本文選題：遼寧絨山羊 + 副結(jié)核分支桿菌�。� 參考：《錦州醫(yī)科大學》2017年碩士論文

【摘要】：目的副結(jié)核分支桿菌(Mycobacterium paratuberculosis)引起的羊副結(jié)核病給遼寧省遼寧絨山羊飼養(yǎng)業(yè)造成的危害日趨明顯,臨床病例不斷增多,一些優(yōu)質(zhì)母羊遭到淘汰,目前對副結(jié)核病的早期診斷容易誤診和漏診,研究出快速鑒別副結(jié)核分支桿菌的方法對羊副結(jié)核病的早期診斷以及治療具有重要意義。本研究采用分離培養(yǎng)和PCR檢測方法對疑似感染羊副結(jié)核分支桿菌病料樣本進行鑒定得到副結(jié)核分支桿菌,并初步探究副結(jié)核分支桿菌MAP0862基因的反應原性,為進一步研究副結(jié)核分支桿菌的亞單位疫苗的開發(fā)奠定了試驗基礎(chǔ)。方法1、副結(jié)核分支桿菌的分離鑒定本研究對采自遼寧省某養(yǎng)殖場飼養(yǎng)遼寧絨山羊疑似感染羊副結(jié)核分支桿菌病料樣本,通過細菌的分離培養(yǎng)、PCR檢測等方法進行鑒定。2、遼寧東南部地區(qū)羊副結(jié)核病血清學調(diào)查采用ELISA檢測方法對遼寧東南部地區(qū)的遼寧絨山羊開展了副結(jié)核病血清學調(diào)查工作。3、副結(jié)核分支桿菌MAP0862基因的克隆與表達針對羊副結(jié)核分支桿菌主要抗原MAP0862基因設計利用DNAstar軟件設計一對特異性引物,引物兩端加入Bam HI和Hind III酶切位點。以分離的羊副結(jié)核分支桿菌DNA為模板進行PCR擴增,并對其產(chǎn)物進行克隆,轉(zhuǎn)化,獲得的T-MAP0862質(zhì)粒,雙酶切后用T4連接酶與表達載體連接,構(gòu)建出原核表達載體PET-28a-MAP0862并轉(zhuǎn)化到表達菌BL21(DE3)感受態(tài)細胞中,經(jīng)IPTG誘導表達4小時后,經(jīng)SDS-PAGE電泳分析蛋白表達情況,用KCl法純化蛋白并用Westernblot檢測蛋白的免疫原活性。結(jié)果1、遼寧絨山羊副結(jié)核分支桿菌分離鑒定細菌分離培養(yǎng)所分離出的菌株為抗酸染色陽性桿菌,參照MAPISMav2的因序列,利用Oligo6.0軟件對全基因組序列進行比對分析,針對全基因組序列保守區(qū)域,設計一對特異性引物,通過PCR方法擴增出來的目的片段大小為246bp,與預期的目的片段大小基本一致。鑒定結(jié)果表明從疑似羊副結(jié)核病羊所分離菌株為羊副結(jié)核桿菌。2、遼寧東南部地區(qū)羊副結(jié)核病血清學調(diào)查羊副結(jié)核病血清學調(diào)查結(jié)果表明,遼寧東南部地區(qū)絨山羊副結(jié)核病平均血清陽性率為12.54%;其中3個規(guī)�；B(yǎng)殖場的平均血清陽性率為16.06%,最高陽性率為18.4%,最低為11.82%;5個散養(yǎng)戶的平均血清陽性率為7.41%,最高陽性率為10.71%,最低為4.44%。3、副結(jié)核分支桿菌MAP0862基因克隆表達(1)采用PCR方法擴增MAP0862基因,所得條帶大小為1080bp,與預期大小基本一致,并成功構(gòu)建了PET-28a-MAP0862表達載體。(2)經(jīng)IPTG誘導,SDS-PAGE電泳在38k Da的位置出現(xiàn)清晰的目的蛋白帶,結(jié)果表明重組基因獲得有效表達,純化后的蛋白僅有唯一的目的蛋白帶。(3)用羊副結(jié)核陽性血清進行Westernblot檢測,證明MAP0862重組蛋白具有良好的反應原性。為進一步研究MAP0862作為研制疫苗和檢測方法建立奠定了基礎(chǔ)。結(jié)論1、成功從患病羊群中分離培養(yǎng)出羊副結(jié)核分支桿菌,通過提取DNA鑒定該分離菌株為副結(jié)核分支桿菌。2、遼寧東南部地區(qū)羊副結(jié)核病血清學調(diào)查結(jié)果,3個規(guī)�；B(yǎng)殖場絨山羊的平均陽性率16.06%,5個散養(yǎng)戶的平均陽性率為8.64%,表明該地區(qū)規(guī)�；B(yǎng)殖場副結(jié)核病的感染率要明顯高于散養(yǎng)絨山羊。3、成功構(gòu)建了PET-28a-MAP0862重組表達載體,用表達菌BL21(DE3)表達獲得38k Da(含標簽)目的蛋白,經(jīng)Western-blot檢測該蛋白具有較好反應原性。
[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.
【學位授予單位】：錦州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.618

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