Detection on antiserum of Yersinia pestis phage in Marmota himalayana blood in the natural plague foci of Qinghai-Tibet Plateau / 中国热带医学

@#Abstract: Objective　To detect and analyze the antiserum of Yersinia pestis phage in Marmota himalayana blood from the natural plague foci of Qinghai-Tibet Plateau by micro-bolus technique, to provide a theoretical basis for interaction between phages and mammalian immunology, phage therapy and interaction between bacteriophage and ecology in future. Methods　Using diagnostic Yersinia pestis phage and 3 wild plague phages from Qinghai-Tibet Plateau Natural Plague Foci as antigens, 847 serums of Marmota Himalayana blood, from Tongde, Guinan, Gonghe, Xinghai, Tianjun foci counties in Qinghai Plateau, were collected from July to September in 2020, 2021 and determined on antiserum of Yersinia pestis phage by microplate method and double agar plate method. Results　The neutralization reaction experiment lasted for 24 hours between 4 phage and 847 serums by microplate method independently. These mixtures were tested by double agar plate method. All results were negative on antiserum of Yersinia pestis bacteriophage. Conclusions　The positive antiserum of Yersinia pestis phage in Marmota himalayana were not found the natural plague foci of Qinghai-Tibet Plateau, which agreed with plague epidemiology in 5 foci counties in Qinghai plateau from 2020-2021, that was a characteristic of the resting period. In other words, it was in the absence of plague pathogen. It also showed indirectly that the absence or weak presence of Yersinia pestis bacteriophage in the plague foci. It showed a lower frequency on host animals coming into contact with phages naturally. The antiserum of Yersinia pestis phage may be related to the form of plague infection and the intensity of the disease.

Respuesta anticlases mayores de anticuerpos humanos séricos en antisueros hiperinmunes obtenidos en carneros / Characterization of hyperimmune anti-human serum produced in sheep

Introducción: La inmunoelectroforesis es una técnica de precipitación que permite la caracterización de muestras biológicas complejas. En el Departamento de Inmunología del Instituto de Ciencias Básicas y Preclínicas Victoria de Girón se cuenta con un antisuero hiperinmune obtenido por inmunizaciones de carneros contra proteínas totales séricas humanas y con otro antisuero anti IgA de calostro humano. Objetivo: Identificar IgG, IgM e IgA en suero humano y determinar respuesta anti IgM humana en el antisuero anti IgA de calostro humano obtenido en carnero. Material y Métodos: Se realizó un estudio observacional, descriptivo y transversal desde noviembre de 2017 hasta junio de 2018. Se desarrolló una inmunoelectroforesis de suero humano normal empleando el antisuero hiperinmune. Resultados: Se identificaron IgG, IgM e IgA además de albúmina y otras fracciones proteicas y se determinó respuesta anti IgM humana en el antisuero anti IgA de calostro humano obtenido en carnero. Conclusiones: Este trabajo permitió identificar y determinar la respuesta anticlases mayores de inmunoglobulinas en la muestra de estudio(AU)

Introduction: Immunoelectrophoresis is a precipitation technique that allows the characterization of complex biological samples. The Immunology Department of the Institute of Basic and Pre-Clinical Sciences Victoria de Girón has a hyperimmune antiserum obtained by immunization of sheep against human serum total proteins and it also has an anti-human IgA antiserum obtained from human colostrum. Objective: The aim of this study was to identify IgG, IgM and IgA in human serum and to determine response to anti-human IgM in human colostral IgA with antiserum obtained in sheep. Material and Methods: An observational descriptive cross-sectional study was conducted from November 2017 to June 2018. Immunoelectrophoresis of normal human serum was performed using hyperimmune antiserum. Results: These procedures allowed to identify IgG, IgM and IgA in addition to albumin and other protein fractions and to determine response to anti-human IgM in human colostral IgA with antiserum obtained in sheep. Conclusions: This work allowed us to identify and determine significant anti-class responses of immunoglobulins in the sample studied(AU)

