Partial purification and characterization of bovine fibroblast interferon

Bovine fibroblast interferon (BoF-IFN), produced in primary bovine embryonic kidney cell cultures after priming and infection with bluetongue virus, was purified by controlled pore glass (CPG) chromatography to a specific activity of 10(6) U/mg of protein, with 40% recovery of the original activity. The crude IFN was concentrated more than sevenfold during purification. This proved to be a relatively simple, practical method of obtaining sufficient quantities of partially purified natural BoF-IFN for further studies. The CPG-purified BoF-IFN was further concentrated by sequential ultrafiltration and was analyzed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis (SDS-PAGE). Interferon, recovered from denaturing conditions either by dialysis against phosphate-buffered saline solution or by dilution in cell culture medium containing 10% fetal bovine serum, migrated as a single stainable protein with molecular weight of 21,000 on analytic SDS-PAGE gels. Recovered IFN activity from preparative SDS-PAGE totalled 8.7% of that applied. Attempts to further purify CPG-purified BoF-IFN by zinc chelate affinity chromatography were unsuccessful.     

Beaded agarose affinity chromatography of bovine fibroblast interferon

Bovine fibroblast interferon (BoF-IFN), produced in bovine embryonic kidney cell cultures by priming and infection with bluetongue virus, was partially purified by controlled pore glass chromatography. The partially purified BoF-IFN then was subjected to beaded agarose affinity chromatography in 2 distinct fractions--1 after the addition of 1M NaCl and the other one after the addition of 1.5M NaCl containing 50% ethylene glycol. Analysis of fractions by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis revealed a broad molecular weight range (14,900 to 27,900) for IFN eluted by 1M NaCl, and 2 discrete molecular weight ranges (16,000 to 19,500 and 28,300 to 34,000) for IFN eluted by 1.5M NaCl containing 50% ethylene glycol. The specific activity of the IFN eluted with 1.5M NaCl containing ethylene glycol was 2.85 X 10(6) U/mg of protein, compared with 5.7 X 10(5) U/mg of protein in the controlled pore glass-purified IFN.     

Purification of the third component of canine complement

A rapid, simple procedure has been developed for the purification of the third component (C3) of canine complement. Dog plasma was initially fractionated by precipitation with 4% (w/v) polyethylene glycol (PEG) 4000. The supernatant was depleted of plasminogen using a Sepharose 4B-lysine column, and the effluent was again fractionated with PEG 4000 at 16% (w/v). The precipitate was resuspended and passed over a DEAE-Sephacel column. The fractions containing hemolytically active C3 were pooled, concentrated, and passed over a CM-Sepharose CL-6B column to yield the final purified product. Rabbit anti-whole dog serum identified only one protein in the purified material on immunoelectrophoresis. When injected into a rabbit, the purified product raised an antisera which reacted with only one protein in both whole dog serum and the purified product. Analysis by SDS-PAGE revealed a single band of MW 179,000 +/- 7,000 (+/- 1 S.D.) daltons which, upon reduction with 2-mercaptoethanol, resulted in 2 bands of 114,000 +/- 6,000 daltons and 65,000 +/- 3,500 daltons. Final recovery was 18% with respect to C3 antigen and 9% with respect to C3 hemolytic activity.     

Bovine placental protease specificity toward muscle connective tissue proteins

Enzymes currently used to tenderize meat are not substrate-specific, resulting in extensive myofibrillar protein degradation that often produces an undesirable texture. Bovine placental metalloproteases, which selectively hydrolyze connective tissue proteins while leaving myofibrillar proteins intact, may tenderize meat without causing texture problems. Therefore, our objective was to extract and crudely purify bovine metalloproteases from bovine placenta for possible use as tenderizers in meat systems. Enzymes were extracted from homogenized tissue and purified by ammonium sulfate precipitation. Samples were collected before (crude enzyme) and after gel filtration on a Sephadex G-100 column. Spectrophotometric analysis identified one major peak (filtered enzyme). Gelatin, casein, and type I acid-soluble collagen zymography were used to determine substrate specificity. Beef myofibrillar proteins were incubated with crude and filtered enzyme fractions, enzymes quenched, and substrate degradation visualized using SDS-PAGE. Active gelatinases and collagenases exhibiting molecular weights of 57 to 65 kDa were detected on zymograms. Banding patterns from crude enzyme indicated two enzymes with both gelatinase and collagenase activity and a third enzyme with gelatinase activity only. Banding patterns from filtered enzyme indicated two enzymes with both gelatinase and collagenase activity. Proteolytic activity was not detected with casein, actin, or myosin heavy-chain substrates. Due to specificity for collagen and gelatin, these enzymes may be capable of improving the tenderness of certain cuts relatively high in connective tissue, while avoiding myofibrillar protein hydrolysis.     

