Establishment And Preliminary Application Of An Indirect ELISA Method For Detection Of Chlamydia Abortus Antibody Based On OmpA Protein

Chlamydia abortus（C.abortus）is the main pathogen causing endemic abortion of ewes（EAE）in sheep.It is a member of the genus Chlamydia,it is a gram-negative organism that is strictly parasitic in cells.The disease can infect goats,sheeps,cows and other animals through the placenta and cause abortion and stillbirth of female livestock.Pregnant women can also be infected by direct contact with infected livestock,resulting in abortion.Because animals infected with C.abortus dosen’t show any symptoms in the early stage.They are often overlooked or easily misdiagnosed.At present,the widely used clinical detection method is IHA,which has poor sensitivity,so it has a certain degree of early diagnosis of the disease.Therefore,it is necessary to establish a method for detecting C.abortus antibodies in clinical practice,so as to achieve the purpose of screening infected sheeps in time.In this experiment,the prokaryotic expression of the ompA gene of goat C.abortus from Shaanxi isolates was carried out,and then an indirect ELISA method for detecting antibodies was established using the purified ompA protein,and the established method was used for preliminary clinical detection.The results of the research are as follows:1.Abortion occurred in a dairy goat farm in Shaanxi Province.Laboratory tests confirmed that the abortion was caused by C.abortus infection.On this basis,ompA（1 503 bp）and CPAF（1 506 bp）genes of C.abortus in Goats were successfully cloned.The ompA gene is 99.54%-99.91%homologous to the ompA gene sequence of other sheep C.abortus genomes,and the CPAF gene is 91%-100%homologous of other sheep C.abortus genomes.This two genes are all have highly conserved and intraspecific conservation.The genetic evolution tree was constructed to show that the ompA gene of the Shaanxi isolate was closely related to the Dutch isolate DQ435300.1.The CPAF gene is closely related to the British isolate CR848038.1.2.The recombinant prokaryotic expression plasmid p ET-28a-ompA was constructed.After 1 mmol/L IPTG induced expression for 7 h,the protein expression was the largest,and the ompA protein with a size of about 38 ku was obtained after purification by Ni column.The Western blot test showed that the ompA protein could bind to the positive serum of C.abortus and had reactogenicity.3.An indirect ELISA method was developed to detect serum antibodies to C.abortus in goats used ompA protein as coating antigen.Protein coated 6μg,the serum to be examined for 1:200 dilution,the enzyme standardⅡantibody was diluted 1:5000,and the time of TMB was 30 min,the critical value was 0.324;This method can only be combined with the positive serum of C.abortus,and has strong specificity.When the positive serum was diluted 1:1 280,OD_450nmwas still above the critical value of 0.324,and the sensitivity was high.The intra-batch coefficient of variation ranged from 4.70%-8.02%,and the inter-batch coefficient of variation ranged from2.43%-9.13%,had a good repeatability.The indirect ELISA method was used to detect 574 serum samples from dairy goats in six different areas of Shaanxi province,and the overall positive rate was 14.98%.In conclusion,laboratory diagnosis of abortion in a dairy goat farm in Shaanxi province was carried out in this study,ompA and CPAF genes of Shaanxi strain of chlamydia abortus were cloned and analyzed.On this basis,an indirect ELISA method based on ompA protein was established and applied preliminarily.A method for detection of C.abortus antibody was established,and clinical data were provided for the prevalence of C.abortus in Shaanxi province.