Serviço de convivência e fortalecimento de vínculos: reXistências por via do acontecimento / A public service for youth: community identity through resistance / Un service public pour la jeunesse: l'identité communautaire par la résistance / Un servicio público para los jóvenes: identidad comunitaria por vía de la resistencia

Este texto traz um relato de experiência advindo das atividades de uma orientadora social realizado no Serviço de Convivência e Fortalecimento de Vínculos (SCFV) de um Centro de Referência de Assistência Social (Cras) situado na Grande Florianópolis (SC). O serviço integrou 20 jovens com idade entre 12 e 18 anos no espaço de uma instituição de formação policial. Este trabalho analisará: (1) um acontecimento ocorrido na instituição policial; (2) quais foram os seus desdobramentos; e (3) como a orientadora social atuou na mediação do grupo. A análise da do relato de experiência e a mediação da orientadora social foram embasadas no conceito de acontecimento de Gilles Deleuze. No decorrer dos encontros, a instituição parceira do projeto adotou medidas disciplinares que desqualificavam a cultura proveniente dos cotidiano dos(as) jovens participantes, o que levou o grupo a constituir uma identidade coletiva a partir da resistência à violência simbólica. Assim, diante da situação-problema, tornou-se possível uma experiência de fortalecimentos de laços comunitários

This paper discusses a situation that occurred in a public youth service for young people living in vulnerable city areas. The service worked with 20 young people aged between 12 and 18 years and the meetings took place at a police training institution. Inspired by Gilles Deleuze's theory, this text analyzes 1) an event that occurred at the police institution, 2) their consequences and 3) how the professional responsible dealt with the event in a group context. At some point in the meetings, the police training institution adopted disciplinary measures that disqualified the participants' culture, leading the group to create a collective identity to resist symbolic violence. Thus, a community gathering experience emerged in the face of the conflicted situation

Le texte discute une situation survenue dans une fonction publique désignée pour accueillir des jeunes vivant dans des quartiers vulnérables. Le service comprenait 20 jeunes de 12 à 18 ans et les réunions se sont déroulées dans un établissement de formation policière. Inspirées par la théorie de Gilles Deleuze, ce texte analyse 1) un événement survenu dans l'institution policière, 2) qu'elles en ont été ses conséquences et 3) comment le professionnel responsable a agi pour faire face à l'événement dans un contexte de groupe. À un moment donné au cours des réunions, l'institution de formation a adopté des mesures disciplinaires qui disqualifiaient la culture des jeunes participants, ce qui a conduit le groupe à se forger une identité collective basée sur la résistance à la violence symbolique. Par conséquent, une expérience de rassemblement communautaire a émergé face à la situation conflictuelle

El texto se refiere a una situación ocurrida en un servicio público designado para trabajar con jóvenes que viven en zonas vulnerables de la ciudad. El servicio incluyó a 20 jóvenes de entre 12 y 18 años y las reuniones se ubicaron en una institución de capacitación policial. Este texto analiza 1) un suceso que ocurrió en la institución policial, 2) cuáles fueron sus consecuencias y 3) cómo actuó el profesional responsable del servicio para enfrentar el suceso en un contexto grupal. El análisis de esta situación y la referencia teórica que apoyó los actos profesionales se inspiraron en la teoría de Gilles Deleuze. En algún momento de los encuentros, la institución de capacitación policial adoptó medidas disciplinarias que descalificaron la cultura de los jóvenes participantes, lo que llevó al grupo a constituir una identidad colectiva basada en la resistencia contra la violencia simbólica. Por lo tanto, ante la situación conflictiva, fue posible una experiencia de reunión comunitaria

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.

Interdomain Disulfide Bond Enhances the Stability of Single-chain Antibody Against Pro-gastrin-releasing Peptide / 中国生物化学与分子生物学报

Pro-gastrin-releasing peptide (ProGRP) is a specific marker of small cell lung cancer.

