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.

Construction and quality analysis of phage display library for random mutagenesis of camel nanobody / 国际生物医学工程杂志

Objective To construct phage display antibody library of artificial mutation to compare with the sequence of the natural phage display antibody library. To scientifically evaluate the quality of the artificial mutation of phage display library, and provide some references for the further transformation of the nanobody. Methods Using random mutation method, NNY fixed-point santuration mutation was performed on combine the follicle-stimulating hormone receptor (FSHR) of human nanobody. The mutant DNA sequence was connected to the vector pMECS to construct the phage display library of VHH06-CDR3 random mutation. By sequencing and analysis of DNA sequences, the diversity of the library and the amino acid distribution of CDR3 were compared between mutation library and the immune library of FSHR. The degree of enrichment of cloning was determined by six rounds of affinity screening. Results According to the NNY mutation rule ,the CDR3 regions with 16 amino acids by random mutations was synthesized and the VHH-CDR3 random mutant phage display library was constructed . The phage display library of VHH06-CDR3 random mutant size was 7.36×108 cfu/ml. Polyclonal and monoclonal phage ELISA showed that after six rounds of screening, the output phage and the combination of FSHR showed obvious enrichment, but there was no clone combined with FSHR. Conclusions Although the VHH06-CDR3 mutant phage display library has sequence diversity, it is not conducive to obtaining target antibodies in affinity screening due to the lack of functional diversity of CDR3.

Construction and screening of phage display library for TIM-3 nanobody / 药学学报

Immunotherapy is a new strategy for cancer treatment that has the potential to treat all types of cancer. T cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3) is a key negative regulator of T cell activation. TIM-3 blockage using anti-TIM-3 monoclonal antibody therapy has a great appeal and special advantages. Nanobodies, derived from heavy chain fragment in camelid animals, are now proving clinical values in the development of antibody drugs. In this study, we have immunized camel with TIM-3 antigens and then constructed phage display library. Moreover, 29 nanobodies with different complementarity-determining regions sequences have been screened from the phage display library by phage display technology. In addition, we successfully constructed the cell line stably expressing TIM-3, and screened 10 TIM-3 nanobodies with high specificity and high affinity using flow cytometry. Our study will lay the foundation for the future screening and development of anti-TIM-3 whole humanized functional nanobody.

Construction and screening of nanobody targeting macrophage membrane receptor Vsig4 / 西安交通大学学报(医学版)

ABSTRACT:Objective To construct V-set and immunoglobulin domain containing 4 (Vsig4)nanobodies (Nbs) as specific macrophage probes so as to use them as molecular probes of macrophagocytes.Methods A nanobody phage library was generated by using peripheral blood lymphocytes isolated from an alpaca immunized with recombinant Vsig4 protein.After three rounds of selection against recombinant Vsig4.The Nbs were subjected to sequencing and genome alignment to obtain VHH sequence.Nbs were isolated and tested for Vsig4 specificity in an ELISA using recombinant Vsig4.The affinity capacity of Nbs was verified by the cell line stably expressing Vsig4. Results A nanobody phage library with an estimated 7.27 × 107 clones with 70% insertion was successfully constructed.Totally 1 3 6 Vsig4-positive clones were sequenced and aligned according to different CDR3 sequences. In summary,1 5 Vsig4 nanobodies were obtained and grouped into 3 different CDR3 epitopes.The affinity of representing nanobody and Vsig4 was analyzed via ELISA;Nb1 1 9 showed the highest affinity against both recombinant and native Vsig4.Conclusion We successfully constructed and screened Vsig4 specific nanobody number 1 1 9 with high affinity and specificity.It can help with macrophage detection and in vivo monitoring.

Construction of Human ScFv Phage Display Library against Ovarian Tumor / 华中科技大学学报（医学）（英德文版）

In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E.coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 × 109 cfu/μg was obtained. After amplification with helper phage, the titer of antibody library reached 5 × 1012 cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.

