Splicing By Overlap Extension PCR Research Articles

Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase.

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents.

Genomic Structure of Two Kv1.3 Channel Blockers from Scorpion Mesobuthus eupeus and Sea Anemone Stichodactyla haddoni and Construction of their Chimeric Peptide as a Novel Blocker.

Different toxins acting on Kv1.3 channel have been isolated from animal venom. MeuKTX toxin from Mesobuthus eupeus phillipsi scorpion and shtx-k toxin from Stichodactyla haddoni sea anemone have been identified as two effective Kv1.3 channel blockers. In this work, we characterized the genomic organization of both toxins. MeuKTX gene contains one intron and two exons, similar to the most scorpion toxins. There are a few reports of genomic structure of sea anemone toxins acting on Kv channels. The sequence encoding mature peptide of shtx-k was located in an exon separated by an intron from the coding exon of the propeptide and signal region. In order to make a peptide with more affinity for Kv1.3 channel and greater stability, the shtx-k/ MeuKTX chimeric peptide was designed and constructed using splicing by overlap extension-PCR (SOE-PCR) method. MeuKTX, shtx-k, and shtx-k/MeuKTX were cloned and the expression of the soluble proteins in E. coli was determined. Molecular docking studies indicated more inhibitory effect of shtx-k/MeuKTX on Kv1.3 channel compared to shtx-k and MeuKTX toxins. Key amino acids binding channel from both toxins, also involved in interaction of chimeric peptide with channel. Our results showed that the fusion peptide, shtx-k/MeuKTX could be an effective agent to target Kv1.3 channel.

Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells.

Background KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins.MethodsThis study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.ResultsThe VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.DiscussionThese results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.

Multiplexed Competition in a Synthetic Squid Light Organ Microbiome Using Barcode-Tagged Gene Deletions.

Beneficial symbioses between microbes and their eukaryotic hosts are ubiquitous and have widespread impacts on host health and development. The binary symbiosis between the bioluminescent bacterium Vibrio fischeri and its squid host Euprymna scolopes serves as a model system to study molecular mechanisms at the microbe-animal interface. To identify colonization factors in this system, our lab previously conducted a global transposon insertion sequencing (INSeq) screen and identified over 300 putative novel squid colonization factors in V. fischeri To pursue mechanistic studies on these candidate genes, we present an approach to quickly generate barcode-tagged gene deletions and perform high-throughput squid competition experiments with detection of the proportion of each strain in the mixture by barcode sequencing (BarSeq). Our deletion approach improves on previous techniques based on splicing by overlap extension PCR (SOE-PCR) and tfoX-based natural transformation by incorporating a randomized barcode that results in unique DNA sequences within each deletion scar. Amplicon sequencing of the pool of barcoded strains before and after colonization faithfully reports on known colonization factors and provides increased sensitivity over colony counting methods. BarSeq enables rapid and sensitive characterization of the molecular factors involved in establishing the Vibrio-squid symbiosis and provides a valuable tool to interrogate the molecular dialogue at microbe-animal host interfaces.IMPORTANCE Beneficial microbes play essential roles in the health and development of their hosts. However, the complexity of animal microbiomes and general genetic intractability of their symbionts have made it difficult to study the coevolved mechanisms for establishing and maintaining specificity at the microbe-animal host interface. Model symbioses are therefore invaluable for studying the mechanisms of beneficial microbe-host interactions. Here, we present a combined barcode-tagged deletion and BarSeq approach to interrogate the molecular dialogue that ensures specific and reproducible colonization of the Hawaiian bobtail squid by Vibrio fischeri The ability to precisely manipulate the bacterial genome, combined with multiplex colonization assays, will accelerate the use of this valuable model system for mechanistic studies of how environmental microbes-both beneficial and pathogenic-colonize specific animal hosts.

Construction and Activity Analyses of Single Functional Mouse Peroxiredoxin 6 (Prdx6).

IntroductionPeroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase activity and phospholipase A2 activity. Previous studies have shown a significant positive correlation between the intracellular survival ability of Brucella and Prdx6. Here, the Prdx6 enzyme with a single activity was constructed to facilitate study of the relationship between the single function of Prdx6 and Brucella infection.Material and MethodsThe target open reading frame (ORF) DNAs of Prdx6 with a single active centre were prepared using gene splicing by overlap extension PCR (SOE-PCR), and the recombinant eukaryotic expression plasmids inserted by Prdx6 with the single activity centre were constructed and transfected into murine Raw264.7 macrophages. The glutathione peroxidase activity and phospholipase A2 activity of the constructed Prdx6 were examined.ResultsThe core centres (Ser32 and Cys47) of Prdx6 were successfully mutated by changing the 94th nucleotide from T to G and the 140th nucleotide from G to C in the two enzyme activity cores, respectively. The constructed recombinant plasmids of Prdx6 with the single active centre were transfected into murine macrophages showing the expected single functional enzyme activity, which MJ33 or mercaptosuccinate inhibitors were able to inhibit.ConclusionThe constructed mutants of Prdx6 with the single activity cores will be a benefit to further study of the biological function of Prdx6 with different enzyme activity.

Construction, expression, and characterization of a single-chain variable fragment (ScFv) antibody targeting to the encephalomyocarditis virus.

Encephalomyocarditis virus (EMCV) is as a potential zoonotic agent with a wide host range. Here, applying gene splicing by overlap extension PCR (SOE-PCR), we describe a simple method for producing single-chain variable fragment (scFv) antibody against EMCV that configurates in the orientation of VH-(GGGGS)4 -VL. DNA template was resverse transcribed by total RNA that derived from hyperimmunized antibody positive mice spleen after inoculation inactivated EMCV-PV21 as antigen. Using the degenerate primers designed for the variable regions of IgG of murine antibody, the 417 bp of gene encoding VH-linker (VHL) and 360 bp of gene encoding linker-VL (LVL) of the anti-EMCV was individually amplified from DNA template by PCR, repectively. The 762 bp gene encoding anti-EMCV scFv was constructed by SOE-PCR when the mixed VHL and LVL genes were used as the template. The amplified gene subcloned into pGEX-6P1 to yield pGEX-6P1/EMCV-scFv. Recombinant vector transformed into the Escherichia coli BL21 (DE3) and a 53 KDa GST-scFv fusion protein was obtained by SDS-PAGE electrophoresis. Animal experiment results showed that the pretective rate of mice in group A which challenged 500 μL 104 TCID50 EMCV per mouse for 7 d post-inoculation scFv 3 d (0.5 mg purified recombinant scFv per mouse) was 91.67% (11/12). The serum anti-EMCV antibody titer in group A mice was most significantly higher than that in positive control mouse (P < 0.01), coversely the serum relative mRNA copies were significantly lower than that in positive control mouse (P < 0.05). These findings indicated that recombinant anti-EMCV scFv has remarkable anti-EMCV effect in mice.

Selection of single-chain variable fragments specific for Mycobacterium tuberculosis ESAT-6 antigen using ribosome display.

Objective(s):Tuberculosis (TB) is still one of the problematic infectious diseases in developing countries, especially in Iran. In the present study, we applied ribosome display technique to select single chain variable fragments (scFvs) specific for the 6-kDa early secretory antigenic target (ESAT-6) antigen of Mycobacterium tuberculosis from a mouse scFv library.Materials and Methods:The gene encoding ESAT-6 was cloned into pET22b(+) plasmid and expressed in Escherichia coli BL21 (DE3). The purified recombinant ESAT-6 protein was injected into female BALB/c mice for immunization, and then m-RNA was extracted from the spleen of immunized mice. The anti-ESAT-6 VH/k chain library was assembled by joining of VH and k into the VH/k chain with a 72-bp DNA linker by SOE (splicing by overlap extension) PCR. The scFv library was panned against ESAT-6 using a single round of ribosome display via a rabbit reticulocyte lysate system.Results:ELISA assay showed that one of the selected scFvs had higher affinity against the recombinant ESAT-6 protein. The affinity of the candidate scFv was ~ 3.74×108 M-1.Conclusion:It could be proposed that the isolated scFv in this study may be useful for the diagnosis of TB.

The Regulatory Roles of ncRNA Rli60 in Adaptability of Listeria monocytogenes to Environmental Stress and Biofilm Formation.

