Jianguo Zhu

Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9

This article describes a proposed method for convenient and efficient detection of Newcastle diseases virus (NDV) that uses the fusion of single-chain variable fragment (scFv) and pOPE101 vector. In order to select the single chain variable fragment (scFv) against NDV F48E9, the total RNA was extracted from the spleen of immunized chicken, and then was converted into cDNA via the reverse transcription. The scFv was spliced by using splice-overlap extension polymerase chain reaction (SOE-PCR). The scFv gene was cloned into a pOPE101 vector and expressed in E. coli. Under the optimized conditions, antibody affinity was studied by indirect ELISA. One positive clone was selected by ELISA screening, named ZL.6. Based on the positive clone and the germline sequence, the results of sequence analysis showed that there are more variation in CDR of VH and VL. In addition, BHK21 cell culture was conducted to examine the potential antiviral activity of ZL.6. The experimental result demonstrated that ZL.6 was able to neutralize NDV F48E9 which infected BHK21 cells. So ZL.6 will be proved useful for further characterization of NDV as potential diagnostic tool and therapeutic agent.

Construction of scFv that bind both fibronectin-binding protein A and clumping factor A of Stapylococcus aureus

Bovine mastitis (BM) causes significant losses to the dairy industry. Vaccines against the causative agent of BM, Staphylococcus aureus, do not confer adequate protection. Because passive immunization with antibodies permits disease prevention, we constructed a recombinant single-chain antibody (scFv) against fibronectin-binding protein A (FnBPA) and clumping factor A (ClfA), two important virulence factors in S. aureus infection. The DNA coding sequences of the variable heavy (VH) and variable light (VL) domains of antibodies produced in the peripheral blood lymphocytes of cows with S. aureus-induced mastitis were obtained using reverse transcription and polymerase chain reaction, and the VH and VL cDNAs were assembled in-tandem using a DNA sequence encoding a (Gly4Ser)3 peptide linker. The scFv cDNAs were cloned into the pOPE101 plasmid for the expression of soluble scFv protein in Escherichia coli. The binding of the scFvs to both FnBPA and ClfA was confirmed using an indirect ELISA and Western blotting. The DNA sequences of the framework regions of the VH and VL domains were highly conserved, and the complementarity-determining regions displayed significant diversity, especially in CDR3 of the VH domain. These novel bovine antibody fragments may be useful as a therapeutic candidate for the prevention and treatment of S. aureus-induced bovine mastitis.

Selection and characterization of single-chain recombinant antibodies against phosphoprotein of newcastle disease virus

Phosphoprotein (P), involved in virus RNA replication and transcription, had become a new target for the research on treating Newcastle disease virus (NDV). Here we described the cloning and expression of phosphoprotein from NDV, and then screened the anti-P antibodies from the chicken single chain fragment variable (scFv) library, which were generated from chickens immunized with the ND vaccines. As a first step, the recombinant expression vector pET28a-P was successfully constructed. In a following step, two anti-P positive scFv clones from the scFv library were selected by indirect enzyme-linked immunosorbent assay (ELISA) method. The sequence analysis of two positive clones showed that there were more variation in complementary determine region (CDR) of VH and VL, and the CDR3 in VH exhibited a significant change in amino acid number and type. In another experiment, the purified scFv antibodies used in the assay was shown to be specific for NDV-P by western blot. The results indicated that the strategy we used in this experiment proved to be convenient way for screening scFv antibody, which paved a new way for the immunization diagnosis and the exploration of integrated control of NDV.

Production and properties of single domain antibody fragments

Single domain antibody (sdAb) is the smallest fully functional antigen-binding fragment. The use of a recombinant sdAb offers a number of benefits in medical and biotechnological applications. This article offers a mini review of the generation and selection of specific sdAbs from a display library accompanied by improving their affinity, stability, and expression; antibody engineering technologies provide an extraordinary opportunity to design new molecules and tailor antibody fragments with increasing efficiency; sdAbs offer an attractive alternative to traditional immunoassays due to their valuable intrinsic properties and their therapeutic applications.

THE PRIMARY USE IN INDIRECT ELISA OF SECRETED PROTEINS MB1761C AND MB2277 OF M. BOVIS

Disease monitoring on bovine tuberculosis (BTB) has been proven to be a capable strategy to control this disease, which has significantly progressed in developed countries. However, such strategies require a prompt, accurate live animal test, which has thus far been lacking in developing countries, such as China. The two secreted proteins, Mb1761c and Mb2277, were selected from the complete genome of M. bovis by using a bioinformatics method. After having been proven by the reactogenicity by Western blot, the two expressed prokaryotic proteins served as coat antigens in ELISA plates to evaluate their ability to detect the antibodies against M. bovis. The results showed that the sensitivity of the indirect ELISA method coated with Mb2277 as antigen was as same as that coated with Mb1761c. To explore the possibility of increasing the sensitivity of the ELISA test, the two proteins were further mixed in a different ratio and coated as antigen in ELISA. The results showed that the sensitivity of the combination of the coated reconstructive proteins (1:1) was stronger than that of single coated protein.