Innovative approaches to improve serodiagnosis of Strongyloides stercoralis infection

ABSTRACT Strongyloidiasis is a helminthiasis of neglected condition that has no gold standard parasitological diagnosis due to the intermittent release of larvae in feces. This study aimed to use an scFv (single chain variable fragment) obtained by Phage Display, previously validated to detect immune complexes in serum samples from individuals infected with Strongyloides stercoralis by enzyme-linked immunosorbent assay (ELISA). Now the ability of scFv to detect the immune complexes was verified by immunofluorescence, flow cytometry using magnetic beads and surface plasmon resonance (SPR). As ELISA, the SPR, immunofluorescence and flow cytometry demonstrated the ability of scFv to detect immune complexes in sera from individuals with strongyloidiasis and discriminate them from sera of individuals with other parasitic diseases and healthy individuals. Besides de conventional ELISA, the novel approaches can also be promptly applied as auxiliary diagnostic tools to the existing parasitological method for accurate diagnosis of human strongyloidiasis.

Preparation of anti-GPC3 single chain antibody for targeted detection of hepatocellular carcinoma / 生物工程学报

Glypican-3 (GPC3) is a key member of Glypican family and plays an important role in the development, angiogenesis and metastasis of hepatocellular carcinoma (HCC). Most HCC overexpresses GPC3, but GPC3 is hardly detected in normal adult liver and benign liver lesions, so it is regarded as a highly specific diagnostic marker and an ideal therapeutic target for HCC. In this study, we cloned the heavy and light chain variable region gene from the monoclonal antibody targeted to GPC3 screened in the previous stage, and linked it with a segment of flexible peptide (Linker) to obtain the single chain antibody against GPC3. The single chain antibody gene was cloned into vector for prokaryotic expression and purified to obtain high purity protein. Detection shows that the single-chain antibody produced by us has the same binding activity with the full-length antibody, and can accurately target the tumor site of Huh7 tumor-bearing model mice after coupling Cy5.5 fluorescence, suggesting that the single-chain antibody has the potential to realize multi-directional liver cancer precise surgical navigation under the guidance of a probe.

The improvement of anti-HER2 scFv soluble expression in Escherichia coli

The relationship between the expression of HER2 and malignity of breast tumors has led to the generation of antibodies targeting HER2+ tumors. In addition, the expression of scFvs, as the smallest antigen-binding region of antibody containing two disulfide bonds in Escherichia coli often results in accumulating non-functional protein in the cytoplasm. A redox-modified strain of E. coli such as Origami (DE3) may facilitate the formation of proper disulfide bond in cytoplasm. The present study aimed to optimize the expression of anti-HER2 scFv in Origami and evaluate the influence of induction temperature, and host strain on the solubility of the protein. To this aim, chemicallysynthesized anti-HER2 scFv of Trastuzumab was cloned in pET-22b (+). The results demonstrated that anti-HER2 scFv is expressed in Origami, purified by using Ni-NTA column, and detected by anti-His antibody in Western blot analysis. The highest anti-HER2 scFv expression in Origami was achieved 24 h after IPTG induction (1 mM) at 37 ºC. Further, the total anti-HER2 scFv expression level was higher in BL21, compared to Origami strain. However, the ratio of soluble/insoluble forms of anti-HER2 scFv increased in Origami strain. Furthermore, higher soluble expression was achieved when the culture of recombinant Origami was conducted at lower temperature (25 ºC).

