羊副结核分支杆菌分离鉴定及MAP0862基因克隆表达

发布时间：2018-06-19 02:00

本文选题：辽宁绒山羊 + 副结核分支杆菌　；参考：《锦州医科大学》2017年硕士论文

【摘要】：目的副结核分支杆菌(Mycobacterium paratuberculosis)引起的羊副结核病给辽宁省辽宁绒山羊饲养业造成的危害日趋明显,临床病例不断增多,一些优质母羊遭到淘汰,目前对副结核病的早期诊断容易误诊和漏诊,研究出快速鉴别副结核分支杆菌的方法对羊副结核病的早期诊断以及治疗具有重要意义。本研究采用分离培养和PCR检测方法对疑似感染羊副结核分支杆菌病料样本进行鉴定得到副结核分支杆菌,并初步探究副结核分支杆菌MAP0862基因的反应原性,为进一步研究副结核分支杆菌的亚单位疫苗的开发奠定了试验基础。方法1、副结核分支杆菌的分离鉴定本研究对采自辽宁省某养殖场饲养辽宁绒山羊疑似感染羊副结核分支杆菌病料样本,通过细菌的分离培养、PCR检测等方法进行鉴定。2、辽宁东南部地区羊副结核病血清学调查采用ELISA检测方法对辽宁东南部地区的辽宁绒山羊开展了副结核病血清学调查工作。3、副结核分支杆菌MAP0862基因的克隆与表达针对羊副结核分支杆菌主要抗原MAP0862基因设计利用DNAstar软件设计一对特异性引物,引物两端加入Bam HI和Hind III酶切位点。以分离的羊副结核分支杆菌DNA为模板进行PCR扩增,并对其产物进行克隆,转化,获得的T-MAP0862质粒,双酶切后用T4连接酶与表达载体连接,构建出原核表达载体PET-28a-MAP0862并转化到表达菌BL21(DE3)感受态细胞中,经IPTG诱导表达4小时后,经SDS-PAGE电泳分析蛋白表达情况,用KCl法纯化蛋白并用Westernblot检测蛋白的免疫原活性。结果1、辽宁绒山羊副结核分支杆菌分离鉴定细菌分离培养所分离出的菌株为抗酸染色阳性杆菌,参照MAPISMav2的因序列,利用Oligo6.0软件对全基因组序列进行比对分析,针对全基因组序列保守区域,设计一对特异性引物,通过PCR方法扩增出来的目的片段大小为246bp,与预期的目的片段大小基本一致。鉴定结果表明从疑似羊副结核病羊所分离菌株为羊副结核杆菌。2、辽宁东南部地区羊副结核病血清学调查羊副结核病血清学调查结果表明,辽宁东南部地区绒山羊副结核病平均血清阳性率为12.54%;其中3个规模化养殖场的平均血清阳性率为16.06%,最高阳性率为18.4%,最低为11.82%;5个散养户的平均血清阳性率为7.41%,最高阳性率为10.71%,最低为4.44%。3、副结核分支杆菌MAP0862基因克隆表达(1)采用PCR方法扩增MAP0862基因,所得条带大小为1080bp,与预期大小基本一致,并成功构建了PET-28a-MAP0862表达载体。(2)经IPTG诱导,SDS-PAGE电泳在38k Da的位置出现清晰的目的蛋白带,结果表明重组基因获得有效表达,纯化后的蛋白仅有唯一的目的蛋白带。(3)用羊副结核阳性血清进行Westernblot检测,证明MAP0862重组蛋白具有良好的反应原性。为进一步研究MAP0862作为研制疫苗和检测方法建立奠定了基础。结论1、成功从患病羊群中分离培养出羊副结核分支杆菌,通过提取DNA鉴定该分离菌株为副结核分支杆菌。2、辽宁东南部地区羊副结核病血清学调查结果,3个规模化养殖场绒山羊的平均阳性率16.06%,5个散养户的平均阳性率为8.64%,表明该地区规模化养殖场副结核病的感染率要明显高于散养绒山羊。3、成功构建了PET-28a-MAP0862重组表达载体,用表达菌BL21(DE3)表达获得38k Da(含标签)目的蛋白,经Western-blot检测该蛋白具有较好反应原性。
[Abstract]:Objective the harm of sheep by Mycobacterium tuberculosis (Mycobacterium paratuberculosis) to the cashmere goat breeding industry in Liaoning of Liaoning is becoming more and more obvious, the clinical cases are increasing, some high quality ewes are eliminated, and the early diagnosis of the secondary tuberculosis is easy to be misdiagnosed and missed. The method of bacilli is of great significance to the early diagnosis and treatment of sheep accessory tuberculosis. This study uses the method of isolation and culture and PCR detection to identify Mycobacterium subtubercular Mycobacterium tuberculosis samples from suspected infected sheep, and explore the preliminary study on the reactivity of MAP0862 gene of Mycobacterium subtuberculosis. The development of subunit vaccine of Mycobacterium subtubercular Bacillus laid the experimental basis. Method 1, the isolation and identification of Mycobacterium subtubercular Mycobacterium tuberculosis in a culture farm of Liaoning Province, Liaoning cashmere goat suspected to be infected with Mycobacterium tumefaciens samples, identified by isolation and culture of bacteria, PCR detection and other methods to identify.2, Southeast Liaoning The serological investigation of the local sheep accessory tuberculosis (ELISA) was used to carry out a serological investigation of the Liaoning cashmere goats in the southeast of Liaoning. The cloning and expression of the MAP0862 gene of the Mycobacterium accessory tuberculosis (MTB) was designed to design a pair of specificity against the MAP0862 gene design of the main antigen of the Mycobacterium tumefaciens using DNAstar software. Primers, both ends of the primers were added to the Bam HI and Hind III enzyme cutting sites. The isolated Mycobacterium tuberculosis DNA was used as a template for PCR