Listeria monocytogenes is a facultative anaerobic Gram-positive bacterium. It is well adapted to external environments and able to infect both humans and animals. To understand the impacts of ncRNA Rli60 on the adaptability of L. monocytogenes to environmental stresses and biofilm formation, a rli60 deletion strain of L. monocytogenes (LM-Δrli60) was constructed using splicing by overlap extension PCR (SOE-PCR) and homologous recombination and then compared it with wild-type strain L. monocytogenes EGD-e in the aspects of adaptability to environmental stresses by measuring their growth under stresses of different temperatures, and acidic, alkaline, hypertonic and alcoholic conditions, and capability of biofilm formation by using crystal violet staining, as well as the transcriptional levels of genes (gltB and gltC) related to the biofilm formation by real-time quantitative PCR (qRT-PCR). The results showed that (1) the growth of LM-Δrli60 strain was significantly slower under environmental stresses of low temperature (30°C), high temperature (42°C), as well as alkaline and alcoholic conditions, (2) the amount of biofilm formed by LM-Δrli60 was attenuated, and (3) the transcriptional levels of gltB and gltC genes at 24h and 48h in LM-Δrli60 revealed a significant reduction. Overall, the results confirmed that ncRNA Rli60 plays important roles in regulating the adaptability of L. monocytogenes to environmental stresses and biofilm formation possibly through impacting the expression of gltB and gltC genes.

Construction of mutant strains of Klebsiella pneumoniae with ampG gene deletion

Objective To construct mutant strains of Klebsiella pneumoniae with ampG gene deletion by homologous recombination and to evaluate the role of ampG gene in inducing the expression of AmpC enzyme. Methods Polymerase chain reaction (PCR) was used to amplify the upstream and downstream fragments of ampG gene. The gene splicing by overlap extension PCR (SOE-PCR) technique was used to construct the fusion fragment, which was then ligated into the temperature sensitive suicide vector pKO3-km after enzyme digestion as pKO3-km-ΔampG. To achieve allelic exchange, the plasmid pKO3-km-ΔampG was introduced into Kp1 and Kp NTUH-K2044 strains by electroporation. The mutant strains of Klebsiella pneumoniae with ampG gene deletion were screened out. The plasmid pACYC184-ampCR was introduced into the Kp NTUH-K2044 wild-type strain and its mutant strain with ampG gene deletion to make them harbor the gene encoding AmpC enzyme. The disk diffusion method was used to evaluate the effects of ampG gene on the expression of AmpC enzyme in Klebsiella pneumoniae strains with cefoxitin as the inducer. Results The recombinant plasmid pKO3-km-ΔampG was constructed successfully. The mutant strains of Klebsiella pneumoniae with ampG gene deletion were constructed as verified by PCR and DNA sequencing. Compared with the Kp1 wild type strain, no AmpC enzyme was produced by the ampG gene knock-out Kp1 strain. The Kp NTUH-K2044 strain could produce AmpC enzyme, while the mutant strain of Kp NTUH-K2044 with ampG gene deletion could not after introduced the pACYC184-ampCR plasmid. Conclusion The mutant strain of Klebsiella pneumoniae with ampG gene deletion was successfully constructed. The Klebsiella pneumonia strain without the ampG gene could not produce the AmpC enzyme. Key words: Klebsiella pneumoniae; ampG gene; Homologous recombination; Gene knock-out; AmpC enzyme

RP215 single chain fragment variable and single domain recombinant antibodies induce cell cycle arrest at G0/G1 phase in breast cancer

BackgroundTargeted therapy is an attractive approach to avoid the side effects of cancer treatment. Based on antibody-targeted superantigens, single chain variable fragment (scFv) and single domain (sdAb) antibodies, characterized by a low molecular weight, low immunogenicity and a high tumor penetration compared to monoclonal antibodies (mAb), have been increasingly used in gene-targeted therapy for cancer. In the present study, we aimed to develop the novel recombinant scFv-RP215 and sdAb-RP215 antibodies based on the variable regions of the RP215 monoclonal antibody (RP215-mAb) against CA215, a pan cancer marker expressed in various human tumor tissues, and to examine their biological activity in breast cancer cell lines. MethodsThe VH and VL genes were amplified from hybridoma cells secreting RP215-mAb by RT-PCR and joined with a linker using splicing by overlap extension PCR (SOE-PCR) to obtain the RP215-scFv gene, whereas the VH gene was used to generate the RP215-sdAb. Gene fragments of antibodies were subcloned into the pET32a(+) vector and expressed in Escherichia coli BL21. Western blot, indirect immunofluorescence (IF), ELISA and competitive ELISA were used to detect the immunoreactivity of scFv-RP215, sdAb-RP215, and RP215-mAb. The CCK-8 assay and cell cycle analysis were used to assess antibodies function. ResultsThe novel recombinant scFv-RP215 and sdAb-RP215 antibodies were successfully developed based on the variable regions of the monoclonal antibody RP215 (RP215-mAb) against CA215. We verified that scFv-RP215 and sdAb-RP215 recognize CA215 on the surface of breast cancer cells (MB231, MCF7, MB468, SK-BR-3 and BT549) and characterized their activity and specificity. Our findings also indicate that scFv-RP215 and sdAb-RP215 induce cell cycle arrest at the G0/G1 phase in breast cancer cells. ConclusionOur results showed that scFv-RP215 and sdAb-RP215 have excellent immunoreactivity and localize accurately to breast cancer cells in membrane-bound form, suggesting their potential as tumor targeting antibodies for breast cancer therapy.

