[Construction of recombinant adenovirus expressing EGFRvIII extracellular domain gene and preparation of single domain antibody].

The aim of this study was to construct a recombinant adenovirus expressing extracellular domain gene of human epidermal growth factor receptor variant Ⅲ (EGFRvIII ECD), and to prepare single domain antibody targeting EGFRvIII ECD by immunizing camels and constructing phage display antibody library. Total RNA was extracted from human prostate cancer cell line PC-3 cells and reversely transcribed into cDNA. EGFRvIII ECD gene was amplified using cDNA as template, and ligated into pAdTrack-CMV plasmid vector and transformed into E. coli BJ5183 competent cells containing pAdEasy-1 plasmid for homologous recombination. The recombinant adenovirus expressing EGFRvIII ECD was obtained through transfecting the plasmid into HEK293A cells. The recombinant adenovirus was used to immunize Bactrian camel to construct EGFRvIII ECD specific single domain antibody library. The single domain antibody was obtained by screening the library with EGFRvIII protein and the antibody was expressed, purified and identified. The results showed that recombinant adenovirus expressing EGFRvIII ECD was obtained. The capacity of EGFRvIII specific phage single domain antibody library was 1.4×109. After three rounds of enrichment and screening, thirty-one positive clones binding to EGFRvIII ECD were obtained by phage-ELISA, and the recombinant single domain antibody E14 with highest OD450 value was expressed and purified. The recombinant E14 antibody can react with EGFRvIII ECD with high affinity in ELISA assessment. The results indicated that the EGFRvIII specific single domain antibody library with high capacity and diversity was constructed and the single domain antibody with binding activity to EGFRvIII was obtained by screening the library. This study may facilitate the diagnosis and treatment of EGFRvIII targeted malignant tumors in the future.

Development of a Bispecific Nanobody Targeting CD20 on B-Cell Lymphoma Cells and CD3 on T Cells.

B-cell lymphoma is a group of malignant proliferative diseases originating from lymphoid tissue with different clinical manifestations and biological characteristics. It can occur in any part of the body, accounting for more than 80% of all lymphomas. The present study aimed to construct bispecific single-domain antibodies against CD20 and CD3 and to evaluate their function in killing tumor cells in vitro. A Bactrian camel was immunized with a human CD20 extracellular peptide, and the VHH gene was cloned and ligated into a phagemid vector to construct the phage antibody display library. A phage antibody library with a size of 1.2 × 108 was successfully constructed, and the VHH gene insertion rate was 91.7%. Ninety-two individual clones were randomly picked and screened by phage ELISA. Six strains with the high binding ability to human CD20 were named 11, 30, 71, 72, 83, and 92, and induced expression and purification were performed to obtain soluble CD20 single-domain antibodies. The obtained single-domain antibodies could specifically bind to human CD20 polypeptide and cell surface-expressed CD20 molecules in ELISA, Western blot, and cell immunofluorescence assays. The anti-CD20/CD3 bispecific nanobody (BsNb) was successfully constructed by fusing the anti-CD20 VHH gene with the anti-CD3 VHH and the bispecific single-domain antibody was expressed, purified, and validated. Anti-CD20/CD3 BsNb can specifically bind CD20 molecules on the surface of human lymphoma Raji cells and CD3 molecules on the surface of T cells in flow cytometry analysis and effectively mediate peripheral blood mononuclear cells (PBMCs) target Raji cells with a killing efficiency of up to 30.4%, as measured by the lactate dehydrogenase (LDH) method. The release of hIFN-γ from PBMCs during incubation with anti-CD20/CD3 BsNb was significantly higher than that of the control group (p < 0.01). The anti-CD20/CD3 BsNb could maintain 80% binding activity after incubation with human serum at 37 °C for 48 h. These results indicated the strong antitumor effect of the constructed anti-CD20/CD3 BsNb and laid the foundation for the further development of antitumor agents and the clinical application of anti-CD20/CD3 BsNb.

Preparation and identification of a single domain antibody specific for adenovirus vectors and its application to the immunoaffinity purification of adenoviruses.

