Genome Sequencing Of Mycoplasma Mycoides Str.PG3and Mycoplasma Ovipneumoniae From Inner Mongolia And Cloning And Expression Of Variable Surface Lipoprotein Genes

Mycoplasmal pneumonia of sheep andgoats （MPSG） is a highly contagious disease that is caused by one of Mycoplasma mycoides subsp.capri（Mmc）, Mycoplasma ovipneumoniae （Movi） and Mycoplasma capricolum subsp. capricolum （Mcc） that has not been discovered in our country. Which are characterized by fever, cough, gasp, gradual wasting, hyperplastic inflammation of pulmonary interstitial tissue, serous and fibrinous pleuropneumonia in sheep and goats. Since this disease was discovered, it spread in those less developed countries and caused important economic losses in industrialized sheep and goat production. Because the infection has not well responded to medication and the traditional vaccine itself has some problems that hard to overcome, Investigators are focusing on development of new type drug and vaccine. Up to date, there are almost no reports on major protective antigen gene that can be use to develop new type vaccine. So, based on isolation and identification of pathogen from samples in Inner Mongolia, genome sequencing and analysis was performed for looking for protective antigen gene. Aapplying recombinant technique, variable lipoprotein genes from Mycoplasma mycoides subsp.capri str.PG3were expressed, then constructed recombinant subunit vaccines and assessed their protective efficacy in this study. We obtained the following results:1.67samples, including lung, trachea, liver, hydrothorax and pericardial effusion, were collected from sheep and goats suspected to suffer from MPSG at Baotou, Ulanqab and Ordos regions in Inner Mongolia, China.18isolates with cultural characteristics of mycoplasma were isolated from lung, trachea and pericardial effusion samples collected from Ulanqab and Ordos regions. According to their morphology, physiological and biochemical characteristics, analysis of16S rDNA sequence and growth inhibition tests, isolates EF1, EF2, EQ1, EQ2, EF26, EQ22, WF1, WF2and WXB1were indentified as Mycoplasma ovipneumoniae. Among which, isolates EF2, EQ2, WF1, WF2and WXB1came from the sheep cases with MPSG, and isolates EF1, EQ1, EF26and EQ22came from the goat cases with MPSG. Isolates EF9, EQ9, EF12, EF13, EF19, and EF21were indentified as Mycoplasma arginini; isolate EF22was indentified as Mycoplasma mycoides subsp.capri; isolate EF4was indentified as Ureaplasma urealyticum. The results showed that Mycoplasma ovipneumoniae was the main pathogen of MPSG in Inner Mongolia.2. The high flux Illumina technology was used for genome sequencing and annotation of and Mycoplasma ovipneumoniae str. WXB1. Mycoplasma mycoides subsp.capri str.PG3genome consists of a circular chromosome of1,025,065bp. The chromosome has a G+C content of only23.61%.3rRNAs,28tRNAs and12sRNAs can be predicted. Genome annotation revealed846putative CDS. There are no Insertion Sequences （IS） and Integrative Conjugative Elements （ICE）. The specific maltodextrin/maltose gene cluster involved in carbohydrate metabolism and more specifically starch/glycogen and maltose utilization. Mycoplasma ovipneumoniae str. WXB1genome consists of a circular chromosome of1,084,159bp. The chromosome has a G+C content of only29.14%.3rRNAs,30tRNAs and11sRNAs can be predicted. Genome annotation revealed888putative CDS. The most of genes encoding proteins participate in protein translation, ribosome synthesis and structure, DNA replication and repair, glycometabolism and environment transfer and conversion.3. It is generally believed that variable lipoproteins could be involved in pathogens colonization and adaption to the host tissue environment at various stages of infection. They have been shown to play a role in adhesion, immunomodulation, and substrate binding. Three genes （GL₀00459;000461;000462） were identified as variable lipoprotein genes in Mycoplasma mycoides subsp.capri str.PG3genome by genomic information analysis. Variable lipoproteins genes GL₀00459, GL₀00461and GL₀00462were cloned. We constructed the protein expression systems and expressed the mature peptide portion of the three proteins in Escherichia coli. Three fusion proteins expected were properly expressed. SDS-PAGE showed that the molecular weights of them were31kDa、40kDa、41kDa respectively. The fusion proteins were immunogenic by Western blot analysis.4. Furthermore, mice were immunized three times with purified fusion proteins. The serum antibody titers of vaccinated mice were tested by the methods of ELISA. The serum antibody titers of fusion proteins immunization group were highly significantly different from that of the control group （P<0.05）; the antiserum was used for opsonophagocytic assay and growth inhibition tests. The results of opsonophagocytic assay proved that the bactericidal capability of whole blood of mice immunized by fusion proteins were highly significantly different from that of the control group （P<0.05）; the results of growth inhibition tests are weak positive. The levels of four cytokines in the sera of the mice immunized with His-tag-GL000459, His-tag-GL000461and His-tag-GL000462all were higher than that of control group. Immunization with His-tag-GL000459significantly increased the production of IL-2and IL-4（p<0.05）, and slightly increased that of IL-10and IFN-γ （p>0.05）. Immunization with His-tag-GL000461significantly increased that of all these cytokines （p<0.05）. Immunization with His-tag-GL000462significantly increased that of IL-2, IL-4and IFN-y （p<0.05）, and slightly increased that of IL-10（p>0.05）. Among three treated groups, His-tag-GL000461-immunized group showed the highest levels of cytokines. Therefore, these findings may indicate that three recombinant proteins exert both cellular and humoral immunity response to them in mice by increasing the levels of several cytokines, including IL-2, IL-4, IL-10and IFN-y. The results of T lymphocyte Proliferation assay revealed that T lymphocyte Proliferation ability of fusion proteins immunization groups were highly significantly different from that of the control group（P<0.05）.In conclusion, the results showed three lipoproteins induced significant humoral and cellular immune responses to them in the immunized mice. More importantly, the results of whole blood opsonophagocitic in vitro assay indicated the antibodies produced by immunized groups produced can neutralize strain PG3and the three variable lipoproteins could be the major surface antigens in Mycoplasma mycoides subsp.capri str.PG3. This study provided theoretical basis and experimental evidence for developing genetically-engineered subunit vaccine against MPSG infected by Mycoplasma mycoides subsp. capri.