Construction of a single-chain variable fragment antibody against daidzin and its potential use in an enzyme-linked immunosorbent assay

Daidzin is one of the major isoflavonoids found in legumes. Several studies has reported estrogenic effects of this class of compounds. To develop an immunoassay to determine the concentration of daidzin in plant samples, a single-chain variable fragment antibody (scFv) against daidzin was constructed. The variable heavy and light chain genes were cloned from cDNA of hybridoma cell line. Then they were assembled together by splicing by overlap extension PCR (SOE-PCR) using specific primers designed to have flexible peptide (Gly(4)Ser)(3) as linker. The constructed scFv gene was ligated into the pET28a expression vector and transformed into E. coli BL21 (DE3). The expressed scFv containing His6-tag at its N-termini was purified by immobilized metal ion affinity chromatography (IMAC). The purified scFv was characterized and applied to use in enzyme-linked immunosorbent assay (ELISA).

A simple and universal ligation mediated fusion of genes based on hetero-staggered PCR for generating immunodominant chimeric proteins

We developed a simple T4 DNA ligase mediated strategy for inframe splicing of two or more cohesive genes generated by hetero-staggered PCR and directionally cloning the spliced product bearing sticky overhangs in to a correspondingly cut vector. For this, two pairs of primers are used in two different parallel PCRs, for generation of each cohesive gene product. We exemplified this strategy by splicing two major super-antigen genes of Staphylococcus aureus, namely, staphylococcal enterotoxin A (sea), and toxic shock syndrome toxin (tsst-1) followed by its directional cloning into pre-digested pRSET A vector. The fusion gene encoding chimeric recombinant SEA-TSST protein (32kDa) was expressed in E. coli BL21(DE3) host strain. The recombinant chimeric protein retained the antigenicity of both toxins as observed by the strong immunoreactivity with commercial antibodies against both SEA and TSST-1 toxin components by Western blot analysis. We observed that the present method for gene splicing with cohesive ends is simple since it does not require elaborate standardization and a single fusion product is obtained consistently during nested PCR with forward primer of first gene and reverse primer of second gene. For comparison, we fused the same genes using splicing by overlap extension PCR (SOE-PCR) and consistently obtained DNA smearing and multiple non-specific bands even after several rounds of PCRs from gel excised product. Moreover, the newly described method requires only two to six complimentary sticky ends between the genes to be spliced, in contrast to long stretch of overlapping nucleotides in case of SOE-PCR.

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

BackgroundJapanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease.Methodology/Principal FindingsThe full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, K m and k cat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency k cat /K m was found for Pyr-RTKR-amc (k cat/K m: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (k cat/K m: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro.Conclusions/SignificanceA simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery.

Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system

Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0×10(7) recombinant phage clones, and the titer of the amplified library was 3.6×10(9)pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described.

Construction of scFv phage display library with hapten-specific repertories and characterization of anti-ivermectin fragment isolated from the library

A specific phage display library was developed for screening antibodies against micro-molecular substances with 16-membered macrocyclic backbone. Through mRNA extraction, RT-PCR and gene splicing by overlap extension PCR (SOE-PCR), single-chain fragment variable (scFv) gene fragments about 750 bp were generated and ligated with the phagemid pCANTAB5E and were transformed into competent cells. A phage display library containing 2.4 × 106 clones was constructed from milbemycin oxime-bovine serum albumin (MILO-BSA) immunized mice. The screening was carried out by ivermectin-bovine serum albumin (IVM-BSA) with different concentration levels. Furthermore, scFv phage clones were isolated within the four round library panning and screening by phage- ELISA, 10 positive clones were obtained finally. These positive clones were then sequenced, and their soluble type antibodies were identified and showed significant binding activity.