绵羊肺炎支原体新疆塔城流行株的分离鉴定及油佐剂灭活苗免疫原性初步研究

发布时间：2018-05-26 01:42

本文选题：绵羊肺炎支原体 + 塔城分离株　；参考：《石河子大学》2015年硕士论文

【摘要】：绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)引起绵羊和山羊发生间质性肺炎的常见病原。羊感染后在临床上主要表现为呼吸障碍、腹泻、消瘦、生长发育迟缓等慢性非进行性肺炎症状,MO已成为影响养羊业发展的重要病原之一。自1963年国外首次分离MO后,我国在1978年从国外引进种羊体内发现该病原,随后相继在全国各地有MO感染的病例报道。新疆是我国养羊业的主要地区,该病在羔羊上的发病率和死亡率较高。目前,尚无有效商品化疫苗来预防绵羊支原体肺炎的发生。因此,在新疆地区开展MO的分离鉴定,应用分离的主要流行株研发预防该病的油佐剂灭活疫苗具有重要的现实意义。本论文开展的主要工作和研究结果如下:1.绵羊肺炎支原体塔城流行株的分离与鉴定从新疆塔城地区采集的疑似MO感染的病死羊肺脏25份病料,用支原体培养基进行MO的分离与鉴定研究。结果成功分离得到15株支原体疑似分离株,分离株菌落呈典型的乳头状,菌体呈多形性。用Dienes染液染色,菌落中心被染成深蓝色、边缘染色浅。14株支原体均水解葡萄糖、吸附红细胞,只有1株支原体水解尿素且不吸附红细胞。15株支原体均使美兰牛乳褪色和发生溶血。通过对分离株16S r RNA基因序列PCR扩增、测序和遗传进化分析,发现14株分离株与Y98株核苷酸同源性98.82%-100%之间,并且MO SC01株和Y98株聚为一支,证实14个分离株均为绵羊肺炎支原体。2.绵羊肺炎支原体塔城分离株抗原基因的克隆、遗传变异分析及表达根据MO SC01株基因组序列设计特异性引物,分别通过PCR扩增HSP 70、黏附素、溶血素A和溶血素C四个基因、克隆测序后进行遗传进化分析,并对重组蛋白HSP70进行免疫性研究。成功克隆了HSP70、黏附素、溶血素A和溶血素C四个基因,大小分别为747 bp、3426 bp、777 bp和1239 bp。MO塔城分离株S3与SC01相比,HSP70基因核苷酸同源性为85.24%,氨基酸同源性为83.24%,有64处核苷酸发生变异,24处氨基酸位点发生突变。黏附素基因核苷酸同源率为93.56%,氨基酸的同源性为92.19%,有180个核苷酸位点发生变异,导致62氨基酸发生突变。溶血素A基因核苷酸同源性为95.24%,氨基酸同源性为93.64%,有32个核苷酸位点发生变异,20个氨基酸位点发生突变。溶血素C基因核苷酸同源性为89.31%,氨基酸同源性为85.23%,有45处核苷酸发生突变,23氨基酸位点发生突变。遗传进化树分析显示,MO塔城分离株与MO SC01株、猪肺炎支原体J、猪肺炎支原体232亲缘关系较近。抗原表位分析结果发现,MO塔城分离株HSP 70、黏附素、溶血素A和溶血素C抗原与SC01株相应的抗原表位存在一定的差异。SDS-PAGE和Western blot分析显示,用大肠杆菌中表达的重组HSP70分子量为53 k Da,可与抗MO多克隆抗体发生反应,诱导小鼠产生MO抗体,具有良好的免疫原性。3.绵羊肺炎支原体油佐剂灭活疫苗的制备及免疫原性研究以MO塔城分离株S3作为菌种,培养MO生长滴度达到1×109 CCU/m L时进行收菌,浓缩20倍,抗原稀释为0.5mg/m L和0.25mg/m L;甲醛37℃灭活48 h,再与油佐剂1:1混匀乳化,制备油佐剂灭活疫苗,并对制备的灭活疫苗进行无菌和安全性检测。选择MO抗体阴性的健康绵羊27只,分为F1、F2和对照组3组,每组9只。F1组和F2组分别经颈部皮下接种抗原浓度为0.5mg/m L和0.25mg/m L的疫苗2m L/只,免疫2次,每次间隔15 d;对照组羊只按照相同剂量和方法注射生理盐水。分别在第0、15、30、45、60、75、90和105d采集血液,分离血清进行间接血凝抗体效价测定。免疫后90d,用S3菌株进行攻毒试验,测量体温、观察临床症状,并于攻毒后35d剖检,观察病理变化。结果显示,F1和F2试验组的羊只经两次免疫后血液中均产生了抗MO特异性抗体,在第45d时抗体效价在1:16-1:64之间,而对照组抗体效价均为阴性。攻毒保护试验结果表明,F1和F2试验组免疫保护率均为88.9%(8/9),而对照组羊100%发病。进一步分析发现,攻毒前间接血凝抗体效价≥8的羊只可获得免疫保护,证实该灭活疫苗具有较好的免疫原性。
[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.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.26

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