Screen of human single chain antibody against N protein of SARS-CoV / 基础医学与临床

Objective To prepare the single chain antibody against N protein of SARS-CoV. Methods With N protein of SARS-CoV expressed in E.coli as antigen, we obtained the single chain antibody against N protein by screening the phage display library of human single chain antibodies. Results The anti-N protein antibody didn’t cross-interacte with BSA and the short peptide containing 6 histidines. The specific interaction between the antibody and N protein was inhibited by the anti-N protein monoclone antibody from immunized mice. ConclusionThe single chain antibody we got is specific to N protein of SARS-CoV,it can be a candidate antibody for fast detection of N protein of SARS-CoV and SARS virus particles in clinical trial study of SARS pathogenesis.

Radiolabeling of Filamentous Phage Peptide Library With 99mTc and Its Biodistribution in Normal Mice / 生物化学与生物物理进展

Numerous peptides that bind to a given target have been selected by phage display technology. However, some peptides isolated to date do not bind with high affinity to tumor or organ sites, even peptides were selected in vivo. Therefore, the biodistribution of 99mTc-labeled filamentous phage peptide library via MAG3 (mercaptoacetyltriglycine) were investigated to gain a better understanding of phage circulation in vivo. The experimental results showed that the liver and spleen were the organs of the greatest accumulation, while heart, muscle, pancreas and brain retained less radioactivity. In opposite to other tissues and organs, the radioactivity in stomach, intestine and bone gradually went up with time. The clearance of 99mTc-labeled phage in blood was very fast from 5 min to 30 min and then slowed down. When phage in vivo circulated at enough long period of time, some phage particles could extravasate in some organs or tissues and internalized there. In conclusion, the circulation time of phage in vivo should be experimentally determined beforehand according to the targeted organs and the specific location of target peptides in order to panning a peptide with high specificity and affinity to that target.

Isolation and Characterization of Human scFv Molecules Specific for Recombinant Human Heat Shock Protein (HSP) 70.1

BACKGROUND: The heat shock proteins (HSPs) play an important role in cellular protection mechanisms against physical or chemical stresses. In this study scFv antibodies specific for human HSP70.1 were isolated from a semi-synthetic human scFv library with the ultimate goal of developing anti-HSP70.1 intracellular antibody (intrabody) that may offer an attractive alternative to gene targeting to study the function of the protein in cells. METHODS: A semi-synthetic human scFv display library (5 X 10(8) size) was constructed using pCANTAB-5E vector and the selection of the library against bacterially expressed recombinant human HSP70.1 was attempted by panning. RESULTS: Three positive clones specific for recombinant HSP70.1 were identified. All three clones used V(H) subgroup III. On the other hand, V(L) of two clones belonged to the kappa light chain subgroup I, but the other utilized V(k) subgroup IV Interestingly, these scFv molecules specifically reacted to the recombinant HSP70.1, yet failed to recognize native HSP70 induced in U937 human monocytic cells by heat treatment. CONCLUSION: Our results indicated that affinity selection of an scFv phage display library using recombinant antigens produced in E. coli might not guarantee the isolation of scFv antibody molecules specific for a native form of the antigen. Therefore, the source of target antigens needs to be chosen carefully in order to isolate biofunctional antibody molecules.

Selection and prokaryotic expression of MUC1 mimic epitope / 第三军医大学学报

Objective To screen MUC1 antigen mimic epitope by phage display peptide library technology and to construct a recombinant plasmid expressing MUC1 antigen mimic epitope.Methods MUC1 mimic epitope was screened by Biopanning,DNA sequencing and amino acid sequence comparison.The gene was constructed into PET-31b(+) expression vector and expressed in Escherichia coli BL21(DE3) after transformation and induction by IPTG.The complete protein of the host bacteria was extracted for SDS-PAGE.The recombinant protein was purified by affinity chromatography on a Ni~(2+)-sepharose column and detected by Western blotting.Results The selected MUC1 mimic epitope could specifically combine with MUC1 monoclonal antibody.The recombinant expression vector PET-31b(+)-MUC1 was constructed successfully after the fusion protein was induced with IPTG.A specific protein band was shown on SDS-PAGE profile and detected by Western blotting.Conclusion An MUC1 mimic epitope was screened out.The epitope fusion protein was successfully expressed for the development of tumor vaccines targeting MUC1.

