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Single chain variable fragment against a? expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

ABSTRACT: Alzheimer's disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-? (A?) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an A? N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits A? fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single chain fragment variable (scFv) without the Fc fragment is capable of regulating either A? aggregation or disaggregation in vitro. Here, a model of cell-free A? "on-pathway" aggregation was established and identified using PCR, Western blot, ELISA, transmission electron microscopy (TEM) and thioflavin T (ThT) binding analyses. His-tagged A8 scFvs was cloned and solubly expressed in baculovirus. Our data demonstrated that the Ni-NTA agarose affinity-purified A8 scFv inhibited the forward reaction of "on-pathway" aggregation and A? fibril maturation. The effect of A8 scFv on A? fibrillogenesis was markedly more significant when administered at the start of the A? folding reaction. Furthermore, the results also showed that pre-formed A? fibrils could be disaggregated via incubation with purified A8 scFv, which suggested that A8 scFv is involved in the reverse reaction of A? aggregation. Therefore, A8 scFv was capable of both inhibiting fibrillogenesis and disaggregating matured fibrils. Our present study provides valuable insight into the regulators of ultrastructural dynamics of cell-free "on-pathway" A? aggregation and will assist in the development of therapeutic strategies for AD.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC4412524 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Single chain variable fragment against aβ expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

Zhang Ying Y Yang Hai-Qiang HQ Fang Fang F Song Lin-Lin LL Jiao Yue-Ying YY Wang He H Peng Xiang-Lei XL Zheng Yan-Peng YP Wang Jun J He Jin-Sheng JS Hung Tao T

PloS one 20150428 4

Alzheimer's disease (AD) is the most common form of age-related dementia, and the most urgent problem is that it is currently incurable. Amyloid-β (Aβ) peptide is believed to play a major role in the pathogenesis of AD. We previously reported that an Aβ N-terminal amino acid targeting monoclonal antibody (MAb), A8, inhibits Aβ fibril formation and has potential as an immunotherapy for AD based on a mouse model. To further study the underlying mechanisms, we tested our hypothesis that the single ...[more]

PMID: 25919299

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Project description:As the second wave of COVID-19 launched, various variants of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have emerged with a dramatic global spread amongst millions of people causing unprecedented case fatalities and economic shut-downs. That initiated a necessity for developing specific diagnostics and therapeutics along with vaccines to control such a pandemic. This endeavor describes generation of murine derived recombinant single-chain fragment variable (scFv) as a monoclonal antibody (MAb) platform targeting the receptor binding domain (RBD) of Spike protein of SARS-CoV-2. A specific synthesized RBD coding sequence was cloned and expressed in Baculovirus expression system. The recombinant RBD (rRBD) was ascertained to be at the proper encoding size of ∼ 600bp and expressed protein of the molecular weight of ∼ 21KDa. Purified rRBD was proved genuinely antigenic and immunogenic, exhibiting specific reactivity to anti-SARS-CoV-2 antibody in an indirect enzyme-linked immunosorbent assay (ELISA), and inducing strong seroconversion in immunized mice. The scFv phage display library against rRBD was successfully constructed, revealing ∼ 90 % recombination frequency, and great enriching factor reaching 88 % and 25 % in polyclonal Ab-based and MAb-based ELISAs, respectively. Typically, three unique scFvs were generated, selected, purified and molecularly identified. That was manifested by their: accurate structure, close relation to the mouse immunoglobulin (Ig) superfamily, right anchored six complementarily-determining regions (CDRs) as three within variable heavy (vH) and variable light (vL) regions each, and proper configuration of the three-dimensional (3D) structure. Besides, their expression downstream in a non-suppressive amber codon of E. coli strain SS32 created a distinct protein band at an apparent molecular weight of ∼ 27KDa. Moreover, the purified scFvs showed authentic immunoreactivity and specificity to both rRBD and SARS-CoV-2 in western blot and ELISA. Accordingly, these developed scFvs platform might be a functional candidate for research, inexpensive diagnostics and therapeutics, mitigating spread of COVID-19.

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Single chain variable fragment against a? expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

Publications

Single chain variable fragment against aβ expressed in baculovirus inhibits abeta fibril elongation and promotes its disaggregation.

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