Porcine proliferative enteropathy(PPE),commonly known as porcine ileitis(PI),is caused by Lawsonia intracellularis(LI)infection.In this study,a recombinant expression plasmid pET28a-LI0841 containing the LI outer membrane protein gene(LI0841)was constructed,sequenced and then transformed into BL21(DE3)host bacteria.IPTG was added to induce expression,and the expression form of recombinant LI0841 protein(rLI0841)was determined by SDS-PAGE.After purification by Ni column,the expressed protein was identified by western blot.The results of SDS-PAGE showed that the target band appeared at 32 ku,and it was mainly expressed in a soluble form.The western blot results showed that rLI0841 could react specifically with the rabbit LI polyclonal antibody,and thus could be used as a coating antigen to establish an indirect ELISA detection method.The optimization results of each reaction condition of the indirect ELISA method as follows:the optimal coating concentration of rLI0841 was 4.38ng/well at 4℃overnight;the best dilution of serum was 1∶100 and the duration of the incubation was 0.5 hour at 37℃;the goat anti-pig IgG-HRP was 1∶10000 dilution and the duration of the incubation was 0.5 hour at 37℃;TMB substrate developed color at 37℃for 15 minutes.The established method could specifically detect LI antibody and had no cross-reactivity with PRRSV,PRV,HPS,SS,APP pig positive serum.The coefficients of variation(CV)of intra-and inter-repeatability tests were both less than 10%,indicating good specificity,sensitivity and repeatability.Compared with AsurDxTM Lawsonia Intracellularis antibody test kit,the compliance rate of the two methods was 91.35%.The established indirect ELISA method was used to detect 413 clinical pig serum samples from Heilongjiang,Jilin and other regions.The positive detection rate of LI was 59.81%(247/413),indicating that LI was prevalent in pig herds in Heilongjiang,Jilin and other regions.This study established a specific ELISA antibody detection method against LI,and provided a method for monitoring the prevalence of PPE in China and scientifically preventing and controlling the disease.
Lawsonia intracellularisindirect ELISArecombinant LI0841 protein
NETL
NSTL
维普
万方数据