Isolation of Human scFv Antibodies Specific for House Dust Mite Antigens from an Asthma Patient by Using a Phage Display Library

BACKGROUND: In order to characterize human antibodies with specificity for mite allergens at the molecular level, a scFv phage display library was constructed using peripheral blood mononuclear lymphocytes from an asthma patient allergic to mite as Ig gene sources. METHODS: Immunoglobulin VH and V gene fragments were obtained by polymerase chain reaction, and randomly combined in pCANTAB-5E vector. The resulting human scFv phage display library had 3 X 10(4) independent clones, and biopanning was performed with house dust mite extracts. RESULTS: Four scFv clones specific for house dust mite extract were isolated. Immunoblot assay showed that our clones reacted to 25 kDa and 50~60 kDa proteins with unknown identity in mite extracts. Sequence analysis indicated that two clones (b7 and c15) are identical, and all clones belong to human VH3 subgroup. On the other hand, light chain usage was different in that two clones (a2 and b7/c15) belonging to V kappa 4 subgroup, but a4 used V kappa 1 light chain gene. CONCLUSION: Our approach should facilitate provision of useful information on the antibody responses against allergens at the molecular level in humans.

Randomization of P2/NC protease cleavage site of HIV-1 Gag protein and and its phage display / 第二军医大学学报

Objective:To construct the phage display library of randomized P2/NC protease cleavage sequences in HIV-1 Gag protein,so as to provide evidences for studying the effect of drug resistance-associated mutations on susceptible cleavage sequences of HIV-1 PR.Methods: CAP2 fragments and NC fragments of HIV-1 gag were generated by PCR.StuⅠ restriction site was introduced to the 5′ terminal of CAP2 fragments and the protease cleavage sequences of 3′ terminal of CAP2 fragments were randomized.The overlapping CAP2 sequence was introduced to the 5′ terminal of NC fragments and Sal Ⅰ restriction site was introduced to the 3′ terminal.CAP2 and NC fragments were linked by overlapping PCR method and the fused CAP2 and NC fragments were cloned into pCANTAB5S-LD3 to construct the phage display library of the P2/NC cleavage site in HIV-1 Gag protein.Results: As much as 2.1?10~6 clones were obtained in the phage library and the titer was 3.0?10~(12)TU/ml.There were 52.1% clones containing inserted CAP2/NC fragments.Sequence analysis of 12 samples showed that nucleotide acids and amino acids were randomly distributed at randomized PR cleavage sites.Nine of 10 positive monoclonal phages specifically bound to human IgG.Conclusion: CAP2/NC protease cleavage sequences of HIV-1 Gag protein have been successfully randomized and its phage display library has been constructed,which may help to screen the phages containing susceptible cleavage sequences of PR with drug resistance-associated mutations and establish the phage model for in vitro screening of PR inhibitor.

Immunity against Schistosoma japonicum Induced by Phage Display Peptides Mimicking Antigenic Epitopes of Trichinella spiralis / 中国寄生虫学与寄生虫病杂志

Objective To obtain the short peptides mimicking antigenic epitopes of Trichinella spiralis ( T/^ ), and explore their cross protective immunity against Schistosoma japonicum ( S^j. ) in mice. Methods IgG antibodies were purified from sera of mice infected with T/^ . The purified IgG was used to immunoscreen a phage random peptide library of 7 amino\|acid residues displayed as a fusion to protein of filamentous phage. Positive clones were obtained by affinity selection, the reactivity of each clone binding to specific IgG was detected by ELISA. Kunming mice were immunized subcutaneously three times with mixed phage clones. The mice were sacrificed 45 days after challenge. The worms and the liver eggs were counted. Results After three rounds of panning, the relevant phages had been enriched approximately 150 times in production as compared to those from the first round. Of 24 phage clones randomly selected from the third round biopanning, 21 clones were shown to actually bind to the specific IgG. As compared with the control group, the worm and the liver egg reduction rates in vaccination group were 42^8% and 66^3% ( P

