Isolation And Identification Of Shigella From Chicken And Clone、prokaryotic Expression And Polyclonal Antibody Preparation Of IpaC Gene

Shigellae are one of the most important pathogens which cause infectious diarrhea in human.They can also infect primates(except human).People think that livestock and birds weren’t infected by shigellae in the past,in fact,pig,cattle,rabbit, duck,chicken can be infected and die.Chicken shigellosis found some years ago is an acute infectious disease. This disease is widespread and harmfull,can cause chicken with different species and ages diarrhea and even lead to chickling die resulting in enormous economy loss for poultry industry in our country.Because of aleopation among people and poultry caused by shigella,it leads to grave hidden danger to public health in mankind.Therefore, the study and research on chicken shigellosis has important significance not only for veterinary surgeon hygiology but also for public medical science hygiology.In this research shigella was isolated with tissues from diarrheic chicken, IpaC gene from this shigella was cloned,analyzed,expressed in protocaryon cell and used to prepare polyclonal antibody. The concrete research results were showed as follow:1.Isolation and identification of shigella from diarrheic chicken through isolating culture,test under microscope after smearing,biochemistry test,PCR test and animal experiment.The result showed that a strain of shigella was isolated from diarrheic chicken’s heart in Zhumadian area;this strain had a serious pathogenicity, morbidity of chickling was a hundred percent(10/10),mortality was fifty percent(5/10) in test group, sick chicken clinical situation was bloody stools,even purulent dysentery;chicken which was sick or mortuary intestines showed that intestines become thin and had ecchymosis, the liver intumesced and bled, the lung bled and congested,the spleen bled, the kidney intumesced; pathological sections were tested under microscope showed that epithelium which lied intestinal mucosa sphaceled and fell off, villus intestinalis disintegrated, tunica propria bled, crypts of lieberkuhn shrinked back, central veins and sinusoid in the liver expanded and congested, sinus lienis congested, alveolus,bronchus and respiratory bronchus congested.2. According to two sides of conservative domain of IpaC gene from human shigella published on GenBurk. a pair of primer was designed. IpaC was amplified from a strain of chicken shigella in Zhumadian by PCR method.cloned to carrier pMDl8-T.and sequenced. The result showed that compared to shigella from people.two bases of this strain were mutation,the position757transformed from T to C, the position936transformed from G to A, the mutation of the position757changed arg to gly,but hadn’t a big influence on protein expression; the homology of IpaC genes from different strains was very high, the nucleotide sequences homology were from97.3%to99.9%, the homologous amino acid sequences homology were96.2%to99.7%.Among the total, IpaC gene of chicken shigella had the highest nucleotide sequence and homologous amino acid sequence homology with shigella flexneri from Japan(X15319), the homology was99.8%、99.7%in order.This gene’s homology which had the lowest nucleotide sequence homology with shigella boydii from American(CP001062) was97.3%, which had the lowest homologous amino acid sequence homology with Shigella dysenteriae from Australia(AY206439) and. shigella boydii from American(CP001062) was96.4%;the isolated strain had the highest similarity to reference strain AL391753,they were on the same branch.3. IpaC gene from shigella in Zhumadian was cloned to carrier pFT32a(+).prokaryotic expression carrier pET32a(+)-IpaC was constructed.Then carrier pHT32a(+)-lpaC was transformed host bacterium BL21(DH3) which was induced by IPTG. SDS-PAGE indicated recombination protein was expressed. The molecular mass of recombination protein was63kDa, the amount of recombination protein held21%of all. Cultivation time before inducing.IPTG concentration and induction time was optimized,the final result was that cultivation time before, inducing was3h (OD result was0.6), IPTG final concentration was1.0mM, induction time was4h. Recombination protein was expressed with large scale, bacteriums were clearaged, supernate and sediment was detected with SDS-PAFE,the consequence demonstrated recombination protein was not only in supernatant,but also in sediment.The supernate was purified with Ni-Agarose His tag purification kit. purity of recombination protein was above90%. Western Blot analysis result demonstrated that recombination protein had specific reaction with ZMD-01shigella masccline. Recombination protein purified was immuned to cock,the highest potency of serum reached to1:12800explaining recombination protein had favourable immunogenicity.