Purification of infectious bronchitis coronavirus by Sephacryl S-1000 gel chromatography

A procedure was developed to purify infectious bronchitis virus (IBV) by gel chromatography (GC) with a Sephacryl S-1000 column. Virus samples concentrated by centrifugation were applied to a Sephacryl S-1000 column and eluted by 0.02 M phosphate buffer (pH 7.2) containing 0.15 M NaCl. Virus particles were recovered mainly in the first peak. Purity of the samples was evaluated by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy. Using electron microscopy, it was found that there were more spike-rich particles in the virus samples purified by GC than in those purified by sucrose density gradient centrifugation (SDGC). In addition, the hemagglutination unit [log10 (infectivity titer/hemagglutination titer)] of GC-purified virus samples was approximately 10 times lower than that of SDGC-purified virus samples. These results indicate that Sephacryl S-1000 gel chromatography is useful for purification of IBV.     

金黄色葡萄球菌肠毒素A在枯草芽孢杆菌中的表达及其活性分析

利用优化后的SEA基因片段与带有枯草芽孢杆菌启动子的表达载体pBC38c,成功构建了含有SEA基因的重组表达质粒pBC38c-SEA。通过电转化的方法,将重组质粒pBC38c-SEA转入枯草芽孢杆菌1A751中。对目的蛋白进行表达,采用硫酸铵沉淀法浓缩上清中的蛋白,超声波裂解法裂解菌体沉淀,SDS-PAGE凝胶电泳法检测蛋白表达情况,Western-blot对表达的目的蛋白进行活性分析,并对表达条件与硫酸铵浓度进行优化。结果表明,成功构建了枯草芽孢杆菌表达质粒pBC38c-SEA,并在枯草芽孢杆菌中获得了表达,目的蛋白存在于培养基上清中,且具有抗原性。随着培养时间的延长,目的蛋白表达产量增多,在35h时达到最多,同时硫酸铵终浓度为50%,目的蛋白得到最好沉淀。本试验结果表明,SEA基因得以分泌表达,为进一步研究其生物活性并作为佐剂在临床中应用奠定了基础。     

两种方法纯化猪血清免疫球蛋白IgG的比较

为了从猪血清中纯化免疫球蛋白IgG,得到纯度更高的样品,采用饱和硫酸铵分级沉淀和Protein G柱吸附分离两种方法进行纯化,将获得的IgG经SDS-PAGE凝胶电泳纯度鉴定和HPLC浓度测定,结果表明,利用Protein G柱吸附分离纯化出的样品在纯度和浓度方面都要高于盐析法,Protein G柱吸附分离纯化IgG更适合于实验室小规模的操作,能够获得纯度高的样品。     

兔病毒性出血症病毒的提纯及部分特性鉴定

应用建立的SDS—蔗糖密度梯度超速离心法,纯化了从兔肝组织中粗提的兔病毒性出血症病毒。通过血凝、电镜观察、SDS-PAGE、琼脂双向免疫扩散、免疫印迹等试验,证明SDS—蔗糖梯度柱分离的病毒纯度高,具有4种结构多肽。     

Concentration of infectious aquatic rhabdoviruses from freshwater and seawater using ultrafiltration

Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus, and spring viremia of carp virus were concentrated and detected from freshwater and seawater samples by using hollow-fiber ultrafiltration. Within 60 min, virus in a 50-L freshwater or saltwater sample was concentrated more than 70-fold, and virus retention efficiencies were consistently greater than 88%. Retention efficiency was highly dependent upon concentrations of column blocking and sample stabilization solutions. A large column with a surface area of 1.15 m2 and a filtration capacity of 5-200 L exhibited optimal viral retention when blocked with 2% fetal bovine serum (FBS) and when the samples were supplemented with 0.1% FBS. Conversely, a small column with 100-fold less surface area and a filtering capacity of 0.5-2.0 L was optimized when blocked with 1% FBS and when the samples were supplemented with 0.1% FBS. The optimized ultrafiltration procedure was further validated with water from a tank that contained IHNV-exposed juvenile sockeye salmon Oncorhynchus nerka, resulting in an average virus retention efficiency of 91.6 +/- 4.1% (mean +/- SE). Virus quantification of concentrated samples demonstrated that IHNV shedding in sockeye salmon preceded mortality; shedding of the virus was observed to increase significantly as early as 7 d postchallenge and peaked at day 14, when virus levels reached 4.87 x 10(3) plaque-forming units/mL. We conclude that ultrafiltration is a reliable and effective method for concentrating viable aquatic rhabdoviruses from large volumes of water and has application for the analysis of environmental water samples.     

