Rho GTPase activating protein 21-mediated regulation of prostate cancer associated 3 gene in prostate cancer cell

The overexpression of the prostate cancer antigen 3 (PCA3) gene is well-defined as a marker for prostate cancer (PCa) diagnosis. Although widely used in clinical research, PCA3 molecular mechanisms remain unknown. Herein we used phage display technology to identify putative molecules that bind to the promoter region of PCA3 gene and regulate its expression. The most frequent peptide PCA3p1 (80%) was similar to the Rho GTPase activating protein 21 (ARHGAP21) and its binding affinity was confirmed using Phage Bead ELISA. We showed that ARHGAP21 silencing in LNCaP prostate cancer cells decreased PCA3 and androgen receptor (AR) transcriptional levels and increased prune homolog 2 (PRUNE2) coding gene expression, indicating effective involvement of ARHGAP21 in androgen-dependent tumor pathway. Chromatin immunoprecipitation assay confirmed the interaction between PCA3 promoter region and ARHGAP21. This is the first study that described the role of ARHGAP21 in regulating the PCA3 gene under the androgenic pathway, standing out as a new mechanism of gene regulatory control during prostatic oncogenesis.

Advances in the Mechanism of Phage Resistance to Bacterial Biofilms and Strategies for Its Application / 现代检验医学杂志

Bacterial biofilms(BF)are complex microbial communities formed by bacteria on living or abiotic surfaces.Their formation significantly enhances bacterial virulence and drug resistance and is associated with a high proportion of chronic bacterial infections,posing a serious threat to human health.The ability of traditional antibiotics and commonly used disinfectants to clear biofilms is limited,and an effective new strategy to treat BF is urgently needed.Bacteriophage,as a kind of virus that can infect and lyse bacteria,has high safety and specificity,and is considered as a promising alternative method for the treatment of BF.In this paper,the mechanism of bacteriophage anti-bacterial biofilm and the application strategies based on bacteriophage and its derivatives in the prevention and control of bacteriophage biofilm formation were reviewed,which provided new ideas for the development of efficient bacteriophage anti-bacterial biofilm methods.

Construction of a genomic database for Group A Streptococcus and the analysis of prophage distribution / 中华实用儿科临床杂志

Objective:To illustrate the characteristics of the distribution of prophages among the Group A Streptococcus(GAS) by mining the existing whole genome sequencing of the GAS, performing bioinformatic analyses, extracting data about prophages, and analyzing the state of prophages in the genome and genetic composition of some prophages. Methods:It was a retrospective study.Genome assembly sequences of GAS reported in GenBank till May 2020 were collected, and the important background information of these strains was sorted out to create a local genomic database.A phylogenetic tree of the whole genome of GAS was conducted using the bioinformatics software.The core genome was analyzed, and potential prophages and their integrity in the genome were predicted to obtain the characteristics of the distribution of prophages.Genotype types, number of core genes, and number, length and carrying rate of prophages in the database for GAS were analyzed.Results:A database containing the genome sequence of 2 529 GAS strains was established, involving 140 emm genotypes.These strains were isolated from 19 countries from East Asia, Europe, America and Oceania.Stratified by the disease background, these strains were mainly divided into invasive infection, non-invasive infection and immune sequelae.Prophage analysis of 1 798 genomes showed that at least one complete prophage was detected in 1 366 (76.0%) genomes.The number of complete prophages of each strain ranged from 0 to 6, and the length ranged from 32.8 to 62.6 kb, which was mainly 30-40 kb in length.The phiHKUssa, phiHKUvir and phiHKU488 were the most common prophages present in dominant clones circulated in China in recent years, which mainly carried virulence genes like the speC, spd1 and ssa. Conclusions:Prophages are widely distributed in the genome of GAS, which are of great significance in the evolution and expansion of dominating clones and thus reshape the population structure within the emm genotype.The establishment of a local genome database provides important baseline data for molecular epidemiological surveillance.