Adenovirus belongs to the family of Adenoviridae. As a vaccine carrier, it has high safety and stimulates the body to produce cellular immunity and humoral immunity. This study prepared an adenoviral vector-specific single-domain antibody for use in adenovirus identification and purification. We successfully constructed a single domain antibody phage display library with a capacity of 1.8 × 109 by immunizing and cloning the VHH gene from Bactrian camel. After the second round of biopanning, clones specific for adenovirus were screened using phage ELISA. Twenty-two positive clones were obtained, and two clones with the highest binding affinity from ELISA were selected and named sdAb 5 and sdAb 31 for further application. The recombinant single-domain antibody was solublely expressed in E. coli and specifically bound to adenoviruses rAd26, ChAd63 and HAd5 in ELISA and live cell immunofluorescence assays. We established an effective method for immunoaffinity purification of adenovirus by immobilizing the single domain antibody to Sepharose beads, and it may be used to selectively capture adenoviruses from cell culture medium. The preparation of the adenovirus-specific single-domain antibody lays a foundation for the one-step immunoaffinity purification and identification of adenoviruses.

Preparation and characterization of a single-domain antibody specific for the porcine epidemic diarrhea virus spike protein.

Porcine epidemic diarrhea (PED) is a diarrheal disease of swine caused by porcine epidemic diarrhea virus (PEDV). It is characterized by acute watery diarrhea, dehydration and vomiting in swine of all ages and is especially fatal for neonatal and postweaning piglets. The spike protein of PEDV plays an important role in mediating virus attachment and fusion to target cells, and recent studies also reported that the neutralizing epitopes of the spike protein were mainly located in the S1 subunit, which makes it a candidate for vaccine development and clinical diagnosis. In this study, we successfully constructed an immune phage display single-domain antibody library with a library size of 3.4 × 106. A single-domain antibody, named S7, specific for the spike protein of PEDV was identified from the phage display single-domain antibody library. S7 could be expressed in a soluble form in E. coli, bound to the spike protein of PEDV in ELISA and stained the PEDV virus in Vero cells, but it showed no neutralization activity on PEDV. These results indicated the potent application of the S7 antibody as an imaging probe or as a candidate for the development of a diagnostic assay.

Effect of nisin on microbiome-brain-gut axis neurochemicals by Escherichia coli-induced diarrhea in mice.

The effects of nisin on the neurochemicals, Aquaporin-3 (AQP-3) and intestinal microorganisms in the brain-gut axis of mice were analyzed by using enzyme linked immune sorbent assay (ELISA) and high throughput sequencing in this investigation, to further revealed the relationship between intestinal flora abundance in mice and neurochemicals in the brain-gut axis. Using HE staining found damage of structure of small intestine villi in the model group (Escherichia coli O1, E. coli O1). Compared with normal control and ciprofloxacin groups, using ELISA showed that nisin increased the highest norepinephrine (NE) expression in the brain, expression of 5-hydroxytryptamine (5-HT) and dopamine (DA) in the duodenum, and increased the expression of AQP-3 in jejunum. Using high-throughput sequencing showed the highest diversity of cecal microflora in nisin group (ACE-index = 1417.25, Chao1-index = 1378.45), but the cecal microflora in the negative control group (ACE-index = 969.54, Chao1-index = 340.29) exhibited the lowest species diversity. Our data indicated that nisin regulates neurochemicals, AQP-3 and cecal microflora imbalance in mice.

Lipopolysaccharide induces SBD-1 expression via the P38 MAPK signaling pathway in ovine oviduct epithelial cells.

BACKGROUND: Beta defensins are secreted from ovine oviduct epithelial cells (OOECs) in response to microbial infection, and are potential alternatives to antibiotic agents in the treatment of microorganism infection, particularly given the abuse of antibiotic agents and the increasing number of drug-resistant bacteria. The aberrant expression of defensins may result in disorders involving organ and oviduct inflammation, such as salpingitis. METHODS: In the present study, we investigated the effects of LPS on the mRNA expression levels of sheep ß-defensin-1 (SBD-1) in ovine oviduct epithelial cells. The OOECs in vitro culturing system were established and treated with different concentrations of LPS for indicated time. In addition, MAPK inhibitors and TLR4 antibodies were pretreated to investigate the potential mechanism which involves in LPS regulating SBD-1 expression. RESULTS: LPS markedly upregulated SBD-1 expression in a concentration- and time-dependent manner. Treatment with 100 ng/mL LPS resulted in the phosphorylation of JNK, ERK and P38 MAPK. Interestingly, the LPS stimulated SBD-1 expression was attenuated by pretreatment with the P38 MAPK inhibitors SB203580 and SB202190 but not the JNK inhibitor SP600125, while the ERK inhibitor PD98059 had a minor effect. Furthermore, treatment with a Toll-like receptor 4 (TLR4) neutralizing antibody significantly decreased P38 MAPK phosphorylation and LPS induced SBD-1 expression. CONCLUSIONS: Together, these findings suggest that SBD-1 is upregulated by LPS via the TLR4 receptor, mainly through the P38 MAPK signaling pathway in ovine oviduct epithelial cells to protect the ovine oviduct epithelium from pathogen invasion.