Desarrollo de un producto anti-toxina shiga para la prevención del síndrome urémico hemolítico / Development of a product anti-Shiga toxin for prevention of the hemolytic uremic syndrome

El síndrome urémico hemolítico (SUH) típico es una enfermedad huérfana causada por cepas de Escherichia coli productoras de toxina Shiga (Stx) y caracterizada por daño renal agudo, anemia hemolítica microangiopática y plaquetopenia. Es endémico en Argentina, el país con mayor incidencia de SUH en el mundo. Debido al rol fundamental de la Stx en su patogenia, se puede considerar que, como otras toxemias conocidas, el SUH podría ser tratado con anticuerpos. Este trabajo describe el desarrollo de un nuevo tratamiento capaz de neutralizar el efecto tóxico de distintas variantes de la Stx. El tratamiento consiste en fragmentos F(ab')2 provenientes de un antisuero equino cuya eficacia y potencia contra Stx1 y Stx2 se comprobó en diferentes modelos preclínicos. El producto mostró ser seguro en animales, presentó la farmacocinética descripta para compuestos similares y se pudo establecer una posible ventana terapéutica para su adecuada administración. En conjunto, los resultados preclínicos obtenidos validan la realización de un estudio clínico de primer uso en humanos. En dicho estudio, que se realizará en el Hospital Italiano de Buenos Aires, se analizará la seguridad y la farmacocinética del producto en voluntarios adultos sanos. Estos resultados sentarán las bases para la realización del estudio clínico fase II en pacientes pediátricos con infección por cepas de E. coli productoras de Stx.

The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) -producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.

Methodology establishment and application of therapeutic treatment on rats with anti-glomerular basement membrane nephritis / 中草药

Objective To establish a suitable model for therapeutic treatment on rats of anti-glomerular basement membrane nephritis. Methods Rabbit with rat glomerular basement membrane and freund’s adjuvant were immuned by multi-point subcutaneous injection biweekly, five times totally, and the antiserum was prepared. Experimental model for therapeutic treatment was established. Serum level of immune complex (CIC), intensity of immunofluorescence of glomerular basement membrane, lesion of kidney and glomerular basement membrane was measured by multiple intravenous injection of antiserum to rats to determine suitable experiment conditions including titer of antiserum, dose of antiserum, method and time of antiserum injection. The model was observed the efficacy using Gushen 2 as therapeutic drug. Results Rats with 4 mL antiserum (titer 1:4) by intravenous injection were immuned 4 times 3 d. Rat anti-glomerular basement membrane nephritis form on day 6, 8, and 10 since immunization; Pathogenic condition were relatively mild on day 6 since immunization and basically stable on day 8 and 10 since immunization. Verification tests showed that, Gushen 2 significantly reduced the 24 h urine protein excretion, relieved kidney lesion for 30 consecutive days, which had obvious therapeutic effect on rats with anti-glomerular basement membrane of nephritis. Conclusion Rats immuned with 4 mL antiserum (titer 1:4) by intravenous, immune points up to 4 times 3 d, rat anti-glomerular basement membrane nephritis model form which are suitable for therapeutic treatment of drug on day 8 since immunization.

A Novel Polyclonal Antiserum against Toxoplasma gondii Sodium Hydrogen Exchanger 1

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.

Preparation of the polyclonal antibodies of CDPK 5 gene from toxoplasma gondii and the identification of its functions / 重庆医学