Evaluation of Mannheimia haemolytica leukotoxin prepared in nonsupplemented and BSA or FBS supplemented RPMI 1640 medium

Mannheimia haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Supernatant preparations containing Lkt produced by M. haemolytica serotype 1, grown in RPMI 1640 medium supplemented with BSA or FBS and without supplements were evaluated during this study. Analysis of obtained Lkt showed presence of 105 kDa antigen (SDS-PAGE electrophoresis). The obtained bacterial protein fraction estimated as Lkt was detected by Western blotting with mouse monoclonal (Mab 605 and Mab 601) anti-Lkt antibodies. No significant differences were found in obtained leukotoxin between wildtype and reference M. haemolytica strains. Our studies showed that growth in media supplemented with BSA or FBS had no significant influence on leukotoxin production. When BSA or FBS supplements were used, additional protein fractions in electrophoregrams SDS-PAGE were observed. These protein bands did not react with Mab 605 and/or Mab 601 in Western blotting analysis. Lkt immunogenicity was detected by immunoblotting with sera from Lkt immunized rabbits and calves.     

Identification of bovine herpesvirus-1 polypeptides involved in serum neutralization

Bovine herpesvirus (infectious bovine rhinotracheitis virus)-infected cell antigens were solubilized with Nonidet P-40. The crude antigen extract was separated by reaction with bovine hyperimmune serum in line immunoelectrophoresis; individual immunoprecipitates were used to immunize rabbits. Rabbit sera possessing serum neutralizing activity were analyzed by reaction with crude antigen extract in immunoprecipitation sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Four virus-specified glycopeptides, with molecular weights of 69-75K, 77-81K, 82-92K and 108-115K, appeared to be involved in inducing serum neutralizing antibody.     

Antiviral activity against transmissible gastroenteritis virus, and cytotoxicity, of natural porcine interferons alpha and beta.

Porcine interferon (POIFN)-alpha prepared in primed peripheral blood leukocyte cultures induced with Newcastle disease virus and POIFN-beta from PK-15 cell cultures induced with polyinosinic:polycytidylic acid were partially purified by precipitation with potassium thiocyanate and anion exchange chromatography. Mean purification factors in terms of units of POIFN per mg of protein, of 37 and 12 were obtained for POIFN-alpha and POIFN-beta respectively. In yield reduction assays in swine testis and pig kidney cell cultures, POIFN-alpha and POIFN-beta had greater antiviral activity against vesicular stomatitis virus than against transmissible gastroenteritis virus (TGEV). The antiviral effects were greater at higher concentrations of interferon (IFN), and when the IFN treatments were continued postinfection. Porcine interferon-beta showed greater antiviral activity against TGEV than POIFN-alpha, but this may have been partly due to cytotoxicity. There were no major differences in the antiviral activities of crude and partially purified IFN preparations. Both types of IFN showed antiviral activity against TGEV in yield reduction assays in porcine intestinal explant and intestinal epithelial cell cultures. Crude POIFN-beta was found to be rapidly cytotoxic, especially in porcine cells, and some fractions of partially purified POIFN-beta were also cytotoxic. The cytotoxicity of POIFN-beta was partially neutralized by antibodies against human IFN-beta, but human IFN-beta was not cytotoxic for porcine or bovine cells.     

Concentration of Infectious Aquatic Rhabdoviruses from Freshwater and Seawater Using Ultrafiltration

