本文選題：綿羊肺炎支原體 + 塔城分離株　； 參考：《石河子大學(xué)》2015年碩士論文

【摘要】：綿羊肺炎支原體(Mycoplasma ovipneumoniae,MO)引起綿羊和山羊發(fā)生間質(zhì)性肺炎的常見病原。羊感染后在臨床上主要表現(xiàn)為呼吸障礙、腹瀉、消瘦、生長(zhǎng)發(fā)育遲緩等慢性非進(jìn)行性肺炎癥狀,MO已成為影響?zhàn)B羊業(yè)發(fā)展的重要病原之一。自1963年國(guó)外首次分離MO后,我國(guó)在1978年從國(guó)外引進(jìn)種羊體內(nèi)發(fā)現(xiàn)該病原,隨后相繼在全國(guó)各地有MO感染的病例報(bào)道。新疆是我國(guó)養(yǎng)羊業(yè)的主要地區(qū),該病在羔羊上的發(fā)病率和死亡率較高。目前,尚無(wú)有效商品化疫苗來(lái)預(yù)防綿羊支原體肺炎的發(fā)生。因此,在新疆地區(qū)開展MO的分離鑒定,應(yīng)用分離的主要流行株研發(fā)預(yù)防該病的油佐劑滅活疫苗具有重要的現(xiàn)實(shí)意義。本論文開展的主要工作和研究結(jié)果如下:1.綿羊肺炎支原體塔城流行株的分離與鑒定從新疆塔城地區(qū)采集的疑似MO感染的病死羊肺臟25份病料,用支原體培養(yǎng)基進(jìn)行MO的分離與鑒定研究。結(jié)果成功分離得到15株支原體疑似分離株,分離株菌落呈典型的乳頭狀,菌體呈多形性。用Dienes染液染色,菌落中心被染成深藍(lán)色、邊緣染色淺。14株支原體均水解葡萄糖、吸附紅細(xì)胞,只有1株支原體水解尿素且不吸附紅細(xì)胞。15株支原體均使美蘭牛乳褪色和發(fā)生溶血。通過(guò)對(duì)分離株16S r RNA基因序列PCR擴(kuò)增、測(cè)序和遺傳進(jìn)化分析,發(fā)現(xiàn)14株分離株與Y98株核苷酸同源性98.82%-100%之間,并且MO SC01株和Y98株聚為一支,證實(shí)14個(gè)分離株均為綿羊肺炎支原體。2.綿羊肺炎支原體塔城分離株抗原基因的克隆、遺傳變異分析及表達(dá)根據(jù)MO SC01株基因組序列設(shè)計(jì)特異性引物,分別通過(guò)PCR擴(kuò)增HSP 70、黏附素、溶血素A和溶血素C四個(gè)基因、克隆測(cè)序后進(jìn)行遺傳進(jìn)化分析,并對(duì)重組蛋白HSP70進(jìn)行免疫性研究。成功克隆了HSP70、黏附素、溶血素A和溶血素C四個(gè)基因,大小分別為747 bp、3426 bp、777 bp和1239 bp。MO塔城分離株S3與SC01相比,HSP70基因核苷酸同源性為85.24%,氨基酸同源性為83.24%,有64處核苷酸發(fā)生變異,24處氨基酸位點(diǎn)發(fā)生突變。黏附素基因核苷酸同源率為93.56%,氨基酸的同源性為92.19%,有180個(gè)核苷酸位點(diǎn)發(fā)生變異,導(dǎo)致62氨基酸發(fā)生突變。溶血素A基因核苷酸同源性為95.24%,氨基酸同源性為93.64%,有32個(gè)核苷酸位點(diǎn)發(fā)生變異,20個(gè)氨基酸位點(diǎn)發(fā)生突變。溶血素C基因核苷酸同源性為89.31%,氨基酸同源性為85.23%,有45處核苷酸發(fā)生突變,23氨基酸位點(diǎn)發(fā)生突變。遺傳進(jìn)化樹分析顯示,MO塔城分離株與MO SC01株、豬肺炎支原體J、豬肺炎支原體232親緣關(guān)系較近�？乖砦环治鼋Y(jié)果發(fā)現(xiàn),MO塔城分離株HSP 70、黏附素、溶血素A和溶血素C抗原與SC01株相應(yīng)的抗原表位存在一定的差異。SDS-PAGE和Western blot分析顯示,用大腸桿菌中表達(dá)的重組HSP70分子量為53 k Da,可與抗MO多克隆抗體發(fā)生反應(yīng),誘導(dǎo)小鼠產(chǎn)生MO抗體,具有良好的免疫原性。3.綿羊肺炎支原體油佐劑滅活疫苗的制備及免疫原性研究以MO塔城分離株S3作為菌種,培養(yǎng)MO生長(zhǎng)滴度達(dá)到1×109 CCU/m L時(shí)進(jìn)行收菌,濃縮20倍,抗原稀釋為0.5mg/m L和0.25mg/m L;甲醛37℃滅活48 h,再與油佐劑1:1混勻乳化,制備油佐劑滅活疫苗,并對(duì)制備的滅活疫苗進(jìn)行無(wú)菌和安全性檢測(cè)。選擇MO抗體陰性的健康綿羊27只,分為F1、F2和對(duì)照組3組,每組9只。F1組和F2組分別經(jīng)頸部皮下接種抗原濃度為0.5mg/m L和0.25mg/m L的疫苗2m L/只,免疫2次,每次間隔15 d;對(duì)照組羊只按照相同劑量和方法注射生理鹽水。分別在第0、15、30、45、60、75、90和105d采集血液,分離血清進(jìn)行間接血凝抗體效價(jià)測(cè)定。免疫后90d,用S3菌株進(jìn)行攻毒試驗(yàn),測(cè)量體溫、觀察臨床癥狀,并于攻毒后35d剖檢,觀察病理變化。結(jié)果顯示,F1和F2試驗(yàn)組的羊只經(jīng)兩次免疫后血液中均產(chǎn)生了抗MO特異性抗體,在第45d時(shí)抗體效價(jià)在1:16-1:64之間,而對(duì)照組抗體效價(jià)均為陰性。攻毒保護(hù)試驗(yàn)結(jié)果表明,F1和F2試驗(yàn)組免疫保護(hù)率均為88.9%(8/9),而對(duì)照組羊100%發(fā)病。進(jìn)一步分析發(fā)現(xiàn),攻毒前間接血凝抗體效價(jià)≥8的羊只可獲得免疫保護(hù),證實(shí)該滅活疫苗具有較好的免疫原性。
