Expression Of The Major Outer Membrane Protein And Establishment Of ELISA For Diagnosing Swine Chlamydophila Abortus

Swine Chlamydophila abortus was caused by Chlamydophila abortus(CA)and the mainly syndromes of it were abortions of female animals and orchitis of male animals. In this thesis, The major outer membrane protein (MOMP)gene of Swine CA from QingHai was cloned, analyzed and expressed in E.coli. Then Indirect ELISA for diagnosising it was established by using recombinant MOMP. Results were obtained as follows:1. Cloning and analyzed of the major outer membrane protein coding gene of Swine CA. Genomic DNA was extracted from the Chick embryo yolk sac infected with Swine CA. According to the published complete sequence of MOMP in GenBank, four primers specific to MOMP gene were designed. The MOMP gene amplified by nested PCR was cloned into pMD18-T vector to sequence and compare with other CA strains. Sequencing analysis results indicated that the ORF was 1170 bp and encoded a polypeptide of 389 amino acid residues with the predicted molecular mass of 41.8 ku. Then SignaIP 3.0 Server analysis showed that amino acid residues from the 1st to the 19th could represent the signal peptide sequences.2. Prokaryotic expression of the MOMP gene of Swine CA. According to MOMP gene sequence, a pair of primers specific to the target gene were designed. The 1101 bp MOMP fragments without signal peptide sequence was digested by EcoR I and Sal I and cloned into the vector pGEX-4T-1.After being ligated, it was transformed into Escherichia coli. The insert position and the size of the ORF were indentified by PCR, enzyme digestion and the sequence analysis. The results showed that the prokaryotic expression vector was constructed successfully. By transform pGEX4T-MOMP into BL21(DE3), the recombinant strain BL21(DE3) was obtained. After being induced by IPTG, the expressed proteins were measured by SDS-PAGE and Western-blot. The SDS-PAGE results showed that the genes can express successfully in E.coli. The Western-blot results indicated that the expressed protein can be recognized and combinated with positive serum of swine CA. By extractting, solubing, SDS-PAGE, Target proteins were isolated by cutting the gel slices that contain the right bands. Proteins in gel slices were electronically eluted by running the slices in dialysis bags. The recombinant protein purificated was less lost and could meet the need of antigen and antibody concentration in the tests.3. Establishment of Indirect ELISA for Swine CA.The purificated recombinant proteins were used to coat a 96 well high affinity ELISA plate as antigen. A series trials were performed for the groped optimal reaction conditions of the indirect ELISA and the results were obtained as follow: the optimal concentration of serum was 1:20, the optimal concentration of MOMP was 1:400; the optimal concentration of anti-pig IgG-HRP was 1:80000; the best coating condition was 37℃1h and 4℃overnight; the critical value of the indirect ELISA was 0.381. On this basis, the indirect ELISA was compared with the indirect hemagglutination test kit, detecting 150 of serum samples achieved 96.7%. This method was established for diagnosis and detecting swine chlamydiosis.