Abstract

Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus, and spring viremia of carp virus were concentrated and detected from freshwater and seawater samples by using hollow-fiber ultrafiltration. Within 60 min, virus in a 50-L freshwater or saltwater sample was concentrated more than 70-fold, and virus retention efficiencies were consistently greater than 88%. Retention efficiency was highly dependent upon concentrations of column blocking and sample stabilization solutions. A large column with a surface area of 1.15 m² and a filtration capacity of 5–200 L exhibited optimal viral retention when blocked with 2% fetal bovine serum (FBS) and when the samples were supplemented with 0.1% FBS. Conversely, a small column with 100-fold less surface area and a filtering capacity of 0.5–2.0 L was optimized when blocked with 1% FBS and when the samples were supplemented with 0.1% FBS. The optimized ultrafiltration procedure was further validated with water from a tank that contained IHNV-exposed juvenile sockeye salmon Oncorhynchus nerka, resulting in an average virus retention efficiency of 91.6 ± 4.1% (mean ± SE). Virus quantification of concentrated samples demonstrated that IHNV shedding in sockeye salmon preceded mortality; shedding of the virus was observed to increase significantly as early as 7 d postchallenge and peaked at day 14, when virus levels reached 4.87 × 10³ plaque-forming units/mL. We conclude that ultrafiltration is a reliable and effective method for concentrating viable aquatic rhabdoviruses from large volumes of water and has application for the analysis of environmental water samples.

Received April 22, 2011; accepted August 4, 2011     

Replacement of sodium chloride with dextran or polyethylene glycol for immunoprecipitation of lipopolysaccharide with antibodies in chicken or turkey sera

Dextran or polyethylene glycol could replace sodium chloride in agarose gels for inducing immunoprecipitation of $\text{Pasteurella multocida}$ lipopolysaccharides with antibodies in chicken or turkey sera. Resolution of immunoprecipitates was best when 3% concentrations of either dextran or polyethylene glycol were used. Higher concentrations increased opacity of the gels. Nonspecific precipitation of serum or γ-globulin fractions in gels was caused by the electrophoresis buffer, dextran, and polyethylene glycol. Dialysis of serum or γ-globulin fractions against the electrophoresis buffer and soaking gels in buffers of pH greater than 7.0 that contained 3% polyethylene glycol reduced nonspecific precipitation. Incorporation of dextran or polyethylene glycol into gels enhanced immunoprecipitation in rocket immunoelectrophoresis but resulted in slower mobility of antigen.     

Studies on the purification of the leucocidin of Fusobacterium necrophorum and its neutralization by specific antisera

Leucocidin from several strains of Fusobacterium necrophorum was partially purified by gel filtration on Fractogel HW55 (F), the majority of the activity being present in the 50 ml of filtrate collected after 1.1 void volumes had passed through the column (termed Fraction 1, or #1). The material also contained lipopolysaccharide in 12.5% SDS-PAGE gels run under reducing conditions, but the protein did not migrate into 7.5% PAGE gels run under non-reducing conditions. Rabbit and bovine antisera to the leucocidin possessed antibodies against antigens in concentrated, washed culture supernates from toxigenic F. necrophorum and neutralized the leucocidal activity of such supernates. Absorption of the antisera with homologous, washed F. necrophorum cells reduced ELISA antibody titres by greater than 50%, but decreased neutralization titres by 15%. Absorbed rabbit IgG anti-#1 precipitated a single rocket in crossed immunoelectrophoresis and identified two proteins, of molecular weights (M.W.) 14 000 and 13 000, and 1 protein of M.W. 13 500 in immunoblots from toxigenic and non-toxigenic strains, respectively. An additional protein of M.E. 103 000 was present after SDS-PAGE separation of supernates from toxigenic but not non-toxigenic F. necrophorum and was not present in whole cell components. It was considered that the leucocidin may be present in a dimeric form in culture supernates from toxigenic strains. Antisera to leucocidins from several strains of F. necrophorum exhibited variable neutralization titres against leucocidins from heterologous bacteria.     

Beta-adrenergic receptor binding in crude porcine adipose tissue plasma membranes.

Methods have been detailed to prepare a crude membrane fraction from isolated porcine adipose tissue cells. Adipocytes were obtained after incubation of 5 g of adipose tissue slices with 4,500 units of a selected lot of collagenase in a total volume of 15 mL at 37 degrees C for 90 min. There was no bovine serum albumin present during cell isolation because albumin did not enhance cell yield or yield of lipolytic activity. Isolated cells were lysed by exposure to hypotonic conditions in the presence of 7.5 mM ethylene glycol tetraacetic acid (EGTA) and .8 mM phenylmethylsulfonyl fluoride (PMSF). A 30,000 x g centrifugal pellet was used as the crude membrane preparation. Binding of tritiated dihydroalprenolol (DHA), a beta-adrenergic antagonist, was measured in the presence of 7.5 mM EGTA and .2 mM PMSF, because these protease inhibitors improved specific binding by approximately 50% to greater than 150 fmol/mg of protein and decreased non-specific binding to less than 10% at 2.5 nM DHA.     

