| Citrus canker caused by the bacterial pathogen Xanthomonas axonopodis pv. citri (Xcc), a gram-negative bacterium, is a severe bacterial disease of most comMercial citrus species and cultivars around the world, as well as some citrus relatives. For the lack of resistant cultivars and specific chemicals, the following measures have been adopted to control CBCD for a long time: enhanced construction of non-quarantine area and pHytosanitary system against the spread of Xcc, eradication and mass destruction methods dealing with infected plants. The pathogen is the target of quarantine efforts abroad and domestics, then the development of rapid and reliable procedures for diagnosis and control of this pathogen has been an important priority.Single-chain antibody (single chain antibody, scFv) antibody molecule is retained in the smallest functional antigen binding site fragments of a complete antibody molecule molecular weight of about 1/6, its superiority is expressed by a large number of inclusion bodies, easy to genetic engineering operation In particular, easy to construct antibody fusion protein, based on these characteristics, recombinant antibody prospects in the medical field has caused widespread concern. Genetic Engineering Research Center of Chongqing University using citrus canker citrus canker was prepared by single-chain disulfide-stabilized recombinant antibody and antibody, but the stability of scFv poor, and prone to aggregation, in the preparation process is difficult to obtain a higher concentration the active protein. Disulfide-stabilized Fv antibody (disulfide stabilized Fv fragments, dsFv) is more stable than single-chain antibody, but the antibodies also have very low output and high efficiency recovery of the defects. To this end, using the disulfide-stabilized antibody dsFv be based on the introduction of a linker sequence to link VH and VL gene, the advantages of these two antibodies to develop a new set of antibodies, which received better stability single-chain disulfide stable antibody (single chain disulfide-bond Fv, sc-dsFv) antibody.The purpose of this study is to scFv antibody against citrus canker less stable and dsFv antibody production with very low efficiency and high recovery of the defects, building a kind of stability and refolding efficiency of the new antibodies are high, to overcome The first two defects, and for the future of citrus bacterial canker disease diagnosis and prevention of Xcc imMunity and provides new tools and approaches.The study include the following: in the laboratory has received on the basis of disulfide-stabilized antibody mouse anti this study the surface of citrus bacterial canker disease of disulfide-stabilized antibody dsFv LPS as a template, overlap extension PCR in the heavy chain can be variable region VH and VL light chain variable region between the introduction of a human antibody heavy chain constant region 1 (CH1) of the 5 'end of the 12 amino acid sequence as the linker peptide, the recombinant single-chain Fv antibody gene, to connection into the pET24a (+) prokaryotic expression vector and transformed into expression strain E.coli BL21 (DE3), by the expression, renaturation and purified by high-purity sc-dsFv antibody and its specificity, affinity and stability Determination. The mainly results are as follows:①Respectively dsFv-VL gene and dsFv-VH gene as a template by a PCR amplification, the heavy chain VH and light chain variable region VL of a human antibody are introduced between the heavy chain constant region 1 (CH1) 5 'end of the 12 amino acid sequence as a linker peptide, in another round of overlap extension PCR to light chain gene and heavy chain gene fusion construct recombinant antibody sc-dsFv gene by gene sequencing results showed that the correct sequence, linker sequence was successfully introduced between the two genes.②Construction of good genes connected by the digested expression vector pET24a (+), and transformed into the prokaryotic expression strain BL21 (DE3) in. Induced by IPTG, was inclusion body protein. Of the expressed protein SDS-PAGE electrophoresis and Western blot hybridization, electrophoresis results showed that the target protein molecular weight of 30kDa, Western blot hybridization showed that target protein in the correct and expected ideas consistent with that of recombinant antibody sc-dsFv success in prokaryotic expression vector to form of inclusion bodies, over expression, the calculated production of inclusion bodies per 100 ml broth about 0.2g.③Induced acquired sc-dsFv inclusion bodies were dissolved in dilute GuHCl in refolding buffer, refolding, using Ni-NTA column refolded Xcc-sc-dsFv was purified, the purity of gray-scale analysis shows that about 90%.④Interactions with macromolecules instrument (biacore) of Xcc-sc-dsFv antibody affinity, the results show that the Xcc-sc-dsFv its antigen of lipopolysaccharide LPS Xcc has a high affinity binding constant (ka) was 5.31×106 M-1 ? s-1, dissociation constant (kd) is 5.53×10-2 s-1, affinity constant (KD) was 1.04×10-8M; Dot-blot and ELISA specificity tests show that sc -dsFv antibody specific to the purpose of antigen binding activity of strain Xcc good bacteria on the other ten non-binding activity near the source.⑤The stability of sc-dsFv was detected with Xcc-scFv and dsFv as control. The results showed that in a variety of temperatures, the three kinds of antibodies, scFv storage stability of the lowest, dsFv storage stability of the second, sc-dsFv most stable. Conditions at 37℃, scFv at 12 h, the activity decreased sharply to the initial activity of 23.7%, dsFv part in the activity decreased after 12 h and 72 h, maintaining the original activity of 63.2%, while the sc-dsFv activity has been slightly higher than the dsFv until 48 hours consistent with the basic. scFv were incubated at 35℃for 3 h activity declined rapidly from the initial 17.2%, has been basically inactivation, 45℃after 3 h incubation dsFv still has 58.6% of the activity, were incubated at 55℃for 3 h, almost no activity, sc-dsFv in 45℃-55℃higher than the activity between the dsFv. Frozen at -20℃scFv activity test within a week dropped to about 20% activity in 60 days to disappear. dsFv and sc-dsFv activity over time have decreased in the first 150 days of the former activity decreased to 50%, while the sc-dsFv activity remained at 70%. Thus, the result showed that the relative stability of Xcc-dsFv was higher than that of scFv. Compared with Xcc-scFv's, the thermal stability of Xcc-dsFv elevated nearly 20°C. |
