Studies On The Cloning And Expression Of Single-chain Fv Antibody Fragments

Myasthenia gravis (MG) is an autoimmune disease characterized by transmission barriers at neuromuscular junction(NMJ). In 80-85% of myasthenia gravis patients, antibodies to the nicotinic acetylcholine receptor (AChR) can be detected. Many investigations, both in vitro and in passive transfer models, have shown that these antibodies reduce the number of functional AChRs at the postsynaptic membrane by increasing AChR degradation, inducing complement-mediated damage to the postsynaptic membrane, or by blocking AChR function. Myasthenia crisis, defined as respiratory failure requiring mechanical ventilation in MG, is a common life-threatening complication that occurs in approximately 15% to 20% of patients with MG during their life time.Cloning of only the variable genes of an Ig allows the production of the Fv fragment. Nevertheless, this molecule tends to dissociate because of the lack of covalent or disulphide bonds between the two chains. Placing a linker peptide gene between VH and VL genes allows the expression of the single chain Fv (scFv) fragment, which does not exist in nature and is much more stable than Fv, maintaining its antibody specificity, folding, low immunogenicity (they have humanized, smaller molecules), also more easily be labeled genetically with modified elements. These reagents have been developed as therapeutics against cancer and HIV. Since misfolded and accumulated intracellular proteins characterize a wide range of neurodegenerative disorders, they are also potentially useful intrabody targets. Here, we discuss the extension of intrabody technology to the nervous system, in which studies of Huntington's disease have been used to develop the approach, and anti-synuclein and beta-amyloid strategies are in the early stages of development. Research on several other neurodegenerations, including Parkinson's, Alzheimer's, and prion diseases, provides support for the development of intrabodies directed against specific targets, or possibly against more common downstream targets, as novel therapeutics and as drug discovery tools.Univalent antibody fragments directed against the main immunogenic region (MIR) of the human acetylcholine receptor (AChR) are capable of protecting the AChR against loss induced by antibodies from myasthenia gravis (MG) patients. Pasteur Institute constructed and purified the functional scFv fragments of 192 and 195. These fragments showed high affinity (K_D=0.6nM and 0.8 nM) to human AChR. These fragments are two orders of magnitude higher than early constructed scFv198 (K_D=80nM). The scFv192 almost completely protected human AChR against binding of intact anti-MIR mAb. Human AChR was also very efficiently protected (74-84%) by the scFv192 against binding of autoantibodies from MG sera with high anti-alpha subunit antibody fractions. These scFvs are good candidates for protection of MG patients after appropriate genetic modifications. However, they expressed these scFvs using the expression vector plasmid pHEN1, which is a phagemid particle. As a result, the quantity of the soluble antibody fragments isolated from culture supernatants was small.To achieve more scFv fragments, we cloned the DNA fragment that encode the scFv1929# into the prokaryotic expression vector pET32a(+) which could add a Trx tag on the N-terminal of the target protein. The reconstructed vector is then transformed into the expression host BL21(DE3) pLysS. Our main results are as following:Using pHEN1 as template, the scFv1929# DNA with the restriction site of EcoR v and Not I was amplified by polymerase chain reaction (PCR) and cloned into prokaryotic expression plasmid pET-32a(+). Then E. coli BL21(D E3) pLysS cells were transformed with the recombinant expression vector pET-32a(+)-scFv1929#. The size of scFv1929# PCR products from pHEN1 was 730 bp. The length of the scFv1929# gene fragment was 730 bp after digesting the recombinant vector with EcoR v and Not I, and it confirmed that the scFv1929# DNA was inserted into the expression vector correctly. DNA sequences were verified by sequencing.Then E. coli BL21(D E3) pLysS cells were transformed with the recombinant expression vector pET-32a(+) -scFv1929# Two different transformation temperature and IPTG concentration were tried during the expression. The accuracy of inserted gene and specificity of scFv1929# were detected by two enzymes digestion technology, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blot(WB). The expressed proteins had a single expected band of about 44 KD in SDS-PAGE. Though most of the expressed proteins were produced as inclusion bodies, there were many products isolated from culture supernatants as soluble fragments, which can be purified in the future. And the temperature and concentration of IPTG had little effect on the quantities of the soluble products. The specificity of scFv1929# were tested by WB.In summary, this study successfully constructed scFv1929# efficient prokaryotic expression systems. While most are still expressed as target protein inclusion bodies, but the soluble protein in the supernatants become larger, which can be directly used in purification and has good specificity.