Purification and quantitative measurement of bovine serum amyloid-A

Bovine serum amyloid-A (b-SAA) was purified from a pool of acute phase serum using hydrophobic interaction chromatography and gel filtration. Serum was applied at a low salt concentration to a phenyl-sepharose column and SAA was eluted with a gradient of 0 to 6 M guanidine-HCI. Fractions containing SAA were pooled, concentrated and further purified by gel filtration on Superose-12. The concentration of SAA in bovine serum was quantified by an indirect ELISA using rabbit anti-human SAA and horseradish peroxidase conjugated donkey anti-rabbit IgG. Dilutions of an acute phase bovine serum sample were used as working standards. The SAA concentration of this standard was determined by comparison with purified b-SAA on SDS-polyacrylamide gel electrophoresis followed by densitometry at 590 nm. The assay detection limit was 3 μg ml⁻¹; the intra-assay coefficient of variation was 4 per cent and interassay coefficients of variation were 5·5 per cent and 7·2 per cent at 66 and 178 μg ml⁻¹ SAA, respectively. In calves experimentally infected with Pasteurella haemolytica type A1 the ELISA was able to detect a 10-fold increase of SAA within 24 hours of inoculation.     

大麦芽阿魏酸酯酶的分离纯化及其部分酶学性质的测定

本试验采用硫酸铵分级沉淀、透析、DEAE-52离子交换层析对大麦芽中的阿魏酸酯酶(FAE)进行了分离纯化,并测定了其部分酶学性质。结果显示,分离纯化后获得了阿魏酸酯酶纯品,纯化倍数达34倍;经SDS-PAGE电泳显示为单一条带,并测定其相对分子质量约为29.3 ku;最适反应温度为50℃;最适pH为5.5,但阿魏酸酯酶在60℃以下,pH 4.5~7.5之间有较好的稳定性;Zn2+、Cu2+、Fe3+对酶活有很强的抑制作用,Na+和EDTA对酶有一定的激活作用。     

Vitrification of In Vitro-produced Bovine Embryos by Addition of Ethylene Glycol in One-step

The objective of this study was to simplify two-step addition of cryoprotectant for vitrification of bovine embryos by developing a one-step procedure. Survival was calculated as a percentage of non-vitrified controls developed from the same batch of oocytes. In experiment 1, bovine blastocysts were vitrified following one- or two-step addition of cryoprotectant. Exposure of embryos to cryoprotectant in one-step resulted in survival rates not significantly lower (p > 0.1) than those obtained by two-step addition (85% vs 98%, respectively). Based on these results, experiments 2-4 were designed to test one-step addition of cryoprotectant more rigorously. Experiment 2 exposed day 7 blastocysts to 6, 7 or 8 M ethylene glycol for 2.5 or 3.5 min. At 24 h post-vitrification, survival of embryos was similar, irrespective of ethylene glycol concentration or exposure time (6 M 38%, 7 M 51%, 8 M 59%; 2.5 min 54%, 3.5 min 45%). In experiment 3, blastocysts were exposed to 7 M ethylene glycol for shorter times (30 or 60 s); 30 s exposure resulted in decreased survival (8% vs 31%, p < 0.05). Experiment 4 concerned one-step addition of cryoprotectant to day 6 bovine morulae, exposed to 7 M ethylene glycol for 1 or 1.5 min. There was no difference in survival between exposure times of 1 or 1.5 min (28% vs 45%, respectively; p > 0.1). It is unclear why many embryos survive vitrification with one-step addition of cryoprotectant, but others do not. Although, one-step addition of cryoprotectant simplifies the vitrification procedure, survival rates were inadequate for routine cryopreservation of in vitro-produced bovine embryos.     

Bovine interleukin 2: biochemical and biological characterization

Interleukin 2 (IL-2), secreted by bovine peripheral blood mononuclear cells (PBL) on stimulation with concanavalin A (Con A), was purified and characterized by different chromatographic and electrophoretic techniques. The ability of IL-2 to support proliferation of Con A-stimulated bovine lymphoblasts was used to assay and quantitate IL-2 activity. Bovine IL-2 having an apparent MW of 27,000 eluted from a gel-filtration column; from an anion exchange column peak activity was detected at 190 mM NaCl. Binding of bovine IL-2 to phenyl-Sepharose gel and elution with 35-60% ethanediol indicated its hydrophobic nature. Studies on cross-species reactivity revealed that both buffalo and goat lymphocytes respond to cattle IL-2 and detected 35% of activity from a standard cattle IL-2 preparation. Sheep lymphocyte response to cattle IL-2 was negligible.