| Clostridium perfringens type C can infect pigs, horses, cattle, sheep or goat and otherlivestock animals. In particular, newborn piglets that are less than3days old are moresusceptible to this infection, commonly known as piglets red dysentery. This disease is aserious threat to the livestock industry in China because of the high mortality. Clostridiumperfringens type C produces three major toxins namely alpha toxin (CPA), beta1toxin (CPB1)and beta2toxin (CPB2), in which alpha toxin is an important virulence factors possessing thecharacteristics of cytotoxic, hemolytic, lethal and dermonecrotic activities. At present,preventive measures of red dysentery caused by Clostridium perfringens type C are takensuch as vaccination against Clostridium perfringens type C in pregnant sows throughintramuscular route before farrowing, that in-turn helps the piglets to gain passiveimmunization from sows by feeding colostrum. Meanwhile, antibiotics such as penicillin,oxytetracycline and other are also given as a therapeutic measure. Nowadays, some antibodydrugs targeted against Clostridium perfringens type C are also available in the market. In therecent decade, passive immunization by oral administration of pathogen specific chicken eggyolk antibodies (IgY) is an effective approach to provide a rapid and problem basedprotection due to its high specificity, cost-effectiveness, convenience with high yield. IgYantibodies possess strong immunization reaction to the highly conserved protein of mammalswith the advantage of phylogenetic distance between chickens and mammals. Therefore, IgYhas noticeable economic advantages and higher market value for the immuno-prevention andimmunotherapy of the mammalian digestive tract diseases. The purpose of this study was toproduce the IgY antibody against recombinate Clostridium perfringens type C alpha toxinprotein expressed in E. coli, with respect to develop the diagnostic kit and antibody drug fordiagnosis and treatment of the Clostridium perfringens type C infection respectively. Thestudy was divided in three parts:1. Amplification of the Clostridium perfringens Type C alpha toxin sequence. A pair ofprimers was designed by primer prime5.0software based on alpha toxin sequence in thepaper reported by Titball et. al (1989) and commercially synthesized primers were used to amplify the alpha toxin genes. The amplified fragments (1200bp) were inserted into theT-Vector pMD19, then introduced into DH5α. Plasmids of the cultured DH5α were extractedand digested with restriction endonuclease EcoR I and Xho I after blue/white selection. Thepositive clones were selected for sequencing.2. Expression and purification of the alpha toxin protein. The T-Vector pMD19-α andpET-32a both were digested with EcoR I/Xho I and ligated with T4DNA ligase to yieldexpression plasmid pET-32a-α. The pET-32a-α was introduced into BL21(DE3) and inducedwith IPTG after identifying by enzyme analysis and PCR reaction. The concentration of IPTGand the induction time were optimized subsequently. After being analyzed by SDS-PAGE andWestern blot, the alpha toxin protein was purified by Ni+affinity column. The results showedthat the alpha toxin protein was expressed in an inclusion format when induced with0.5mmol/L IPTG for6hours. The alpha toxin protein molecular weight band (61kD) waspurified by Ni+affinity column and exhibited perfect reactivity by Western blot.3. Production of IgY antibody against alpha toxin protein. Anti-α IgY was produced byimmunizing the laying hens with recombinant alpha toxin protein after dialysis. The generatedIgY antibody was extracted from immune eggs by Water Dilution-two-step (NH4)2SO4precipitation method and analyzed by SDS-PAGE. The titer of IgY antibody against alphatoxin protein was monitored by indirect ELISA. Specificity of the IgY antibody against alphatoxin protein was identified by Western blot. Results showed that the IgY antibody consistedtwo parts,65kD and27kD. The titer of the anti-α IgY was found to be1:320000after thefourth immunization, meanwhile the result of Western blot indicated that prepared IgYantibodies interact with recombinate alpha-toxin specifically.In this study, the specific IgY antibody against Clostridium perfringens type C alphatoxin protein was developed and thus could be used as tool to prepare antibody drug for theClostridium perfringens type C infection. |
