| Citrus canker caused by the bacterial pathogen Xanthomonas axonopodis pv. citri (Xac), 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 Xac, 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.Recombination monoclonal antibodies (RMA) are artificial constructions produced by recombining the antibody molecule or synthesizing the gene sequence with gene recombination technology and then transforming the antibody DNA into cells for expression, which hold dual function between therapy and diagnosis. Using antigen-antibodies specific binding principle, it can be used for human disease, plant disease diagnosis, treatment and prevention. Single chain variable fragment (scFv) is artificial construction small molecular antibody produced by genetic engineering, whose heavy chain variable region and light chain variable region are spliced together with a connecting peptide using genetic engineering methods. Because of its low or no immunogenicity, small molecular weight, strong organizations penetrating power, low cost, large-scale production, and other characteristics has been widely used in the medical field. But it is also unstable and prone to aggregation, it is difficult to get high concentration antibody and restricts its actual application. Another way to generate stable recombinant Fv is to connect the VH and VL by an interdomain disulfide bond instead of a linker peptide through introduce cysteines in VH and VL, this results in disulfide-stabilized Fv(dsFv). DsFv, when they can be successfully produced, solve most problems that are associated with scFv; they are very stable, often show full antigen binding activity, and sometimes have better affinity than scFv.The purpose of this study is to resolve the drawback of unstable with scFv screening by ribosome display in our laborary. The research content mainly includes: The genes of VH and VL raised against Xac were mutated by the method of PCR-based mutagenesis, and then cloned into expression plasmid. The recombinant plasmids were transformed into E.coli BL21(DE3) strain and these two genes were expressed, the products mainly exist in the form of inclusioin body. The inclusion body proteins were harvested and dissolved, and then diluted into refolding solution pro rata to form dsFv antibody which was further purified. The products of expression and renaturation were analyzed by SDS-PAGE and western-blot. The affinity of Xac-dsFv to Xac-LPS was determined by BIAcore. The specificity and stability were detected by ELISA. This research provides new reagent for Xac diagnosis, and it can be used to research the recognition mechanisms of Xac to its host, it may provide a new method to biological contrl of Xac. The mainly results are as follows:①Succeed in amplifying VH and VL and clone. Through PCR-based mutagenesis, cysteines were introduced into VH44 and VL100, sequencing indicated genes were correct. The VH and VL mutation genes were cloned into plasmids pET30a(+) and transformed into E. coli BL21(DE3).②VH and VL were expressed induceing by IPTG. The supernatants and inclusion body were analysis by SDS-PAGE. It showed the products of VH and VL existed mainly in the form of inclusion bodies, the relative mass of the targeted protein was approximated 32 kDa. Western-blot was performed to confirm the expression of VH and VL, which showed that the products of expression were correct.③The inclusion bodies of VH and VL were dissolved by guanidine hydrochloride and diluted into refolding buffer pro rata to form Xac-dsFv antibody. After renaturation the Xac-dsFv was analyzed by non-reduced SDS-PAGE, reduced SDS-PAGE and western-blot. It showed that Xac-dsFv was renaturation successful, the relative mass was 46kDa. The Xac-dsFv was purified with HisTrap HP column; grey analysis indicated that the purity of Xac-dsFv was up to 83.27%.④The affinity of Xac-dsFv was analysis by BIAcore, the results demonstrated Xac-dsFv retained high affinity to Xac-LPS with binding constant(ka) of 3.40×10-10M, dissociation constant(kd) of 5.47×10-2 s-1 and affinity constant(KD) of 9.98×10-10M. The Specificity of Xac-dsFv was detected by Dot-blot and ELISA, it showed that Xac-dsFv kept high Specificity to Xac, and had no cross reaction with others ten control bacterium.⑤The stability of Xac-dsFv was detected with Xac-scFv as control. The Xac-dsFv and scFv in PBS were incubated at 37℃for a serious of time and at a serious of temperatures for 3 h. ELISA showed, at the condition of 37℃, in the first 12 h the activity of scFv reduced to 25.6% and nearly lost all activity 24 h late. For Xac-dsFv in the first 12 h the activity reduced slightly to 80.2% and remained relatively stable in later tests, and still remained 61.3% activity 72 h late. The activity of scFv was maintained at 25℃, and when the temperature reached 35℃, the activity reduced to 30.7% and lost all activity at 45℃. The binding activity of Xac-dsFv was retained and showed almost no change at 35℃, 52.6% activity was maintained at 45℃and lost activity at 55℃. Thus, the result showed that the relative stability of Xac-dsFv was higher than that of scFv. Compared with Xac-scFv's, the thermal stability of Xac-dsFv elevated nearly 20℃.
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