Objective Screening the immune polypeptide sequence of toxoplasma (Tg) CDPK5 gene ,which were synthesized and then immunized the New Zealand white rabbit to prepare antiserum ,and identification its function .Methods Bioinformatics a‐nalysis was used to determine the immune peptide of Tg CDPK5 sequence ,which were artificially synthesized to immune white rab‐bit to prepare antiserum .The titers of antibodies were determined by ELISA and the polyclonal antibodies were verified with CD‐KP5 antigen by Western blot .The sub‐cellular localization of Tg CDPK 5 were obtained by immunofluorescence assay .Results 17 bp peptide sequence from the Tg CDPK5 N‐terminal were chosen as immune polypeptide by bioinformatics analysis .Synthetic pep‐tide were used to immune rabbit to obtain polyclonal antiserum .The result showed that the titer of the obtained ployantibody were 1∶640 000 ;Western blot demonstrated that the antiserum could specifically recognize Tg CDPK 5(75 .4 × 103 );Immunofluores‐cence assay revealed this antibody could specifically recognize the endogenous Tg CDPK 5 of Toxoplasma gondii .Conclusion Ac‐cording to the analysis of Tg CDPK5 sequence information ,this study successful obtained Tg CDPK5 polyclonal antibody .

Comparative studies by different dyeing methods on antiserum preparation against hemorrhagins from Agkistrodon acutus venom / 中国免疫学杂志

Objective:In order to look for a good method for preparation of hemorrhagin antiserum. Methods: Three kinds of hemorrhagins including AaH Ⅰ, AaH Ⅱ, and AaH Ⅳ were purified from Agkistrodon acutus venom according to predecessors's methods and crude AaH Ⅰ, AaH Ⅱ and AaH Ⅳ were obtained. Preparation electrophoresis was used to purify AaH Ⅰ,AaH Ⅱand AaH Ⅳ further. As for an hemorrhagin, six different dyeing methods were used to dye PAGE gel and the gel contained hemorrhagin was obtained respectively. The ground gel contained hemorrhagin was used to immune mice and its antiserum was obtained. Antiserums quality was tested through ELISA test and neutralization of the hemorrhagic activities of corresponding hemorrhagin. Results:Effective IgG concentration in different antiserum was different and effective IgG made through non toxic type protein fast stain reagent kit was higher than others. Conclusion:Non toxic type protein fast stain reagent kit is the best dyeing method among the six dyeing methods.

Antisuero AA479: nuevo reactivo para la serotipificación de variantes atípicas se Shigella flexneri / AA479 antiserum: new reagent for the serotype characterization of atypical variants of Shigella flexneri

Shigella flexneri is divided into 13 serotypes based on the combination of antigenic determinants present in the O-antigen. A new O-antigen modification with phosphoethanolamine has been identified. The presence of this antigenic determinant (called E1037) is recognized by monoclonal antibody MASF IV-1. Given the increasing incidence of these new variants and the difficulty in supplying the monoclonal antibody to our country, we produced a polyclonal antiserum (AA479) through immunization with a S. flexneri Xv strain. The antiserum specificity was assessed by slide agglutination against isolates from clinical cases and a culture collection representing all Shigella serotypes. The results obtained demonstrated a 100% correlation between AA479 absorbed antiserum and monoclonal antibody MASF IV-1. The availability of AA479 antiserum in every public hospital in Argentina will allow us to identify atypical S. flexneri isolates in order to strengthen Shigella surveillance in our country and to compare with global epidemiological dat

Shigella flexneri se divide en al menos 13 serotipos sobre la base de la combinación de determinantes antigénicos presentes en el antígeno O. Se identificó una nueva modificación del antígeno O con fosfoetanolamina. La presencia de este determinante antigénico (denominado E1037) es reconocida por el anticuerpo monoclonal MASF IV-1. Teniendo en cuenta la incidencia creciente de estas nuevas variantes y la dificultad en la provisión del anticuerpo monoclonal para nuestro país, se elaboró un antisuero de tipo policlonal (AA479) mediante la inmunización con un cultivo de S. flexneri Xv. La especificidad del antisuero se evaluó por aglutinación en lámina con aislamientos clínicos y cultivos de colección, con lo que quedaron representados todos los serotipos de Shigella. Los resultados obtenidos demostraron una correlación del 100% entre el antisuero AA479 absorbido y el anticuerpo monoclonal MASF IV-1. La disponibilidad del antisuero AA479 en todos los hospitales públicos de Argentina permitirá identificar los aislamientos atípicos de S. flexneri; de esta forma se podrá fortalecer la vigilancia de Shigella en nuestro país y comparar con los datos epidemiológicos a nivel global