Construction and application of EV71 ScFv high-throughput cassette expression system / 中华实验和临床病毒学杂志

Objective@#To construct a method to express ScFv antibody from PCR products, and use it in phage display for high-throughput ScFv expression.@*Methods@#Cytomegalovirus (CMV) promotor, ScFv and BGH-Poly A gene fragments were amplified by PCR. Overlapping PCR was used to form a tandemly linear cassette gene. By transiently transfected into 293T cells, ScFv antibodies expression of cassette gene were tested by Western blot, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescent antibody (IFA). Ninety-six clones of antibody genes in phage library were selected and expressed by cassette expression system. The expression level was evaluated and analyzed.@*Results@#Three fragments were obtained and a cassette expression system formed. Cassette expression system worked successfully in 293T cells, as a Mr.38×103 brand could observed in Western blot assay. The expressed antibody could specifically bind to its antigen by result of ELISA and IFA. This cassette expression system could also be used in phage display for high-throughput panning.@*Conclusions@#The cassette expression system was constructed successfully and high-throughput antibody expression has been achieved.

The improvement effect of scFv17 on the gait of triple-transgenic mice / 中国应用生理学杂志

OBJECTIVE@#To observe the gait changes of Alzheimer's disease PS1M146V/APPswe/tauP301L triple-transgenic (3xTg-AD) mice and to investigate the improvement effect of single chain variable domain antibody fragment 17 (scFv17) on the gait.@*METHODS@#In the present study, a selection of 6-month-old 3xTg-AD mice (=18) and C57BL/6 wild-type mice (=24) was performed. First, we observed their gait changes and found that the gait of 12-month-old 3xTg-AD mice was severely damaged. Then, the two groups of mice were randomly divided into four groups:WT+PBS(=12), WT+scFv17(=12), 3xTg-AD+PBS(=9) and 3xTg-AD+scFv17(=9). The gait behavior test and pathological test were performed after 12 weeks'continuous administration of scFv17 (1.5 mg/kg) or an equal volume of PBS (0.01 mol/L) by nasal gavage twice a week.@*RESULTS@#Compared with the same month old wild type mice, the rear track width of 12 month old 3xTg-AD mice was increased(<0.01), swing time percent was decreased (<0.01), stance time percent was increased(<0.01), so the ability of movement coordination and balance was seriously damaged. ScFv17 could improve the coordination and balance ability of 12 month old 3xTg-AD mice(<0.01). The morphological structure of 3xTg-AD mice cerebellar Purkinje cells was improved. The treatment of scFv17 increased the Nissl body number of the cerebellar Purkinje cells of 3xTg-AD mice (<0.01). scFv17 reduced the amyloid β protein (Aβ) plaques in the cerebellar cortex of 3xTg-AD mice (<0.01), and scFv17 reduced the intracellular neurofibrillary tangles (NFT) of the cerebellar Purkinje cells of the 3xTg-AD mice (<0.01).@*CONCLUSIONS@#The coordination and balance ability of 3xTg-AD mice was significantly impaired. ScFv17 can improve gait behaviour in the 3xTg-AD significantly.The mechanism may be related to the improvement of the structure and protein function of cerebellar Purkinje cells, and the eliminating of the Aβ plaques and the neurofibrillary tangles.

Preparation and identification of anti-human ICAM-1 scFv / 生物工程学报

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.

Application of a single-chain fragment variable (scFv) antibody for the confirmatory diagnosis of hydatid disease in non-endemic areas

Background: Hydatid disease is a serious parasitic disease threatening public health. Because of its rarity in non-endemic coastal areas, determining the nature and origin of a chronic, enlarged liver cystic mass is challenging in these regions. Under these circumstances, physicians need a confirmatory diagnostic tool beyond immunological and radiological examinations. This study investigated a novel human single-chain fragment variable (scFv) antibody for the confirmative diagnosis of 18 atypical hydatid disease cases in non-endemic coastal areas. Results: A scFv antibody against cystic echinococcosis was produced by genetic engineering and then applied to the immunohistochemical diagnosis of 18 cases of cystic echinococcosis presented in non-endemic coastal areas. The diagnosis of these cases by ultrasound and serum-based examinations was inconclusive. The 750 bp scFv antibody gene was expressed in COS-7 cells, and the antibody localized in the cytoplasm. The scFv antibody can detect the germinal layer and protoscolices of actively growing cysts but not of the degenerating protoscolices and has a diagnostic efficiency higher than that of single serum or ultrasound testing (P b 0.05). The combined use of scFv antibodies with serology and ultrasound diagnostics results in a diagnostic efficiency comparable to that of surgery. The scFv antibody can be used as a confirmatory test for the diagnosis of hydatid disease in non-endemic areas, providing a beneficial supplementary diagnostic method that complements traditional immune testing and ultrasonic radiology and thus helping physicians to effectively differentiate hydatid disease.

