Drug Sensitivity And Gene Vaccine Research Of Mycoplasma Ovipneumoniae (Mo) Strain From Goats In Guizhou Province

Mycoplasma ovipneumoniae(Mo)is one of the main pathogen of proliferative interstitial pneumonia which infect goat,sheep and lamb.In goat industry,interstitial pneumonia cause great economic losses.Since 1972,after the first time identification,Mo has been found all over the world.In China,several goat infection by Mo have been reported in shandong,xinjiang and guangxi province.In guizhou,several Mo strains have been isolated in different areas.In goat,the prevention and controlment of respiratory system disease cause by Mo are always difficult,thus these two problems bacome a hot spot in disease research.In this research,clinical prevention and controlment methods have been established,base on drug prevention experiment.Also bioinformatics analysis and vaccine research that aim at Mo membrane protein P30 gene have been studied.1.Drug sensitivity analysis of Mo in Guizhou province Mo guizhou isolates Mo-GZTZ,Mo-GZWN and standard strains Y98 as research objects,treated by 12 kinds of commonly antibiotics include quinolone,tetracycline and 6 kinds of environment disinfectant such as aminoglycoside antibiotic and phenols,quaternary ammonium salt and chlorine.After trace dilution method,enzyme mark instrument test analysis,and the results show ciprofloxacin hydrochloride,and grace,sulfonic acid doxycycline,characters and fumaric acid tai miao cephalosporins and mesylate have strong inhibitory effect of Mo standard strains and two separated strains;Phenol,New Clean out,chlorine dioxide have strong sterilization effect,these three disinfection material can be applied to the prevent and control of clinical disinfectant as Mo environment in guizhou.2.The cloning and bioinformatics analysis of P30 gene of Guizhou strains Total DNA of Mo guizhou has been extracted,amplify P30 gene by PCR,after purification,connect with T gene cloning vector,construct recombinant plasmid p MD19T-P30,positive recombinant plasmid was identified by enzyme digestion and sequencing analysis.The results showed that P30 gene fragments have been obtained by PCR amplification in standard strain and two separation strains.P30 gene nucleic acid sequence is obtained by T cloning and sequencing.Sequence alignment,according to the results of strain and standard strain isolated Mo P30 gene sequence similarity GZHZ respectively: 99.1%;GZKY: 94.9%;GZQX: 99.4%;GZXS: 99.3%,isolates were more locus mutation or missing.Based on the sequence homology analysis showed that Mo isolates and SC01 homology between 72.2% ~ 76.3%;Mo isolates and Mhp homology between 72.5% ~ 78.0%;Mo isolates and Mf homology between 73.5% ~ 79.8%,and other types of mycoplasma homology are below 30.0%.P30 gene is deduced amino acids of bioinformatics analysis shows,P30 gene can encode 285 aa;Secondary structure prediction of its less random curl;According to protein hydrophilic,flexibility,surface polarity and antigen index distribution area,a comprehensive analysis of the three possible antigen epitope,which located in 3 ~ 58,80 ~ 154,183 ~ 268.3.The eukaryotic recombinant plasmid construction of Mo P30 and Hsp70 gene Construction were made use p MD19 T P30,p MD19 T Hsp70 plasmid as a template,according to the principle of Site-directed mutagenesis primer design,application of overlap extension PCR amplification,gene mutation sequence directional cloning to eukaryotic expression vector pc DNA3.1(+)and build eukaryotic recombinant plasmid pc DNA3.1 P30,pc DNA3.1 P30-Hsp70.Set pc DNA3.1-P30 pc DNA3.1 P30-Hsp70 group,blank control group,pc DNA3.1(+)and empty carrier group respectively,immunize mice.ELISA method is applied to analyze the influence of different cells in the immune response in mice into two groups.Results show that the all recombinant plasmid group can cause the mice serum cytokines INF-?,the enhancement of secretion of IL-2 extremely significant difference compared with the blank control group(P < 0.01),compared with the empty vector group significant difference(P < 0.05);At the same time,recombinant plasmid can cause the mice serum cytokine secretion of IL-4 levels decrease compared with blank control group and the empty plasmid group significant difference(P < 0.05);And the blank control group,and no significant difference between the empty plasmid group.And the fusion recombinant plasmid compared with single gene recombinant plasmid significant difference(P < 0.05).