Purification,antiserum preparation and antigenic characters of ClpC2 in Myco-bacterium tuberculosis / 中国免疫学杂志

Objective:To investigate the antigenicity of ClpC 2 and the feasibility of polyclonal antibodies of ClpC 2 as detected antibody.Methods:rClpC2 was induced with IPTG.The rClpC2 was identified by SDS-PAGE and Western blot ,and purified by affinity chromatography ,with which rabbit were immunized and the specificity of rabbit antiserum was detected by Western blot , the titer of rabbit antiserum against ClpC2 was detected by double immunodiffusion and indirect enzyme-linked immunosorbent assay (ELISA).The antigenicity of the rClpC2 was detected by ELISA.The polyclonal antibodies of ClpC 2 were prepared to detect the ClpC 2 in clinical serum of TB patients by ELISA.Results:SDS-PAGE showed specific protein band with a relative molecular mass of 46 kD.The rClpC2 could bind with the antibody in the blood serum of the mouse immuned by MTB.By Bandscan analysis rClpC 2 accounted for about 58.7%of the total bacteria protein ,the purity of rClpC2 was 88.5% after purification.The ClpC2 of BCG could bind with the rabbit antiserum.The titer of antiserum were 1∶32 and 1∶320 000 by double immunodiffusion and ELISA detected respectively.ELISA results showed that clinical serum positive rate of rClpC 2 antigen was 46%in TB patients,the sensitivity of this protein was 46%,and the spe-cificity of this protein was 90%.ELISA results showed that the sensitivity of rabbit antiserum against ClpC 2 was 40%, and the specificity was 90%.Conclusion: Successfully expressed and purified rClpC 2 and high titer polyclonal antibody were successfully prepared,and these results will provide basements for further study on the biological functions of ClpC 2 and its candidate potentiality as serological diagnosis and drug-target and biological functions of antiserum against ClpC 2.

Construction of expression vector and preparation of antiserum for the undefined gene LOC401296 / 军事医学

Objective To clone the full-length CDS sequence of LOC 401296 , an undefined gene that we found recently, and to obtain both its expression in eukaryotic cells or E.coli and antiserum to LOC401296 protein.Methods The full-length CDS sequence of LOC401296 was amplified by polymerase chain reaction (PCR).Then we established the expression vectors pET28B-LOC401296 and pCMV-Myc-LOC401296 by cloning the full-length CDS sequence into vector PET28B and vector pCMV-Myc respectively.Isopropyl Thiogalactoside (IPTG) was used to induce LOC401296 expression in E.coli.Furthermore,the protein purified and refolded was used to immunize BALB /c mice.The titer of the antiserum collected from immunized mice was identified by ELISA assay and Western blot .Results We cloned the full-length CDS sequence of LOC401296 was successfully .Protein LOC401296 was expressed as was expected and the mouse anti-human LOC401296 antiserum was obtained .The antiserum titer reaching 1∶64 000 was identified by ELISA .Besides, Western blot analysis showed that the antiserum could be used to detect protein LOC 401296 .Conclusion The full-length sequence of LOC401296 is obtained and the mouse anti-human LOC401296 antiserum becomes available .This study can contribut to further research on the undefined gene LOC 401296 .

Rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin for single radial immunodiffusion assay / 中华微生物学和免疫学杂志

Objective To establish a method for rapid preparation of antiserum against influenza virus (H7N9) hemagglutinin,and to study the possibility of using it in single radial immunodiffusion (SRID) assay for quantitative detection of antigen in H7N9 influenza vaccine.Methods Hemagglutinin proteins expressed in eukaryotic cells were used to immunize sheep.Serum samples were collected to detect antibody titers by ELISA and double immunodiffusion assay.Different concentrations of antiserum were used in SRID assay to get the optimized concentration.Results After 4 times of immunization,the antiserum titers achieved 1 ∶ 1 000 000 and 1 ∶ 32 as indicated by ELISA and double immunodiffusion assay,respectively.The antiserum could form a clear precipitation line in SRID assay.The detection of antigen in the range of 10 to 40 μg/ml showed good linearity in the standard curve.The antigen titers in six batches of H7N9 vaccine detected by this SRID assay were identical with those by SDS-PAGE assay.Conclusion The antiserum against H7N9 hemagglutinin for SRID assay was developed successfully,and could be used as a reagent for the quantitative detection of antigen in H7N9 influenza vaccine.

Expression and purification of a fusion gene TSO45W-4B-TSOL18 of Taenia solium in Escherichia coli ArcticExpress(DE3) and preparation of rabbit antiserum / 中国地方病学杂志

Objective To construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18 of Taenia solium,to obtain purified recombinant protein TSO45W-4B-TSOL18 and to prepare rabbit antiserum against the recombinant fusion protein.Methods TSO45W-4B and TSOL18 encoding genes were connected with hydrophobic (Gly4Ser)3 linker.TSO45W-4B-TSOL18 fusion gene was synthesized and cloned into an expression vector pGEX-1λT to construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18.The positive recombinants were transformed into Escherichia(E.) coli ArcticExpress (DE3),and the expression of recombinant protein was induced with isopropylβ-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).The expressed recombinant fusion protein was purified through affinity chromatography.The purified recombinant protein was mixed with complete Freunds adjuvant at a dosage of 0.5 mg.Rabbit was intramuscularly and subcutaneously immunized in the hind leg and the back,respectively.After 2 weeks the rabbit was boosted with purified recombinant protein which was mixed with incomplete Freunds adjuvant at the same dosage.Rabbit can be boosted every 10 days until an adequate response was achieved.At the 6th day after the last immunization,blood was collected from the rabbit heart.Serum was separated to purify and prepare the antiserum.ELISA was applied to determine the titer of the antiserum and Western blot assay was used to determine the specificity of the antiserum.Results The size of the synthesized fusion gene TSO45W-4B-TSOL18 was 789 bp.The results of restriction enzyme digestion showed that the recombinant plasmid pGEX-TSO45W-4B-TSOL18 was successfully constructed.DNA sequencing showed that the size of the fusion gene TSO45W-4B-TSOL18 was 789 bp,which was consistent with expected result.As demonstrated by SDS-PAGE,relative molecular mass of the expressed recombinant fusion protein was approximately 55 × 103,and its purity was 85％ after purification with affinity chromatography.Titer of the antiserum against the recombinant protein was 1 ∶ 512 000 in ELISA assay and the specific rabbit antiserum against the purified recombinant protein TSO45W-4B-TSOL18 could specifically bind to the recombinant fusion protein in Western blot assay.The relative molecular mass of the specific band was about 55 × 103,which was consistent with expected size.Conclusions The recombinant plasmid pGEX-TSO45W-4BTSOL18 of Taenia solium is successfully constructed.High quality recombinant fusion protein TSO45W-4B-TSOL18 and high titer rabbit antiserum are successfully prepared.

Quantification of HA in H7N9 influenza vaccine using heterogeneous antiserum of the same HA sub-type virus / 中华微生物学和免疫学杂志