amplification, and the product was cloned and transformed, and the T-MAP0862 plasmid was obtained. After double enzyme digestion, the T4 ligase was connected with the expression vector, and the prokaryotic expression vector was constructed and converted to BL21 (DE3) of the expression bacteria. After 4 hours of IPTG induced expression, the protein expression was analyzed by SDS-PAGE electrophoresis, the protein was purified by KCl method and the immunogenicity of the protein was detected by Westernblot. Results 1, the isolated strains isolated from Liaoning cashmere goat were isolated and identified as acid resistant positive bacilli, referring to MAPISMav2 The whole genome sequence was compared and analyzed by Oligo6.0 software. A pair of specific primers were designed for the conservative region of the whole genome sequence. The size of the target fragment amplified by the PCR method was 246bp, which was basically the same as the expected target fragment size. The identification results showed that the isolated strains isolated from the suspected sheep sub tuberculosis sheep were isolated. The serological survey of the serological investigation of sheep accessory tuberculosis in southeastern Liaoning showed that the positive rate of serological positive rate of cashmere goat accessory tuberculosis in southeastern Liaoning was 12.54%, of which the average positive rate of 3 scale farms was 16.06%, the highest positive rate was 18.4%, the lowest was 11.82%, and the lowest was 11.82%, 5. The average positive rate was 7.41%, the highest positive rate was 7.41%, the highest positive rate was 10.71%, the lowest was 4.44%.3. The clone expression of MAP0862 gene of Mycobacterium accessory tuberculosis (1) amplified MAP0862 gene by PCR method. The band size was 1080bp, and the PET-28a-MAP0862 expression vector was basically consistent with the expected size. (2) SDS-PAGE electricity was induced by IPTG and SDS-PAGE electricity. A clear target protein band in the position of 38K Da showed that the recombinant gene was effectively expressed and the purified protein was only the only target protein band. (3) the positive serum of the positive serum of sheep accessory tuberculosis was detected by Westernblot, which proved that the recombinant protein of MAP0862 has a good reactivity. It is a further study of MAP0862 as a vaccine and a vaccine. The foundation of the detection method was established. Conclusion 1, the Mycobacterium tuberculosis was successfully isolated and cultured from the sick sheep. Through the extraction of DNA, the isolated strain was identified as.2 of Mycobacterium accessory tuberculosis, the serological investigation of sheep accessory tuberculosis in southeastern Liaoning, the average positive rate of 3 large-scale farm Cashmere Goats was 16.06%, and 5 scattered households. The average positive rate was 8.64%, indicating that the infection rate of the sub tuberculosis in the large-scale breeding farm in this area was obviously higher than that of the scattered cashmere goat.3. The recombinant expression vector of PET-28a-MAP0862 was successfully constructed and the expression of 38K Da (labeled) target protein was obtained by the expression of BL21 (DE3), and the protein had good reactivity by Western-blot.
【学位授予单位】：锦州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.618

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