Screening of aldosterone-specific antibodies based on phage display technology / 新乡医学院学报

Objective To screen out specific aldosterone(ALD)antibodies using phage display technology and recom-binant antibody technology,providing raw materials for the research and development of ALD diagnostic kits.Methods Five healthy and clean New Zealand white rabbits were selected and immunized for the first time against the diluted ALD-keyhole limpet hemocyanin antigen(2 mg·L-1)using a multi-point injection method on the back,with a dose of 1 mg per rabbit.Immunization was administered again every 2 weeks,with a 50％reduction in dose.Starting from the third immunization,the ear vein blood of the rabbits was collected one week after each immunization.A chemiluminescent plate coated with 0.25 mg·L-1 ALD-bovine serum albumin antigen was used to measure serum titers via indirect and competitive methods.After the 5th immunization,the rabbit with high serum titers and good specificity was selected,and its spleen and bone marrow were removed.The spleen tissue was grinded,and RNA was extracted using TRIzol reagent in one step to obtain gene sequences in the variable region of light chain(VL)and the variable region of heavy chain(VH).The single-chain variable fragment(ScFv)was connected through the linker and constructed into the bacteriophage vector Pcomb3xss;then,it was carried to Escherichia coli TG1 through electrotransformation,and the ALD ScFv phage display library was constructed accordingly.Three to five rounds of enrichment screening were performed against the library.Monoclonal clones,identified by enzyme-linked immunosorbent assay(ELISA)competitive method,were selected for phage supernatant preparation,and a highly competitive clone sequence was obtained.The screened clone sequence was inserted into the pCMV3 expression vector,and the HEK293 cell was transfected using the transient transfection method after the plasmid was extracted.One week later,the supernatant was collected,and its purity and expression were identified by affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Results After the 5th immunization,the serum titers of 5 rabbits were indirectly tested,and the results showed that the serum titers of 4# and 5# white rabbits were still greater than 10,000 after being diluted by 32,000 times.The test results based on the competitive method showed that the ratio of low to high values in the plasma sample of 5#white rabbit was 2∶1,superior to that of other white rabbits.The 5# white rabbit was selected for phage library construction.The VL and VH gene fragments were amplified by conventional polymerase chain reaction,and then bridged into ScFv(VL+VH).The agar gel electrophoresis analysis showed that the size of the band was about 750 bp,which was consistent with the size of the originally designed fragment.ScFv was cleaved and electroporated into Escherichia coli TG1 to construct a phage library with a storage capacity of 4.73 × 108 cfu·mL-1.After 3 rounds of washing,300 monoclonal clones were selected from the outbound petri dishes to prepare monoclonal bacteriophages.The ELISA results showed a positive rate of 100％among the 300 clones,and 42 clones were tested positive for calibration competition,with a screening rate of 14％.The 42 positive clones were further subjected to clinical sample competition testing,and 16 monoclonal strains that met the requirements were screened.The 16 strains were retested,and the results of the two tests were consistent.After sequencing,6 antibody sequences were selected for construction and expression.After purification,SDS-PAGE reduced gel electrophoresis results showed that there were bands at positions 50,000 on the heavy chain and 25,000 on the light chain.Six highly affinitive and competitive rabbit ALD monoclonal antibodies were obtained.Conclusion Six highly affinitive and competitive rabbit ALD antibodies are successfully screened using phage display technology,which provides a reference method for the discovery of small molecule antibodies.The screened AD1 85 and AD277 antibodies show a competitive advantage twice that of the positive control in the competition of calibration and clinical samples,providing a possibility for the development of raw materials for ALD detection kits.

