Preparation And Application Of Monoclonal Antibody And Immunoproteomic Study Of Mycoplasma Suis In Jilin

Eperythrozoonosis caused by Eperythrozoon-the parasitism prokaryotes in the blood is a new zoonosis which is difficult to be paid attention, but has a potential threat. The disease can reduce quality and yield of animal products and decrease animal reproductive ability, and has the popular, recessive infection, chronic persistent and conditional pathogenic characteristics. Eperythrozoonosis seriously harms to the healthy development of animal husbandry and human health. China is one of the most countries which have seriously epidemic Eperythrozoonosis and almost all of the provinces (autonomous regions) have been reported. Although, some progress has been made about the research of swine Eperythrozoonosis at home and abroad, but so far there is still no effective measures about the prevention and treatment of it. So, in this study, Mycoplasma suis HSPA1gene, and α-enolase gene in Jilin were cloned by PCR;A SYBR Green real-time PCR was developed;Two McAbs against Mycoplasma suis were produced by immunizing with the purified Mycoplasma suis,and McAbs’ antibody subtype,antibody specificity, cellular localization and neutralization properties were identified and analyzed;A double-antibody sandwich ELISA was developed using Mycoplasma suis monoclonal antibody (MAb);Immunogenic proteins of Mycoplasma suis in Jilin were screened by infected serum and monoclonal antibody and identified by mass spectrometry;In order to provide the foundation for Eperythrozoonosis prevention and cure research.1.Cloning and bioinformatics analysis of Mycoplasma suis HSPA1gene and α-enolase gene in JilinTwo pairs of primers based on the sequence of HSPA1gene and a-enolase gene in GenBank, and taking the genomic DNA of M. suis as a template for the PCR method. Then the gene was cloned into the pMD-18T-simple vector, sequenced and analyzed. The result indicated that the HSPA1gene consisted1044nucleotides encoding a protein of333amino acids. The HSPA1gene shared97%homology with the foreign isolates in GenBank (AM265536) and had the highest homology with Mycoplasma haemocanis and Mycoplasma haemofelis (68.4%) based on the phylogenetic tree. The encoded protein isoelectric point of HSPA1gene was4.69. HSPA1gene had transmembrane domains. The sequence of a-enolase gene encoded393aa. The homology of nucleotide sequence was98%and that of amino acid sequence was100%. The gene had the closest relationship with Mycoplasma wenyonii, but distantly related with Mycoplasma. cynos.This study is to understand the biological characteristics of Mycoplasma suis in Jilin from the perspective of molecular, and provide a scientific basis for diagnosis and prevention of Mycoplasma suis.2.Establishment of the SYBR Green real-time PCR for detection of MycoplasmaA SYBR Green I-based real-time PCR was developed for detection of Mycoplasma suis by using a pair of primer which was designed to target a-enolase gene. The product of PCR (119bp) was linked to pMD-18T-simple vector served as standard curve.The sensitivity,specificity and repeatability tests of the method were conducted. Then49clinical samples were tested in parallel by SYBR Green real-time PCR and conventional PCR. The results showed a precise linear relationship with a correlation coefficient of R=0.894;the detection limits was1.44copies/μL of DNA plasmid.The melting curve showed a single peak could only be detected for Mycoplasma suis,and were not detected for DNA of Toxoplasma gondii,Streptococcus suis, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus(PRRSV).The variation coefficient of repeatability tests were less than5%. Clinical samples test showed that the positive rates of Real-time PCR (40.8%) was higher10.2%than the positive rates of conventional PCR(30.6%). The SYBR Green Real-time PCR of Mycoplasma suis a-enolase gene in this article was a method of high sensitivity,strong specificity and good stability. The research provided important technical support for the diagnosis and treatment with Mycoplasma suis.3.Preparation and identification of monoclonal antibody against MycoplasmaThe6weeks old female BALB/c mice were immunized with the purified Mycoplasma suis. McAbs against Mycoplasma suis were produced by fusing SP2/0myeloma cells with spleen cells of BALB/c immunized with purified Mycoplasma suis and were screened by indirect ELISA.Then specificity,sub-class of antibodies and other biological characteristics of McAb against Mycoplasma suis were identified. And use laser scanning confocal technique to observe cellular localization of monoclonal antibody