Identification Of B-Cell Epitopes Of MSG1of Mycoplasma Suis And Development Of A Double Antibody Sandwich Elisa For Detection Of MSG1Protein

Mycoplasmas suis/M.suis was also known as porcine eperythrozoon/PE, which was the pathogen of infectious anemia. It threatens the global pig industry and causes great economic loss in our country. There were at least eight proteins of M.suis were identified. Among of them, MSG1was an important adhesion protein of M.suis, which has glycolytic activity. In this study, five monoclonal antibodies (MAbs) against M.suis MSG1protein were screened and three linear epitopes (251-255aa,268-274aa and291-197aa) were identified with them. It provides the foundation for developing the epitope vaccine against M.suis. Moreover, two MAbs of MSG1protein were used to establish the sandwich ELISA. It provides the foundation for detection of M.suis in the future.The contents of this research contain3parts as following:1. Expression and monoclonal antibodies preparation of MSG1of Mycoplasma SuisThe recombinant plasmid pBAD/His-msgl was transformed into E.coli Top10, and the recombinant bacteria were induced by arabinose. BALB/c mice were immunized with purified rMSGl native protein. The monoclonal antibodies (MAbs) were prepared by fusing mouse myloma cells with spleen cells from BALB/c mice immuned with purified recombinant MSG1protein. Five hybridoma cell lines secreting MAbs against MSG1protein were screened by indirect enzyme-linked immunosorbent assay (ELISA) and named as1C10,2D8,2F10,4G10and10E9, respectively. Western blot and indirect immunofluorescence assy (IFA) results showed that the obtained MAbs reacted specifically with recombinant MSG1. It could be concluded that the specific anti-MSGl protein MAbs were developed and they may be useful in the development of detection assays and provide basis for mapping the epitopes of MSG1protein.2. Identification of B-cell Epitopes of MSG1of Mycoplasma suisIn order to identification of B-cell epitopes of MSG1of Mycoplasma suis,23truncated msgl gene fragments were designed and amplified by PCR. These fragments were cloned into the pET-32a vector and these recombinant proteins were expressed by Escherichia coli system. Epitopes mapping results indicated that MAb4G10and10E9recognized the linear epitope I(268)KDGENE(274), MAb1C10recognized the epitope D(291)THGSVF(297), and MAb2F10reacted with the epitope L(251)CLKI(255). These results will facilitate future investigations into the function of MSG1of Mycoplasma suis and establishment of diagnostic methods for Mycoplasma suis infection.3. Development of a double antibody sandwich ELISA for detection of MSG1protein of Mycoplasmas suisTo develop a method for Mycoplasmas suis detection, a double-antibody sandwich ELISA (DAS-ELISA) was developed using MSG1monoclonal antibody (MAb)2F10as capture antibody and1A7-HRP against MSG1as detecting antibody. The optimized reaction conditions showed that the optimal coating concentration of2F10was4μg/mL, the optimal working dilution of1A7-HRP was1:500. The specificity and sensitivity results showed that the ELISA had no cross-reaction with Streptococcus suis and Haemophilus Parasuis, and at least0.0625μg/mL MSG1can be detected. The coefficient of variation of reproducibility was less than10%. The results revealed that the ELISA method was quick, sensitive and Convenient, which can be used to detect the infection of M. suis.