Effect of HtrA serine peptidase 3 gene on choroidal neovascularization and M2 macrophage polarization / 眼科新进展

Objective To investigate the effect of the HtrA serine peptidase 3(HTRA3)gene on choroidal neovascu-larization(CNV)and M2 macrophage polarization.Methods Fasting venous blood was collected from 30 patients with wet age-related macular degeneration(wAMD group)and 30 healthy subjects(normal group).The serum HTRA3 messen-ger ribonucleic acid(mRNA)level was detected by quantitative reverse transcription polymerase chain reaction(qRT-PCR).RF/6A cells were randomly divided into the control group,NC-sh group and HTRA3-sh group.Lentiviral vectors of NC-shRNA and HTRA3-shRNA were transfected into RF/6A cells in the NC-sh group and HTRA3-sh group by Lipo-fectamine2000.HTRA3 transfection was detected by qRT-PCR and Western blot.Then,the RF/6A cells were randomly di-vided into the N group,H group,H+NC-sh group and H+HTRA3-sh group.After cell transfection,RF/6A cells in the N group were cultured in a RPMI 1640 complete medium at a normoxia state,and cells in other groups were cultured in a RP-MI 1640 medium with 200 mmol·L-1 CoCl2 at a hypoxia state.Tubule formation was measured by Matrigel.The C57BL/6J mice were divided into the control group,CNV group,CNV+NC-sh group and CNV+HTRA3-sh group,with 12 mice in each group.Mice in the control group were unmodeled mice,and mice in the other groups were laser-induced CNV model mice.NC-shRNA and HTRA3-shRNA lentiviral vectors with a titer of 1 × 1011 TU·mL-1 were administered to mice in the CNV+NC-sh group and CNV+HTRA3-sh group via intravitreal injection.Mice in the control group and CNV group were in-jected with phosphate buffered saline.After 7 days of treatment,the mice were examined by fundus fluorescein angiogra-phy,and the eyeballs received hematoxylin & eosin staining.The mRNA levels of HTRA3,chitinase-like protein 3(Ym-1),arginase 1(Arg-1),inducible nitric oxide synthase(iNOS),cyclooxygenase-2(COX-2)and vascular endothelial growth factor(VEGF)in RF/6A cells or choroidal tissues were detected by qRT-PCR.The protein expression levels of HTRA3,VEGF and nuclear factor kappa B(NF-κB)p65 in RF/6A cells or choroidal tissues were detected by Western blot.Re-sults Compared with the normal group,serum HTRA3 mRNA level of patients in the wAMD group increased(t=11.804,P＜0.001).Compared with the control group and NC-sh group,the expressions of HTRA3 mRNA and protein in RF/6A cells in the HTRA3-sh group decreased(all P＜0.05).Compared with the N group,the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF in RF/6A cells in the H group increased(all P＜0.05).Compared with the H+NC-sh group,the number of closed lumen and the mRNA and protein expressions of HTRA3 and VEGF decreased in RF/6A cells in the H+HTRA3-sh group(all P＜0.05).Compared with the control group,the mRNA and protein expression levels of HTRA3 increased,the relative fluorescence intensity of CNV increased,the mRNA levels of Ym-1 and Arg-1 in-creased,the iNOS and COX-2 mRNA levels decreased,and the NF-κB p65 protein expression level increased in mice of the CNV group(all P＜0.05).Compared with the CNV+NC-sh group,the mRNA and protein expression levels of HTRA3 de-creased,the relative fluorescence intensity of CNV decreased,the mRNA levels of Ym-1 and Arg-1 decreased,the mRNA levels of iNOS and COX-2 increased,and the NF-κB p65 protein expression level decreased in mice of the CNV+HTRA3-sh group(all P＜0.05).Conclusion Down-regulation of HTRA3 can inhibit the formation of CNV and the polarization of M2 macrophages.HTRA3 may be an important potential target for the prevention and treatment of wAMD.