Cultivation of Pichia pastoris carrying the scFv anti LDL (-) antibody fragment. Effect of preculture carbon source

Abstract Antibodies and antibody fragments are nowadays among the most important biotechnological products, and Pichia pastoris is one of the most important vectors to produce them as well as other recombinant proteins. The conditions to effectively cultivate a P. pastoris strain previously genetically modified to produce the single-chain variable fragment anti low density lipoprotein (-) under the control of the alcohol oxidase promoter have been investigated in this study. In particular, it was evaluated if, and eventually how, the carbon source (glucose or glycerol) used in the preculture preceding cryopreservation in 20% glycerol influences both cell and antibody fragment productions either in flasks or in bioreactor. Although in flasks the volumetric productivity of the antibody fragment secreted by cells precultured, cryopreserved and reactivated in glycerol was 42.9% higher compared with cells precultured in glucose, the use of glycerol in bioreactor led to a remarkable shortening of the lag phase, thereby increasing it by no less than thrice compared to flasks. These results are quite promising in comparison with those reported in the literature for possible future industrial applications of this cultivation, taking into account that the overall process time was reduced by around 8 h.

Affinity maturation of human scFv anti-TSLP in vitro / 中国免疫学杂志

Objective:To single amino acid mutation of the full human scFvs against TSLP to enhance its affinity.Methods: The specific scFvs against TSLP was screened in our previous study and here the three-dimensional structures of TSLP and anti-TSLP scFvs were simulated by Discovery Studio system,then the molecular docking was made.The amino acids of binding epitope were randomly mutated and the mutated amino acids were selected which could remarkably improve the affinity of scFvs.The primers were designed based on the sequence of mutation amino acids and the scFv sequences were mutated by the overlapping extension PCR.The DNA of mutated scFvs was ligated with the expression vector pLZ16 and transformed into E.coli DH5αF′.Then the scFvs were expressed and the scFvs with improved affinity were selected by ELISA and BIAcore.Results: The five scFvs with single amino acid mutation were screened out by DS system,which could elevate the affinity of scFvs.The mutated anti-TSLP-scFvs were amplified by PCR,which size was about 1 000 bp.The mutated scFvs with correct sequence were expressed,and the mutated scFvs with improved affinity were detected by ELISA and BIAcore.The affinity of selected mutated scFv (M4) has been about 10 times higher than the scFv nonmutation.Conclusion: The affinity of anti-TSLP-scFv has been improved successfully.

Construction and expression of anti IL-4R single antibody of prokaryotic expression vector / 天津医药

Objective To construct anti-IL-4R murine anti-human single-chain variable fragment (scFvs) antibodies through BL21 (DE3) prokaryotic expression system. Methods The anti-IL-4R scFv sequence was optimizated on the basis of previous findings. The optimized scFv sequence was analyzed. The recombinant plasmid pET-32a-scFv was constructed. The recombinant plasmid was detected through enzyme identification, and was turned into BL21 (DE3) prokaryotic expression bacteria to express the pET-32a-scFv recombinant protein in E.coli BL21 (DE3). The purification and renaturation were researched, and SDS-PAGE analysis was studied. The molecular weight of ScFv against IL-4R was analyzed by SDS-PAGE. The expression of the fusion protein was detected by Western-blot assay. Results The length of fusion gene scFv-MLT sequence was 761 bp. The molecular weight of the recombinant expression of proteins of anti-IL-4R single antibody was approximately 45 ku. The recombinant proteins showed high specificity with anti-6 × His-tag antibody. Conclusion This experiment successfully constructs pET-32a-scFv prokaryotic expression system of recombinant protein with high immune reactivity, which provides the basis for further study of anti-IL-4R single chain antibody as drug target.

