| Chicken coccidiosis (Coccidiosis in Chicken) is a very serious harm of common parasitic disease, is composed of Sporozoa, coccidia, Amy otologic various coccidiosis caused by acute epidemic infection. Over the years many scientists of the disease have done lots of research work, and there are a lot of anticoccidial drugs were developed and applied in clinical practice. From Levine found sulfonamides with anticoccidial effect begins, so far have included chemical synthetic drugs and polyether ionophore antibiotics in two categories, more than50kinds of drugs are used in the prevention and cure of chicken coccidiosis,. But as a result of the drug-resistant strains and drug residues harmful to human body and the severity of the problem, drug prevention is limited, so that the immune prevention technology to become the inevitable control of coccidiosis in chickens. Task group has successfully obtained E.tenella specific single-chain antibody gene. This experiment by constructing E.tenella bispecific single chain antibody gene ScFv and Pseudomonas exotoxin PE40recombinant immunotoxins, and recombinant immunotoxins transformed into Escherichia coli expression and purification, obtaining high purity recombinant immunotoxins, which were in vitro of Eimeria tenella killing experiment, the recombinant immunotoxins killing effect was validated, eventually lay the recombinant immunotoxins fundamental to clinical application, as well as for the control of coccidiosis in chickens to provide a favorable theoretical basis.Research methods:This paper constructed in vitro with resistance to Eimeria tenella bispecific single chain antibody gene (ScFv) and Pseudomonas aeruginosa external toxin gene (PE40) of recombinant toxin gene, recombinant toxin gene into the expression vector pET-28a, using CaC12method in Escherichia coli competent cell preparation, will be the recombinant plasmid transformed into competent Escherichia coli cultured overnight,37C, second days out of the Petri dish observation of the colony, by cultured respectively selected morphologically homogeneous colony, extraction of plasmid DNA, using EcoR I and Hind III method to do the double restriction enzyme identification, further IPTG expression induced by SDS-PAGE and Western-blot, using the expressed product was analysis of identification, and determination of protein allergens. Use of metal nickel affinity chromatography purification method will target protein was purified, via in vitro of Eimeria tenella killing experiment on target protein cytotoxicity test, and the result is drawn into a killer curves, thus laying the foundation of in vivo animal experiments, and for the control of coccidiosis in chickens to provide a favorable theoretical basis.Results:in vitro the recombinant plasmid was transformed into PET28ScFv-PE40of competent Escherichia coli, extraction of plasmid, in EcoR ? and Hind ? double enzyme action, by agarose gel electrophoresis method, respectively in5200bp and1200bp appears at a length of and of the same length prior sequence determination of nucleic acid bands, prove that bacteria in the presence of plasmid is for the purpose of plasmid. The E. coli expression induced by IPTG, and through SDS-PAGE analysis showed that ScFv-PE40recombination, gene expression, expression amount in every hour is almost no difference, the expression of the protein with a relative molecular mass of approximately66kDa (3.5kDa pET28peptide and66kDa ScFv-PE40), expression amount in the account for about13.6%of total bacterial proteins. In addition, in the control group did not contain empty plasmid pET28, so no express the same size of protein. For further identification of the expressed product, use PVDF transfer film will express proteins do transfer, the transfer film on Western blot analysis,66kDa visible a distinct protein bands appeared, that target protein with some original reaction. Objective to protein by nickel affinity chromatography purification method and purification, visible with a protein enrichment with approximately66x103, and PET28ScFv-PE40expression protein fusion protein position. Through the SDS-PAGE analysis results indicate that the target protein after purification, the purity can reach more than90%, recovery of the soluble protein in about60%, recovery rate is6mg/L. The target protein was purified after in vitro of Eimeria tenella killing experiment, and is drawn into a killer curves. Killer Curves the results show that, according to the advance of the time, the control group and the treatment group morphological integrity of Eimeria tenella gradually decreased in number, and the use of recombinant toxin group morphological integrity of Eimeria tenella number decreased significantly greater than in the control group (P<0.06), the three Killer Curves for comparison, can be seen as a result of the reorganization of toxin dose reduction, treatment group decreased gradually slowed down, proved that the recombinant toxin on Eimeria tenella killing effect more obvious, but the effect on the dose has a certain dependence. |
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