identified antigen.The neutralizing effect of McAb against Mycoplasma suis in mouse model was evaluated by SYBR Green real-time PCR.The results showed two hybridoma cell strains secreting McAb against Mycoplasma suis were screened,named3F7and2C4. The ELISA titers of2McAbs in mouse ascites were1:16000and1:14000. Identification of antibody subclasses showed that the two monoclonal antibodies were IgG2a· Western-blot analysis showed that the40ku and 33ku antigen of Mycoplasma suis were identified by3F7McAb and2C4McAb. The two McAbs could specifically react to Mycoplasma suis,but not cross react to Toxoplasma gondii,Streptococcus suis,Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus(PRRSV). Laser scanning confocal microscopy showed that the antigens of Mycoplasma suis were recognized by monoclonal antibody.The neutralization test display that3F7McAb had a certain inhibition effect to proliferation of Mycoplasma suis in animal models.The McAbs against Mycoplasma suis was successfully prepared,which laid a foundation of developing a rapid determination method and prevention for Mycoplasma suis.4.Development and application of a double antibody sandwich ELISA for detection of Mycoplasma suisTo develop a method for Mycoplasma suis detection, a double-antibody sandwich ELISA(DAS-ELISA) was developed using Mycoplasma suis monoclonal antibody (MAb) as capture antibody and rabbit polyclonal antibodies against Mycoplasma suis as detecting antibody. The optimized reaction conditions showed that the optimal coating concentration of Mycoplasma suis MAb was5.42mg/L, the optimal working concentration of rabbit polyclonal antibodies against Mycoplasma suis was6.84mg/L, and the optimal working dilution of HRP-labelled goat-anti-rabbit IgG was1:2000with a cutoff value of0.290(OD492nm)-The ELISA had no cross-reaction with Toxoplasma gondii,Streptococcus suis, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus(PRRSV), and at least3.25mg/L antigen of Mycoplasma suis was able to be detected. The coefficient of variation of reproducibility was less than10%. A total of68clinical samples were detected by the ELISA and the blood smear staining method. The positive rate of double-antibody sandwich ELISA for detection of29.4%was higher than the blood smear staining method to detect10.3%. The results revealed that the ELISA method was quick, sensitive and repeatable, which was applicable to the detection of Mycoplasma suis.5.1mmunoproteomic study of Mycoplasma suis in JilinIn order to screen and identify immunogenic proteins of Mycoplasma suis in Jilin. Protein expression profiling of Mycoplasma suis was analyzed by two-dimensional electrophoresis and identified by western blot with infected serum and3F7McAb,and the screened immunogenic proteins of Mycoplasma suis were identified by mass spectrometry. The result indicated that850μg total protein were loaded onto pH3～10IPG strip for2-DE follow by staining with Coomassie Brilliant Blue G-250nitrate. 2-DE image analysis indicated that60±5protein spots were recognized,and protein spots were mainly focused between pI values6to9and molecular weight values45ku-25ku.9protein spots in protein expression profiles of Mycoplasma suis were identified by infected serum and3F7McAb.5protein spots were successfully identified by mass spectrometry.2proteins (ATP-binding protein and choline kinase) of Mycoplasma and3proteins (hypothetical protein Msui05020,hypothetical protein Msui06340and hypothetical protein CAG:472) of Mycoplasma suis were identified using ’Mascot’ server.The ATP-binding protein was identified by3F7McAb.The study will foundation for the further research of diagnostics and vaccine candidate molecular of Mycoplasma suis.Conelusion:1.Mycoplasma suis HSPA1gene and a-enolase gene in Jilin were successfully cloned by Polymerase Chain Reaction(PCR), and bioinformatics features of purpose gene were analyzed.2.A SYBR Green I-based real-time PCR was successfully developed for detection of Mycoplasma suis, and the method was sensitive, specific and repeatable.3.Two McAbs against Mycoplasma suis were successfully produced, and laser scanning confocal microscopy showed that the antigens of Mycoplasma suis were recognized by monoclonal antibody.The3F7McAb had a certain inhibition effect to proliferation of Mycoplasma suis in animal models.4.A double-antibody sandwich ELISA was successfully developed using Mycoplasma suis monoclonal antibody, and the ELISA method was sensitive, specific, inexpensive and quick.5.9protein spots in protein expression profiles of Mycoplasma suis were identified by infected serum and3F7McAb.5protein spots were identified by mass spectrometry.