Screening and identification of human monoclonal antibodies against low-calcium response V antigen of Yersinia pestis / 中国人兽共患病学报

To characterize human antibodies against low-calcium response V(LcrV)antigen of Yersinia pestis,the mono-clonal antibodies were screened and assayed.Antibody gene was derived from peripheral blood mononuclear cells of the vaccin-ees immunized by plague subunit vaccine in phase Ⅱb clinical trial.Human ScFv antibody library was constructed by phage dis-play.After panning library by using recombinant LcrV antigen,antibody variable genes were sequenced and converted into IgG1 format to evaluate its binding specificity and relevant parameters.An anti-plague human ScFv antibody library was estab-lished contained 7.54× 108 independent clones.After panning by LcrV antigen,3 human antibodies named as RV-B4,RV-D1 and RV-E8,respectively,were identified.Using indirect enzyme-linked immunosorbent assay(ELISA)and Western blot(WB),the specific bindings of the mAbs to LcrV antigen were confirmed.The dissociation constant(KD)of them to LcrV is 2.1 nmol/L,1.24 nmol/L and 42 nmol/L,respectively.Minor protective efficacy was found among 3 human antibodies in Y.pestis 141-infected mice.Three anti-LcrV monoclonal antibodies generated from immunized vaccinees were binding specific antibod-ies and could not block plague infection in mice.These antibodies are the potential candidate reagents for basic research of plague immunity and the application of plague diagnosis.

Phage antibody library technology in tumor therapy: a review / 生物工程学报

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.

Single chain antibody fragment display systems: a review / 生物工程学报

Single chain antibody fragment (scFv) is a small molecule composed of a variable region of heavy chain (VH) and a variable region of light chain (VL) of an antibody, and these two chains are connected by a flexible short peptide. scFv is the smallest functional fragment with complete antigen-binding activity, which contains both the antibody-recognizing site and the antigen-binding site. Compared with other antibodies, scFv has the advantages of small molecular weight, strong penetration, low immunogenicity, and easy expression. Currently, the most commonly used display systems for scFv mainly include the phage display system, ribosome display system, mRNA display system, yeast cell surface display system and mammalian cell display system. In recent years, with the development of scFv in the field of medicine, biology, and food safety, they have also attracted much attention in the sectors of biosynthesis and applied research. This review summarizes the advances of scFv display systems in recent years in order to facilitate scFv screening and application.

Identification and optimization of peptide inhibitors to block VISTA/PSGL-1 interaction for cancer immunotherapy

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8+ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity in vivo. The peptide DVS3-Pal was further designed by d-amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8+ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy.

Progresses of phage display technology application in fully human antibody discovery / 免疫学杂志

Phage antibody display technology is currently the most widely used in vitro antibody screening technology,which uses bacteriophages as a vector,and inserts exogenous antibody library genes into phage capsid protein genes,and expresses the capsid protein on the phage surface while also displays the antibody protein.Antibody drugs play an important role in tumor immunity and microbial immunity due to their targeting advantages,which is also an important driving force for them to become a hot spot in the field of pharmaceutical research and development.Therefore,this article reviews the background,basic principles,antibody library types and antibody fragment types of phage display technology,and looks forward to the latest progress and application prospects of fully human antibodies.

Isolation and identification of plague phage from Suncus murinus in Yunnan Province and its epidemiological significance / 中华地方病学杂志

Objective:To study the basic situation of Yunnan Province Suncus murinus carrying plague phage and to explore its epidemiological significance. Methods:From 2015 to 2018, a survey of plague host animals was carried out in 10 investigation sites in the historical plague foci, new plague foci (after 1982) and stubborn plague foci of domestric mouse in Yunnan Province. The plague phage was isolated and cultured from the intestinal specimens of Suncus murinus. The growth of plaque was observed by double-layer plate method, and the morphology and structure of plague phage were observed under electron microscope. At the same time, intestinal samples were taken to detect the structural gene caf1 of F1 antigen of Yersinia pestis. Results:In this study, a total of 157 Suncus murinus were captured and 16 strains of plague phage were isolated, with a total isolation rate of 10.19%. There was no difference in plague phage isolation rate between historical plague foci (10.00%, 1/10) and stubborn plague foci (16.22%, 12/74), new plague foci (4.11%, 3/73, χ 2 = 0.00, P = 0.965; Fisher test, P = 1.000). However, there was a difference in plague phage isolation rate between stubborn plague foci and new plague foci (χ 2 = 5.88, P = 0.015). There was no significant difference in the isolation rate of plague phage among different sex, growth period and habitat ( P > 0.05). The plaque morphology of the isolated plague phage was diverse, of which four strains were myotavirus phages; and all samples were negative for F1 antigen structural gene caf1. Conclusions:Suncus murinus is widely distributed in the domestic mouse plague foci in Yunnan Province, and the animals carry a certain number of plague phage. Regular surveillance of Suncus murinus and their plague phage has a certain guiding significance for the surveillance and early warning of plague in Yunnan Province.

