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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Development of novel-nanobody-based lateral-flow immunochromatographic strip test for rapid detection of recombinant human interferon α2b / 药物分析学报

Recombinant human interferon α2b(rhIFNα2b)is widely used as an antiviral therapy agent for the treatment of hepatitis B and hepatitis C.The current identification test for rhIFNα2b is complex.In this study,an anti-rhIFNα2b nanobody was discovered and used for the development of a rapid lateral flow strip for the identification of rhIFNα2b.RhIFNα2b was used to immunize an alpaca,which established a phage nanobody library.After five steps of enrichment,the nanobody I22,which specifically bound rhIFNα2b,was isolated and inserted into the prokaryotic expression vector pET28a.After subsequent purification,the physicochemical properties of the nanobody were determined.A semiquantitative detection and rapid identification assay of rhIFNα2b was developed using this novel nanobody.To develop a rapid test,the nanobody I22 was coupled with a colloidal gold to produce lateral-flow test strips.The developed rhIFNα2b detection assay had a limit of detection of 1 μg/mL.The isolation of I22 and successful construction of a lateral-flow immunochromatographic test strip demonstrated the feasibility of performing ligand-binding assays on a lateral-flow test strip using recombinant protein products.The principle of this novel assay is generally applicable for the rapid testing of other com-mercial products,with a great potential for routine use in detecting counterfeit recombinant protein products.

Preliminary Study on Screening and Identification of Lewis a Antigen Mimic Epitope in Alpaca Phage Display Nanobody Library / 中国实验血液学杂志

OBJECTIVE@#To establish a new method for synthesizing Lewis blood group antigens, that is, the mimotopes of Lewis blood group antigens were screened by using an alpaca phage display nanobody library.@*METHODS@#We selected mimotopes of the Lewis a (lea) antigen by affinity panning of an alpaca phage display nanobody library using a monoclonal anti-lea antibody. Enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of the positive clones for the monoclonal anti-lea antibody, and the high-affinity positive clones were selected for sequencing and synthesis. Finally, the sensitivity, specificity and reactivity of the synthesized lea mimotope in clinical samples were verified by ELISA.@*RESULTS@#A total of 96 phage clones were randomly selected, and 24 were positive. Fourteen positive clones with the highest affinity were selected for sequencing. The result showed that there were 5 different sequences, among which 3 sequences with the highest frequency, largest difference and highest affinity were selected for expression and synthesis. The sensitivity and specificity of lea mimic antigen by ELISA showed that, the minimum detection limit of gel microcolumn assay (GMA) and ELISA method were 25 times different, and the lea mimic antigen had no cross reacted with the other five unrelated monoclonal antibodies(P<0.001). Finally, 30 clinical plasma samples were analyzed. The mean absorbance of the 15 positive plasma samples was significantly higher than that of the 15 negative plasma samples (P=0.02). However, the positive signal values of the clinical samples were much lower than those of the monoclonal antibodies.@*CONCLUSION@#A new method of screening lea mimic antigen by using alpaca phage nanoantibody library has been established, which is expected to realize the screening of lea mimotopes, thus realizing the application of high-sensitivity detection methods such as ELISA and chemiluminescence in blood group antibody identification.

Phage display technology and its application in antivirals discovery / 药学学报

The COVID-19 outbreak has drawn attention to viral infectious diseases once again, and the development of antiviral drugs for both known and potentially emerging viruses is of great significance. In recent years, peptides and protein drugs are becoming a hot spot in the field of antiviral drug research and development. Phage display technology, as a powerful tool for screening peptides and protein drugs, has been increasingly concerned in the academic and industrial fields. The present review introduced the basic principle of phage display technology, summarized phage display libraries often used in antiviral drug discovery and their applications, discussed the challenges and future direction of antiviral drug research and development based on phage display technology.

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.

Screening and formulation of novel carriers for Xanthomonas bacteriophage to control bacterial leaf blight disease

Aims@#This study was aimed to evaluate the potential of several carriers to formulate the phages and retain their activity under various pH and temperature conditions.@*Methodology and results@#The skim milk, rice flour, corn flour and CalnuXan (calcium and magnesium) as carriers to formulate the isolated phage to maintain its activity under extreme pH and temperature conditions. Two phages formulated with carriers retained their viability at pH 5, pH 7 and pH 9 compared to that of the unformulated phages. Besides, the formulated phages also retained a high titre compared to the unformulated phages when they were exposed to 37 °C and 45 °C. Based on the in vitro study of the formulation, it was applied in the glass house. The plant height, leaf chlorophyll and disease scoring were recorded and analyzed. In the glass house, the rice plant treated with formulated phages showed higher plant height and chlorophyll content than those treated with unformulated or untreated phages. Nonetheless, both formulated and unformulated protected the rice plant, which showed lower disease severity than the untreated group.@*Conclusion, significance and impact of study@#Phage therapy has been used for treating plant diseases caused by pathogenic bacteria. Despite their effectiveness in killing the pathogen in vitro, the results were not reproducible in the field. Bacteriophages (phages) are sensitive to environmental factors and infection efficiency was dropped when exposed to harmful environments. However, this study successfully formulated two novels Xanthomonas phages, as biocontrol agents against bacterial leaf blight (BLB) disease in rice.

Preparation of anti-VirB12 nanoantibodies, a surface protein of type Ⅳ secretory system of Brucella / 国际生物医学工程杂志

Objective:To prepare camel-derived nanoantibodies that can bind to the recombinant protein VirB12 antigen with high affinity and lay the foundation for further research.Methods:Xinjiang Bactrian camels were immunized six times with VirB12 recombinant protein, total RNA was extracted from lymphocytes isolated from peripheral blood, and the VHH gene fragment was amplified by nested PCR to construct a phage VHH display library. ELISA solid-phase affinity and enrichment methods were used for screening. After three rounds of affinity screening, the clones enriched in the second and third rounds were randomly picked out, and the binding of a nanoantibody with soluble expression to VirB12 was analyzed by ELISA. After sequence determination and multiple alignment, repetitive sequences were removed, and finally five non-redundant sequences were obtained, which were named D1, E6, H8, H9, and H10. The five identified nanoantibody genes were transformed into the WK6 strain, and the soluble expression of an intercellular substance was carried out at 16 °C. After purified expression of Ni-NTA, the binding ability and thermal stability of nanoantibodies and the antigen VirB12 protein were detected by Western Blot and ELISA.Results:Five strains of nanoantibodies were expressed in WK6 bacteria in soluble form. SDS-PAGE showed that the purity of five anti-VirB12 nanoantibodies was close to 90%, and they had high antigen-binding activity and obvious antigen-antibody concentration dependence. All four strains of nanoantibodies showed high thermal stability, and after being treated at 90 ℃, they could still retain more than 60% binding activity.Conclusions:In the study, a VHH phage display library with a capacity of 2.8×10 8 cfu/ml was constructed from Xinjiang Bactrian camel lymphocytes immunized with VirB12 recombinant protein. Five anti-VirB12 nanoantibodies with high affinity and thermal stability were obtained through solid-phase screening and enrichment and soluble monoclonal ELISA detection. These results laid the foundation for further development of VirB12 nanoantibodies.

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.

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 to 11.0 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.