Efeitos do fragmento variável de cadeia única anti-LDL eletronegativa vetorizado em nanocápsulas na aterosclerose experimental / Effects of an anti-LDL(-) single chain fragment variable vectorized in nanocapsules in experimental atherosclerosis

As doenças cardiovasculares são a principal causa de mortalidade no mundo. A aterosclerose é a base fisiopatológica dessas doenças, sendo definida como um processo crônico-inflamatório multifatorial, resultando da interação de diferentes células como linfócitos, macrófagos, células endoteliais e células musculares lisas na parede arterial. A lipoproteína de baixa densidade eletronegativa [LDL(-)], uma subfração modificada da LDL nativa, desempenha um papel-chave na aterosclerose, uma vez que as modificações sofridas por esta partícula são capazes de induzir o acúmulo de ésteres de colesterol em macrófagos e a subsequente formação de células espumosas. O sistema imunológico é crucial no processo aterogênico e estratégias terapêuticas direcionadas à imunoregulação deste processo têm sido utilizadas como novas alternativas tanto na prevenção do desenvolvimento quanto da progressão desta doença. Dentre essas estratégias, destaca-se o uso de fragmentos de anticorpos como o scFv (do inglês, single chain fragment variable), que podem ainda estar conjugados a nanopartículas com o intuito de aumentar sua eficiência de ação no organismo. Diante do papel da LDL(-) na aterosclerose, este projeto objetivou avaliar os efeitos in vitro e in vivo de um sistema nanoestruturado contendo fragmentos scFv anti-LDL(-) derivatizados na superfície de nanocápsulas sobre macrófagos murinos e humanos primários e em camundongos knockout para o gene do receptor da LDL (Ldlr-/-) no desenvolvimento e na progressão dessa doença. Demonstrou-se que o tratamento de macrófagos com a formulação scFv anti-LDL(-)-MCMN-Zn diminuiu de forma significativa a captação de LDL(-), assim como a expressão de IL-1ß (mRNA e proteína) e MCP-1 (mRNA). Foi demonstrada a internalização da nanoformulação pelos macrófagos via diferentes mecanismos de endocitose, demonstrando seu potencial uso como carreador de fármacos. In vivo, a nanoformulação diminuiu de forma significativa a área da lesão aterosclerótica em camundongos Ldlr-/- submetidos à avaliação pela técnica de tomografia por emissão de pósitrons (do inglês, PET), utilizando o radiotraçador 18F-FDG (18F-desoxiglicose), associada à tomografia computadorizada (CT) com agente de contraste iodado, além da análise morfométrica das lesões no arco aórtico. O conjunto dos resultados obtidos evidenciou a ação ateroprotetora da formulação scFv anti-LDL(-)-MCMN-Zn, reforçando seu potencial como estratégia terapêutica na aterosclerose