Research progress of Yersinia pestis phage and their receptors / 中华地方病学杂志

Yersinia pestis phage is a virus that is parasitic within Yersinia pestis and can specifically lyses Yersinia pestis. The adsorption sites of phage infesting host bacteria are called receptor binding protein (RBP), including extracellular membrane protein, lipopolysaccharide, teichoteic acid, pili, flagella, capsular polysaccharide, etc., of which extracellular membrane protein and lipopolysaccharide are the receptors of Yersinia pestis phage. RBP plays a decisive role in the process of Yersinia pestis phage infecting Yersinia pestis. Therefore, the classification, isolation and application of Yersinia pestis phage are summarized; the research progress in identification and structure of Yersinia pestis phage receptor is analyzed, which is helpful in understanding the cleavage mechanism of Yersinia pestis phage and the interaction mode with Yersinia pestis from the molecular level, and provide more powerful support for in-depth study on Yersinia pestis phage receptor.

Phenotypic characteristics of wild-type plague phage growth in different experimental environments / 中国热带医学

@#Abstract: Objective To observe the phenotypic characteristics of 3 wild-type plague phages under different experimental environments, providing scientific evidence for the identification of phage biological characteristics and the study of their interaction with host bacteria in the future. Methods The sensitivity of 3 wild-type plague phages were detected by using liquid culture method, emisolid medium method and micro-liquid culture method based on OmniLog TM microbial identification system. Results The growth result based on LB liquid medium showed that the growth of plague phage 476 for 20-24 hours at both 28 ℃ and 37 ℃was better than that of plague phages 087 and 072204 at 37 ℃, and the growth of plague phages 087 was better than that of plague phages 072204 at 37 ℃. With the attenuated plague bacterium EV76 as the host bacterium, phage 476 was able to form visible plaque on double-layer agar medium for 20-20 hours at both 28 ℃ and 37 ℃, phages 087 and 072204 were only able to form opaque plaque on double-layer agar medium for 20-24 hours at 37 ℃. The growth results based on OmniLogTM system showed that when plague phage was lysed in EV76 strain at 33 ℃, the first row appeared as a straight line with a peak of no more than 100 in the 96-well microplate curve chart. As the phage quantity decreased, the dilution plate appeared with growth curve similar to EV76 strain in turn, and the color of tetrazolium dyes in the experimental wells gradually deepened as the phage number decreased and the host bacteria number increased. Therefore, it indicates that phage 476 was sensitively at both 28 ℃ and 37 ℃, while phage 087 and 072204 were temperature-dependent only at 37 ℃ to attenuated plague bacterium EV76. Conclusions The lysing ability of 3 wild-type plague phages are temperature-dependent, and the growth results are consistent under the three experimental conditions.

Research progress of phage display vaccine / 中国生物制品学杂志

@#Bacteriophages are viruses that infect microorganisms such as bacteria,fungi,actinomycetes and spirochetes.Because of the inherent immunogenicity,genetic plasticity,stability,safety and many other advantages,it has unique potential in vaccine research and development. At present,there are countless researches using it to construct vaccine delivery platforms,mainly including three forms,phage display vaccine,phage DNA vaccine and hybrid phage DNA vaccine,of which the phage display vaccine is the most widely studied. Phage display technology is a novel vaccine preparation technology,which is a molecular biology technology using phage as carrier,integrating foreign polypeptide or protein genes into phage genes and displaying them on the surface of phage in the form of fusion protein. This review mainly elaborated the immunological basis of phage display vaccine,the display system and its application in disease prevention,so as to provide a reference for the development and application of phage display vaccine.