Different ß-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity.

In this study, we examined the relationship between various ß-amyloid (Aß) oligomer assemblies in autopsy brain with the levels of fibrillar Aß and cholinergic synaptic function. Brain tissues obtained from the frontal cortex of 14 Alzheimer's disease (AD) patients grouped into early-onset AD (EOAD) and late-onset AD (LOAD) and 12 age-matched control subjects were used to extract and quantify Aß oligomers in soluble (TBS), detergent soluble (TBST), and insoluble (GuHCl) fractions. The predominant oligomeric Aß assemblies detected were dodecamers, decamers, and pentamers, and different patterns of expression were observed between EOAD and LOAD patients. There was no association between any of the detected Aß oligomer assemblies and fibrillar Aß levels measured by N-methyl[(3)H] 2-(40-methylaminophenyl)-6-hydroxy-benzothiazole ([(3)H]PIB) binding. Levels of pentamers in the soluble fraction significantly correlated with a reduction in choline acetyltransferase activity in AD patients. The number of nicotinic acetylcholine receptors negatively correlated with the total amount of Aß oligomers in the insoluble fraction in EOAD patients, and with decamers in the soluble fraction in LOAD patients. These novel findings suggest that distinct Aß oligomers induce impairment of cholinergic neurotransmission in AD pathogenesis.

Administration of amyloid-ß42 oligomer-specific monoclonal antibody improved memory performance in SAMP8 mice.

Amyloid-ß peptide (Aß) is recognized by many as the leading cause of Alzheimer's disease (AD), and Aß oligomers play a major role in the early-onset form of AD. Recently, the application of passive immunization targeting Aß has been investigated as a potential method of AD immunotherapy. We used a strain of monoclonal antibody against Aß42 oligomers, designated A8, as an Aß inhibitor to suppress Aß aggregation and Aß-derived cell toxicity in vitro, and as a passive immunotherapy approach to treat SAMP8 (senescence accelerated mouse sub-line P8) mice, an animal model of AD, in vivo. First, our results showed that pre-incubation of A8 with Aß oligomers inhibited both the maturation of Aß fiber and Aß oligomer toxicity on SH-SY5Y cells. Second, learning and memory was improved through intraperitoneal administration of A8 in SAMP8 mice. Third, Aß pathology was ameliorated with decreased Aß oligomers and phospho-tau levels in SAMP8 mice. Our data suggest that our monoclonal antibody A8 may be a candidate as a potential immunotherapeutic agent in AD.

[Isolation and identification of a human single chain Fv antibody against amyloid-beta 1-42 soluble oligomers from a human phage display library].

To get specific scFv (Single-chain fragment variable) antibody against soluble Abeta1-42(Amyloid-beta) oligomers, we constructed a human single-chain Fv (scFv) antibody library by phage display technology. Using RT-PCR, we amplified the variable heavy (VH) and variable light (VL) genes from peripheral blood lymphocytes (PBL). Then we obtained the scFv fragments through SOE-PCR, and the scFv fragments were cloned into the vector pCANTAB5E and electroporated into competent Escherichia coli TG1 cells. Consequently, a scFv phage display library containing 2.5 x 10(9) clones was constructed. The recombinant phagemids were rescued by reinfection of helper phage M13K07. Recombinant phages specific for Abeta1-42 oligomers were enriched after four rounds of biopanning and the antigen-positive clones were selected from the enriched clones by phage ELISA. Positive clone B19 was used to infect E. coli HB2151 to express soluble scFv antibody. SDS-PAGE and Western blotting analysis showed that the soluble scFv B19 antibody was expressed successfully and could bind specifically to Abeta1-42 trimer and protofiber. The specific scFv against Abeta1-42 oligomers can be used in the therapeutic research on Alzheimer's disease.