Cardiovascular diseases are the leading cause of mortality worldwide. Atherosclerosis is the pathophysiological basis of these diseases, defined as a chronic inflammatory multifactorial process, resulting from the interaction of several cells such as lymphocytes macrophages, endothelial cells and smooth muscle cells within the arterial wall. The electronegative low-density lipoprotein [LDL(-)], a modified subfraction of native LDL, plays a key role in atherosclerosis, since its modifications are capable of inducing the accumulation of cholesteryl esters in macrophages and the subsequent foam cells formation. The immune system is crucial in atherogenic process and therapeutic strategies directed to the immunoregulation of this process have been used as a new alternative in the prevention of the development as well as the progression of this disease. Among these strategies, it is the use of antibody fragments such as scFv (single chain fragment variable), which may be also conjugated to nanoparticles in order to increase their efficiency in the body. Given the role of LDL(-) in atherosclerosis, the aim of this project was to evaluate the in vitro and in vivo effects of a nanostructured system containing scFv anti-LDL(-) fragments derivatized on the surface of nanocapsules on murine and human primary macrophages and in the development and progression of the disease in LDL receptor knockout mice (Ldlr-/-). It was demonstrated that the treatment of macrophages with scFv anti-LDL(-)-MCMN-Zn formulation significantly decreases the uptake of LDL(-) and the expression IL-1ß (mRNA and protein) and MCP-1 (mRNA). Moreover, the internalization of the nanoformulation by macrophages through different endocytosis mechanisms was shown, demonstrating its potential use as a nanocarrier. In vivo, the nanoformulation decreased the area of atherosclerotic lesions in Ldlr-/- mice evaluated by positron emission tomography with 18F-FDG associated with computed tomography with iodinated contrast agent (PET/CT), besides the lesion morphometric analysis at the aortic arch Thus, these data provide evidence of the atheroprotection action of the ateroprotection action of the scFv anti-LDL(-)-MCMN-Zn formulation, suggesting its promising use as a therapeutic strategy for atherosclerosis

Screening and Characterization of Human Phage Antibody to Permethrin / 现代检验医学杂志

Objective To do screening acute lymphoblastic leukemia patients scFv antibody single chain variable region to cre-ate conditions for the expression and obtain further specificity of antibody fragments.Methods In this study,patients with newly diagnosed acute lymphoblastic leukemia serum as coating antigen using phage display technology,screening phage an-tibody specificity from the semi-synthetic human phage antibody libraries,the first to target the immune antigen-coated tab-let,phage library was added,so that with the target antigen-specific binding phage antibody was immobilized on plates immu-nization,could not be specifically bound phages were rinsed.The eluted specific binding phage,E.coli infection.Could get the specific antibody gene containing phagemid.Results After three “adsorption-elution-amplification”screening process,got stronger leukemia patient antigen-specific phage antibody variable region fragment and identification.Conclusion Got better strain affinity antibody fragments,to create the conditions for the next fragment expression,identification and clinical appli-cation.

Selection,soluble expression and identification of full human scFvs against IL-13 / 中国免疫学杂志

Objective:To select specific and neutralizing scFv against IL-13 and soluble expression and identification them.Methods:In our previous study the scFv library were constructed,and here the scFv library was enriched and the positive scFv were screened from the enriched scFv library for three round.The specific positive scFvs with better affinity were ligated with expression vector for expression and identification.Results:After three rounds of enrichment,30%of clones were positive.The two specific scFvs with better affinity and neutralization were selected from almost 500 clones and then ligated with expression vector LZ16 for soluble ex-pression.The expressed scFvs were identified by western blot and biomolecular interaction analysis.Conclusion: The specific scFvs against IL-13 with better affinity and neutralization had been selected successfully.

Expression of a recombinant protein by fusing anti-CD20 ScFv with dominant antigen epitopes and its anti-tumor activity / 中国免疫学杂志

Objective:To artificially design and express a recombinant protein named as ScFv-pLLO by fusing ScFv gene of Rituximab(C2B8)and dominant antigen epitopes from listeriolysin O(LLO),and studying its anti-tumor activity.Methods:VH and VL gene sequences of C2B8 against CD20 were acquired by searching United States Patent database,and ScFv sequence was constructed by linking VL and VH with a short peptide linker.Two CD4+T cell epitopes from LLO were selected and designed to splice ScFv sequence.The recombinant gene of ScFv-pLLO was cloned into prokaryotic expression vector and purified after induction.The capacity of ScFv-pLLO target-binding to B-cell lymphomas was evaluated by flow cytometry ( FCM ) and co-immunoprecipitation ( Co-IP ) .The effects of ScFv-pLLO on B-cell lymphomas proliferation and apoptosis were detected respectively.The immunogenicity of ScFv-pLLO was assessed by lymphocyte proliferation assay.Results: ScFv-pLLO was successfully expressed.It could bind to different B-cell lymphomas cell lines and obviously inhibit the growth of Raji cells as well as inducing apoptosis.Moreover,ScFv-pLLO was able to stimulate proliferation of spleen lymphocytes of immunized mice.Conclusion: The recombinant protein ScFv-pLLO can target-bind to B-cell lymphomas,and perform inhibitory effect and induce apoptosis on Raji cells that indicate ScFv-pLLO retain the capacity of ScFv derived from monoclonal antibody against CD20.Besides, ScFv-pLLO can induce immune response.This study provides a basis for further research about the role of ScFv-pLLO on simulating tumor cell antigens as well as being tumor vaccine adjuvant.

