δ values) in the proteins from Debye plots. Peak frequencies and polarization times were used to determine dielectric signatures for each cytokine and receptor. Peak frequencies and polarization times were obtained for each cytokine and receptor analyzed. Detection of unique dielectric signatures of the proteins will aid development of sensitive dielectric sensors capable of detecting cytokines and soluble cytokine receptors in various human samples for malaria diagnosis.     

Rapid determination of tungsten in rocks using183mW

A rapid method has been developed for the determination of tungsten, especially in rocks. The reaction¹⁸²W(n, γ)^183mW (T=5.3 sec), with a thermal neutron capture cross-section of 0.5 b was used. The samples were irradiated in the fast pneumatic system of the FRM, which is described briefly. The low-energy γ-rays of the isomer^183mW were measured by a high-resolving Ge(Li) detector. The sensitivity of the method is 0.1 mg tungsten with an accuracy of about 5%; the minimum concentration is 0.1–0.2% W in geological samples. The analysis time is 2 min per sample.