Screening and identification of single-chain fragment variable against the receptor-binding domain of spike protein of SARS-CoV-2 / 中国生物制品学杂志

@#Objective To construct a single-chain fragment variable(scFv)phage display library against receptor-binding domain(RBD)of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(S)to screen specific scFv and identify the function.Methods m RNA was extracted from spleen cells of mice immunized with RBD protein and reversely transcribed into c DNA,with which as template,genes of the hight chain fragment of variable(VH)and light chain fragment of variable(VL)of scFv were amplified and then assembled into scFv gene fragment through splicing overlap extension PCR(SOE-PCR).The scFv gene fragment was inserted to phage vector to construct scFv phage display library.After four rounds of biopanning,the scFv gene with strong binding ability to RBD was screened and expressed recombinantly,purified and identified for biological activity.Results The constructed scFv phage library showed a titer of 6.0×10(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(11)pfu/m L.After four rounds of biopanning,four scFv strains with strong binding to RBD were selected,namely scFv11,scFv12,scFv25and scFv28.scFv was mainly expressed in the form of inclusion body with a relative molecular mass of about 27 000,a concentration of 2.4 mg/m L and a purity of about 90%,which bound specifically to mouse monoclonal antibody against His labeled by HRP after purification.All four scFv strains bound specifically to RBD recombinant protein,among which the other 3 scFv strains bound to the S protein of wild type and multiple mutant strains except scFv28.All four strains showed dose-dependent interaction with RBD,with affinity dynamic fitting dissociation constants(K_Ds)8.9,5.92,10.67and 2.36 nmol/L,and steady-state fitting dissociation constants(K_Ds)of 5.3,6.5,8.7 and 5.8 nmol/L,respectively.scFv11,scFv12 and scFv25 simultaneously identified three independent RBD polypeptides,including RBD2(S(334～353)),RBD9(S(334～353)),RBD9(S(439～458))and RBD13(S(439～458))and RBD13(S(499～518)).Homologous model of scFv constructed by online server SWISS-MODEL showed a good quality and was used for molecular docking.The interface at which scFv11 interacted with RBD only partially coincided with the interaction interface of human angiotensin converting enzyme 2(ACE2)and RBD,and the interaction interfaces of scFv12 and scFv25 with RBD were quite different from that of ACE2.Conclusion In this study,scFv specifically bound to SARS-Co V-2 RBD was screened and prepared through constructing scFv phage library against SARS-CoV-2 RBD,which provided experimental basis for further development of anti-SARS-CoV-2 drugs and detection reagents.

Biological characteristics of Escherichia coli phage and Staphylococcus aureus phage isolated from sewage / 西安交通大学学报(医学版)

【Objective】 Escherichia coli phage (ECP) and Staphylococcus aureus phage (SAP) isolated from sewage were used as research objects, and their biological characteristics were analyzed to provide new experimental materials for the application of phages. 【Methods】 ECP and SAP were purified and cultured by double-layer agar method. Then a series of biological characteristics of these two phages were preliminarily analyzed by electron microscope observation, optimal multiplicity of infection (MOI) test, one-step growth curve test, temperature, pH, chloroform and ultraviolet sensitivity tests, respectively. 【Results】 The results of biological characteristics showed that ECP and SAP were both virulent phages, belonging to myoviridac family. Their optimal MOI was 10-1, and they had strong resistance to ultraviolet light. The cleavage volume of ECP was 76.3 PFU/cell, while that of SAP was 8.3 PFU/cell. ECP had a wide range of temperature tolerance and could stably survive at 30-50 ℃, while SAP was more sensitive to temperature and could be completely inactivated at 50 ℃ for 1 h. ECP could maintain a good lysis activity in the range of pH 5-11, while SAP in the range of pH 6-9. ECP had strong resistance to chloroform and was non-membranous phage, while SAP was more sensitive to chloroform and was a membranous phage. 【Conclusion】 ECP and SAP are both virulent phages and have strong resistance to ultraviolet light. The lysability, temperature, pH, and chloroform tolerance of ECP are stronger than those of SAP.

