| According to the encoding gene sequences of M-like protein published on Genbank, a pair of primers using particular software was designed and synthesized.Then target gene was amplified from genome DNA of ATCC35246strain by Polymerase Chain Reaction and was ligated into the expression vector pET-28a(+).The successful construction of recombinant plasmid was confirmed by PCR identification and restriction endonuclease enzyme indentification,and the recombinant plasmid was transformed into competent cell BL21(DE3).Positive clones were picked out,cultured and induced by IPTG under certain temperature.The result of SDS-PAGE demonstrated that the fusion protein had been expressed as soluble form in supernatant,the Molecular weight was identical to the hypothesis.Western-blot showed that the fusion protein could be recongnized by the recovered swine serum collected from pigs infected by SEZ ATCC35246strain,that is, the target protein has relatively good immunogencity and the corresponding antigen epitopes.Using affinity chromatography,ion exchange chromatography and gel filtration chromatography to purify the M-like protein, and the standard curve was drawed by Bradford-albuminimetry,the statistic knowledge could also be used to ascertain the concentration of protein.On this basis,the purified protein was applied as coating antigen and the chessboard method was applied to confirm the optimized concentration of antigen and dilution degree of serum. A series of optimization experiments were also needed for the establishment of indirect ELISA kit. After that,the specificity,sensitivity and repeatability of kit was verified by our tests. Besides,the average value and standard deviation were concluded by the ELISA test for the standard negative serum. And the critical value was drawed according to the empirical fomula.Epidemiology investigation was carried out with the above kit,and the main serum sources were400porcine serums stored in our lab,80porcine serums collected from the pigs’farm in Nanjing or nearby districts,100porcine serums collected from a meat-food factory in Nanjing,and the experiment animal’s serums such as rabbit,ICR mice.Besides,some cattles’serum were also be detected by our kit.The result showed that in porcine serums kept by our lab, the total positive rate of SEZ’s antibody was45.69%,among which the mature pigs’ postive rate was80.14%,immature ones’ was27.50%,the healthy mice’s was30.20%while the healthy rabbits’ was25.16%and the cattles’ was10%... |
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