Objective To study the possibility of using heterogeneous antiserum in single radial immunodiffusion (SRID) for quantitative detection of HA in H7N9 influenza vaccine product when H7N9-specific antiserum is not available in order to establish a testing method for the detection of H 7N9 antigen in any urgent situation.Methods Antisera specific for H7N1, H7N2, H7N3 and H7N7 were obtained from NIBSC and used for SRID assay .Amino acid sequences of hemagglutinins were comparatively analyzed be-tween H7N9 virus and other viruses used to prepare heterogeneous antiserum .The titers of antisera against H7N9 and their homogenous antigens were detected by double immunodiffusion method .Based on the results of homology analysis and cross-reaction, a suitable antiserum was selected and its applicability was further validated by the SRID assay using H7N9 antigen.Results Influenza A virus subtype H7N3 that used for preparation of 07/278 antiserum showed the highest HA homology with H7N9 (97.14%).The titer of 07/278 antiserum against H7N9 antigen was 1 ∶8 as detected by double immunodiffusion assay .The H7N9 anti-gen and the 07/278 antiserum could form a clear precipitation line in SRID assay .The detection of H7N9 antigen in the range of 10 to 40μg/ml showed a good linearity in the standard curve .Conclusion The 07/278 antiserum from NIBSC can be used as an alternative reagent for the quantitative detection of hemaggluti -nin in H7N9 influenza virus vaccine .

Producción de attisueros para la detección de áido indolacético en cultivos dee bacterias promotoras del crecimiento vegetal / Antisera Production to Detect Indoleacetic Acid in Cultures of Plant-Growth Promoting Bacteria

Se obtuvieron antisueros en conejo utilizando como antígeno el AIA adherido a membranas de nitrocelulosa que mostraron un elevado título y especificidad. Mediante la técnica de inmunoadsorción por manchas marcadas con oro coloidal se detectó la producción de esta auxina por cepas de los géneros Gluconacetobacter, Herbaspirillum, Azospirillum, Pseudomonas, Burkholderia y Bacillus empleando como antígenos los sobrenadantes de los cultivos. Para cuantificar la producción de AIA y corroborar los datos obtenidos se empleó la técnica colorimétrica derivada de Salkowski. Los resultados muestran que todos los géneros bacterianos estudiados tienen la capacidad de producir AIA y se demuestra la factibilidad del uso de este antisuero policlonal para la detección de este metabolito. Teniendo en cuenta las potencialidades de estas bacterias, resulta de gran importancia la utilización de antisueros y técnicas serológicas para la detección rápida y sencilla de este tipo de metabolitos en bacterias asociadas a cultivos de interés económico.

Rabbit polyclonal antisera against indoleacetic acid (IAA) bound to nitrocellulose membrane were obtained, which exhibited a high titer and specificity. The dot immunobinding technique with colloidal gold was used to detect auxin production by several strains belonging to Gluconacetobacter, Herbaspirillum, Azospirillum, Pseudomonas, Burkholderia and Bacillus genera, using culture supernatants as antigens. Moreover, auxin production was quantified by the Salkowski's method to corroborate the previous results. It was found that that all the studied microorganisms produce IAA and the feasibility of using these antisera to detect the metabolite was confirmed. Taking into account the potentialities of plant growth promoting bacteria as biofertilizers, the use of these antisera for a rapid and easy detection of IAA in bacteria associated with important crops is thus recommended.

Polyclonal VDAC3 antibody decreases human sperm motility: a novel approach to male contraception.

Background: Voltage dependent anion channels (VDAC) mediate transport of anions, cations and ATP which play an important role in sperm motility. This study was aimed to examine the effect of polyclonal VDAC3 antiserum to human sperm motility. Methods: Polyclonal VDAC3 antiserum used in this study was produced in rabbits by immunization of VDAC3-specific synthetic peptides. Preimmunserum was collected before immunization and used for control experiment. Recognition of VDAC3 antiserum to antigen in human sperm was performed by western blot. Thirty sperm samples obtained from fertile men which had high quality of sperm motility were washed and collected by Percoll gradient. Sperm motility was assessed by means of evaluation of sperm velocity (seconds per 0.1 mm distance) and the number of unmoved sperm (million per ml) which were observed 0 minute, 30 minutes and 60 minutes after addition of VDAC3 antiserum and preimmunserum as a control. Both data were analyzed by SPSS 13.0 software. Results: VDAC3 antiserum recognized VDAC3 protein in human sperm. Statistical analysis demonstrated that there were increasing numbers of unmoved spermatozoa after addition of anti-VDAC3 antiserum in vitro for 60 minutes observation compared with preimmunserum (control). We found also that sperm velocity decreased signifi cantly after giving anti-VDAC3 antiserum in vitro for 0 minute, 30 minutes, and 60 minutes compared with pre-immunee serum (control). Conclusion: VDAC3 antiserum can decrease motility of human sperm. and may provide a novel principle of male contraception in the future.