[Abstract]:Mycoplasma ovipneumoniae (MO) causes a common cause of interstitial pneumonia in sheep and goats. After infection, the main clinical manifestations of sheep are respiratory disorder, diarrhea, emaciation, growth retardation and other chronic non progressive pneumonia. MO has become one of the important pathogens in the development of sheep industry. Since 1963, it has been the first one. After the secondary separation of MO, our country introduced the pathogen in the sheep from abroad in 1978, and then reported the cases of MO infection in various parts of the country. Xinjiang is the main area of the sheep industry in our country. The incidence and mortality of the disease on lambs are high. The isolation and identification of MO in the Xinjiang area and the application of isolated main epidemic strains to develop an inactivated vaccine to prevent the disease are of great practical significance. The main work and research results in this paper are as follows: 1. the isolation and identification of the Tacheng epidemic strains of Mycoplasma pneumoniae from the Tacheng region of Xinjiang were suspected to be infected with MO. 25 parts of the lung of dead sheep were separated and identified by mycoplasma culture. The results were separated and identified by the Mycoplasma MO. The results were successfully separated and isolated from 15 strains of Mycoplasma. The isolates showed typical papillae and polymorphous. The colony center was dyed deep blue with Dienes dye, and the edge edge coloured.14 strains of Mycoplasma hydrolyzed glucose and adsorbed red. Cells, only 1 Mycoplasma hydrolysate urea and did not adsorb the.15 strains of red blood cells to discoloration and hemolysis. By PCR amplification, sequencing and genetic evolution analysis of the 16S R RNA gene sequence of the isolated strain, the nucleotide homology 98.82%-100% between 14 isolates and Y98 strain was found, and MO SC01 strain and Y98 strain were clustered into one branch. The 14 isolates were cloned from the antigen gene of Mycoplasma sheeppneumoniae.2. isolate of Mycoplasma sheeppneumoniae. The genetic variation analysis and expression were designed according to the specific primers of MO SC01 strain. Four genes of HSP 70, adhesion, hemolysin A and hemolysin C were amplified by PCR, and the genetic evolution analysis was carried out after cloned and sequenced. The recombinant protein HSP70 was immunized. Four genes of HSP70, adhesin, hemolysin A and hemolysin C were cloned successfully, the size of 747 BP, 3426 BP, 777 BP and 1239 bp.MO Tacheng isolates S3 compared with SC01, the HSP70 gene nucleotide homology was 85.24%, the amino acid homology was 83.24%, there were 64 nucleotide variations and 24 ammonia. The nucleotide homology is 93.56%, the amino acid homology is 93.56%, the amino acid homology is 92.19%, the 180 nucleotide loci mutates, which leads to the 62 amino acid mutation. The nucleotide homology of the hemolysin A gene is 95.24%, the amino acid homology is 