Screening and identification of CD44v9 specific peptide ligand for gastric cancer detection / 西安交通大学学报(医学版)

【Objective】 To screen and verify a peptide ligand specific for CD44v9. 【Methods】 A 12-mer phage peptide library was screened on CD44v9 coated on solid phase. Candidate sequences emerged after sequencing. Candidate phages were selected using enzyme-linked immunosorbent assay. The best sequence was chosen for further study. Binding of C9-3 to CD44v overexpressed HEK-293 cells was determined using immunofluorescence. Binding affinity and specificity were verified on gastric cancer tissues with immunohistochemistry. 【Results】 Phages significantly were enriched during panning process. After sequencing, nine individual sequences occurred in 30 selected clones. Among the 9 candidate sequences, C9-3 exhibited the highest frequency. Results of ELISA showed that C9-3 had the highest OD value and selectivity. Thus, C9-3 was chosen for peptide probe synthesis. C9-3 probe stained CD44v overexpressed HEK-293 cells, but not empty vector transfected HEK-293 cells. Immunohistochemistry scores of C9-3 were significantly different between gastric cancer and paracancer tissues (t=3.953, P<0.01). A linear positive correlation was observed between C9-3 binding and CD44v9 expression (r=0.823, P<0.01). 【Conclusion】 In this study, peptide ligand of CD44v9 was successfully screened. The peptide can bind to cells and cancer tissues via CD44v9. It has potential for gastric targeting probes.

Targeted innovative design of Bt Cry toxin insecticidal mimics / 生物工程学报

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Isolation and Phenotypic Characterization of Lytic Bacteriophage with Activity against Pseudomonas aeruginosa

Aims: To perform the isolation and phenotypic characterization of bacteriophage with lytic activity against Pseudomonas aeruginosa. To demonstrate that this type of viral agent can be isolated from the environment and used for the biocontrol of resistant bacterial types, such as Pseudomonas aeruginosa. Study Design: This study was an experimental study. Place and Duration of Study: The study was conducted at, Bacteriology and Mycology Laboratory in the Veterinary Hospital at the School of Agricultural Sciences, Innovation and Business of the University of Passo Fundo (ESAN/UPF) and Center for Diagnosis and Research in Animal Health of the University of Passo Fundo (CDSA/UPF), between April 2022 and June 2022. Methodology: Samples of untreated water were inoculated with the host bacterium strain Pseudomonas aeruginosa ATCC 27853 in an enriched media After the incubation period in, a phage filtrate was obtained by centrifugation followed by filtration. We verified the presence of bacteriophages using spot test and we carried out its purification by the method of sterile toothpick plate transfer on bacterial overlay semi-solid agar. Amplification was performed using an SM buffer elution procedure to produce a stock of viral material. Through assays in Petri dishes with bacterial overlay, we performed titration and phenotypic characterization regarding the lysis spectrum and efficiency of phage infection in the host. Results: We managed to isolate a morphologically characterized lytic bacteriophage with approximately 1 mm of diameter, high clarity in the inhibition area, the presence of halo and well-demarcated edges. The bacteriophage, named as Pseudomonas aeruginosa Phage UPF_PaBP1, demonstrated the infection capacity of the target bacteria in all tested dilutions and a stock preparation with a titre of 6.5 x 10? PFU/ml was obtained for future use. Conclusion: The isolated phage showed strong lytic activity against the bacterial host, a finding that nourishes our expectations regarding the use of this phage as a biocontrol agent and phage therapy.

Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.