A new experiment teaching model in pathogenic microbiology / 中华医学教育探索杂志

Based on their inherent relationship,some independent and verifying experiments of medical immunology were intergrated into a series of comprehensive and open experiments,which was embodied in the preparations of antiserum.so as to set up a new experiment teaching system emphasizing the training of student comprehensive capability.Achievements in training students'scientific research capacity,innovative motivation and practical working ability have been obtained and the results could provide valuable experience for innovation and reform of medical immunology experiment teaching model and method.

Herpes Simplex Virus Type 1 ICP27 Protein: Its Expression, Purification and Specific Antiserum Production / 中国病毒学·英文版

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. Coli and purified by nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin column,finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.

Transfection of DNA-PKCS antisense oligodeoxynucleotides in the radiosensitization of lung cancer cell line A549 in vitro / 中华放射肿瘤学杂志

Objective To observe the effect of DNA-PKCS antisense oligodeoxynucleotides (ASODN) on the radiosensitivity of lung cancer cell line A549 through transfecting DNA-PKcs ASODN into A549 cells.Methods DNA-PKCS ASODN and unrelated ODN were transfected into A549 cells (testing group and control group).These cells were irradiated with 0,0.5,1.0,2.0,4.0,6.0 or 8.0 Gy X-ray.Clo nogenic assay was performed to determine the survival fraction.The parameters Do,Dq and N for the multi target single-hit model,as well as the parameters a,13 and SF2 for the linear-quadratic model,were calculated to evaluate cell radiesensitivity.ResultsIn the control group and testing group,the α value was 0.14 and O.31 ,The βvalue was 0.030 and 0.018 ,the SF2 was 0.63 and 0.41 ,the Do was 2.38 and 2.09 ,the Dq was 1.43 and 0.60 ,respectively.In the testing group,the α value of A549 cells was increased,but the β value, SF2,DO and Dq were decreased.Conclusions DNA-PKCS ASODN can enhance the radiosensitivity of A549 cells and is a potential target in treating lung cancer.

Using the serological technique to identify Ascia monuste orseis (Latreille, 1819) (Lepidoptera: Pieridae) - DOI: 10.4025/actascibiolsci.v31i2.515 / Utilização da serologia na identificação de Ascia monuste orseis (Latreille, 1819) (Lepidoptera: Pieridae) - DOI: 10.4025/actascibiolsci.v31i2.515

This study aimed to obtain specific antiserum for Ascia monuste orseis (Latreille, 1819), with the aim of identifying the predators associated with this insect. The preparation of specific antiserum was performed by immunizing a rabbit via a lymph nodule injection containing antigen obtained from the maceration of 4th and 5th instars of A. monuste orseis caterpillars. Serological homologous tests performed using double diffusion in agar gel were positive 21 days after the first antigen inoculation. Eggs, 1th, 2th and 3th instar caterpillars, pupae and adults of A. monuste orseis also reacted positively to the obtained antiserum.

O presente trabalho teve como objetivo obter antissoro específico paraAscia monuste orseis (Latreille, 1819) com a finalidade de identificar seus predadores. Para a obtenção do antissoro, um coelho foi imunizado, via linfonódulo, com uma injeção de antígeno obtido pela maceração de lagartas do 4o e 5o instares de A. monuste orseis. Reações serológicas homólogas, realizadas por meio do método de dupla difusão em ágar, foram positivas após 21 dias da 1a inoculação do antígeno. Ovos, lagartas do 1o, 2o e 3o instares, pupas e adultos de A. monuste orseis também reagiram positivamente ao antissoro obtido.

Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein / 中国病毒学·英文版

The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.