Role of the Trp-disulfide Triads in the UV Light Induced Degradation of a Monoclonal Antibody scFv.

Proteins are targets for photodegradation due to absorption of incident light by endogenous chromophores, e.g aromatic side chains. In this work we study the role of Trp-disulfide triads in the light induced loss of immunoglobulin activity. Study Design: We investigated a single chain variable fragment (scFv) of the Trp-disulfide triad containing monoclonal antibody 82D6A3. The scFv binds to von Willebrand factor (VWF) and upon illumination with near UV-B-light the scFv partially loses its binding capacity to VWF. In order to relate this observed degeneration to the specific Trp-disulfide triads, we mutated W35(VL) and W36(VH) which are in direct contact with the disulfide

Selection and identification of full human scFv against TSLP / 中国免疫学杂志

Objective:Expression of protein TSLP and selection of full human anti-TSLP single chain Fv ( scFv).Methods:The cDNA of TSLP was amplified.The amplified target gene and the expression vector pET 101/D-TOPO were ligated , and then transformed into E.coli BL21.The protein was induced to expression by IPTG and purified and identified.The biotinylated TSLP protein was used as antigen to select of human TSLP scFv from a constructed human scFv library by phage display .Results: The size of amplified cDNA of TSLP was about 423 bp,and that of expressed protein was about 26 kD.Dot blot and Western blot results showed that the expressed protein was correct.The constructed human scFv library was enriched for three rounds using biotinylated TSLP as antigen by phage display.ELISA results showed that 35% scFvs had binding activity with TSLP.The scFvs with good binding activity were selected and identified by Western blot and sequencing.Conclusion: The full human scFvs against for TSLP were selected suc-cessfully.

Construction,expression,and the analysis of antitumor activity of all anthropogenic medicinal anti-EGFR ScFv (HL-L-L) / 中国生化药物杂志

Objective To construct,express and analysis the antitumor activity of anti-EGFR ScFv(HL-L-L),and analyze bioinformatics and characterize correlation spectroscopy.Methods EGFR ScFv(HL-L-L)gene was obtained by OVER LAPING technology and construction of prokaryotic expression vector pET-28a-c(+)-VH-L-L.After induced expression,target protein HL-L-L was purified by Ni column,and its tertiary structures was predicted with SWISS - MODEL;size distribution and zeta potential distribution of the target protein HL-L-L were characterized by NanoZSP;the inhibition effects of HL-L-L on breast cancer cell proliferation were studied with the method of pharmacology.Results Prokaryotic expression vector pET-28a-c(+)-VH-L-L was constructed successfully,high purity of protein HL-L-L was obtained,and successfully predicted its three-dimensional structure;the size was about 489.7 nm,and the Zeta potential was 38.1 ,negatively charged.It had a certain inhibition effect on MCF # 7 proliferation,compared with blank group,and the difference was significant(P<0.01 ).Conclusion This study successfully express all anthropogenic medicinal anti-EGFR ScFv(HL-L-L),which has significant inhibiting effect on breast cancer cell proliferation.It provides the foun dation for further development of small molecules of anti-EGFR antibody drug.