Production of Human Fab Monoclonal Antibody to Surface Protein, preS1, of Hepatitis B Virus using Antibody Phage Display Library / 대한면역학회지

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. By cloning human Ig gene segments from the B cells of volunteer into pComb3 phagemid vector, antibody library was created of filamentous phage particles displaying Fab fragments on their surface after being rescued with M13KO7 helper phages. The size of library was 7x10' pfu. Phage antibodies (phabs) were panned against biotinylated preS1 using streptavidine coated Dynabead. The soluble Fab antibodies were prepared from phagemid colonies and assayed directly for the ability to bind preS1 by ELISA. And then 3DW and SGW specific to preS1 which have both heavy and light chain to form Fab fragment, were selected. The soluble Fab antibody from 3DW was expressed highly at the concentration of 0.1 - 1.0 mM of IPTG, and 5 hours postinduction. The soluble antibodies from 3DW and SGW showed their relative affinities of 2x10' M ', and Sx10 M ', respectively, and the specificities to preS1 on ELISA. Our results suggest that antibody phage display library is very useful method to generate the human monoclonal antibody and that the human Fab monoclonal antibodies specific to preS1 selected in this study open the way to treat hepatitis B as a component of passive irnmunotherapeutics.

Construction and Screening of a Phage Display Library of Repertoire Single Chain Fv Antibody from Mouse Immunized with BoNTB/Hc / 微生物学通报

To produce antibodies capable of neutralizing botulinum neurotoxin type B(BoNT/B),We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin,induced and purifed.The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined as a single-chain Fv(scFv)DNA fragment.These fragment were cloned into the phagemid pCANTAB5E and the phage display library was constructed.Results showed that the high affinity scFv was obtained after 4 rounds of panning,with its DNA sequence conforming to that of mouse antibody.

The functional binding antibodies to novel purinoceptor selected from phage display library / 中国药理学通报

Aim To screen antibodies of novel purinoceptor as a marker for further study of the purinoceptor. Method BALB/c mice were immunized for 4 times with rat aortic endothelial cell. Then the phage display system was used to construct a single-chain Fv fragment (ScFv) cDNA library from the total RNA of immunized mice. The characteristics of novel purinoceptor not existing on vascular smooth muscle cell but on aortic endothelium were used to enrich the aortic endothelium specific antibodies. Induced with IPTG, these antibodies were secreted into the periplasm of E. coli. The functional experiment of novel purinoceptor named organ bath experiment was used to screen out the positive ScFv from the soluble expressed antibodies. Immunohistochemistry experiment was used for positive ScFv identification. Results The total mouse anti-rat endothelium lgG is 1 ∶16 000. 8?106 mouse anti-rat endothelium ScFv cDNA library was successfully constructed. After 4 times of rat endothelium and rat smooth muscle cells screening, 2 500 ScFv cDNA binding membrane of aortic endothelium was enriched. After 4 times of functional screening, a phage-ScFv named B inhibiting the adenosine induced NO dependent construction by 83.4%?21.6% was selected from the expressed antibodies. Immunohistochemistry experiment showed that ScFv-B combined with aortic endothelium specifically and functional experiment showed that ScFv-B did not have any effect on adenosine induced ileum contraction, indicating that ScFv-B specifically binding to the novel purinoceptor. Conclusions ScFv-B binding specifically to the novel purinoceptor was selected by phage display technique and functional screening experiment which provide a good marker for further study of the novel purinoceptor.