93.64%, there are 32 nucleotide loci variation and 20 amino acid loci. The nucleotide homology of the C gene of the hemolysin C gene was 89.31%, the amino acid homology was 85.23%, there were 45 nucleotide mutations and the 23 amino acid site mutation. The genetic evolution tree analysis showed that the MO Tacheng isolate was closely related to the MO SC01 strain, Mycoplasma pneumoniae J, and Mycoplasma porcine 232. The antigen epitope analysis found that the MO tower was found. There were certain differences in the antigen epitopes of the city isolates HSP 70, adhesin, hemolysin A and hemolysin C antigen and SC01 strain,.SDS-PAGE and Western blot analysis showed that the molecular weight of recombinant HSP70 expressed in Escherichia coli was 53 K Da, which could react with anti MO polyclonal antibody and induce mice to produce MO antibodies, which had good immunogenicity. 3. the preparation and immunogenicity of the inactivated vaccine of Mycoplasma oinupneumoniae oil adjuvant used MO Tacheng isolate S3 as a strain. The growth titer of MO was 1 x 109 CCU/m L, the concentration was 20 times, the antigen was diluted to 0.5mg/m L and 0.25mg/m L, and the formaldehyde was inactivated at 37 C, 48 h, and then emulsified with oil adjuvant, and the inactivated vaccine of oil adjuvant was prepared, and 27 healthy sheep with negative MO antibody were divided into 3 groups: F1, F2 and control group. 9.F1 and F2 groups in each group were vaccinated with 0.5mg/m L and 0.25mg/m L for 2m L/, 2 times and 15 intervals at each interval; the control group was only in the same dose and prescription. The blood was collected at 0,15,30,45,60,75,90 and 105d and the titer of indirect hemagglutination antibody was measured in 0,15,30,45,60,75,90 and 105d respectively. After immunization, 90d was tested with S3 strain, the temperature was measured, the clinical symptoms were observed, and the pathological changes were observed after 35d caesarean section. The results showed that the sheep of the F1 and F2 test group were immunized only after two times of immunization. The anti MO specific antibody was produced in the blood and the antibody titer of the control group was negative at the time of 45d, and the antibody titer of the control group were all negative. The result of the attack protection test showed that the immune protection rate of the F1 and F2 group was 88.9% (8/9), while the control group was 100%. Further analysis found that the sheep with the titer of indirect hemagglutination antibody before attack was only more than 8 The immune protection showed that the inactivated vaccine had good immunogenicity.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.26

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