【摘要】：目的:建立一種用于評價(jià)結(jié)核分支桿菌感染后機(jī)體細(xì)胞免疫水平的檢測技術(shù),從而為結(jié)核病的特異性診斷、治療及療效監(jiān)測提供一種新手段。方法:1.設(shè)計(jì)并合成結(jié)核分枝桿菌PPE36、TB10.4基因片段引物,用結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)標(biāo)準(zhǔn)菌株(H37Rv)DNA做模板進(jìn)行PCR反應(yīng)擴(kuò)增。將擴(kuò)增產(chǎn)物與克隆載體連接、轉(zhuǎn)化至大腸桿菌E.coli DH5a培養(yǎng)后,提取質(zhì)粒進(jìn)行鑒定和保存。2.將質(zhì)粒與表達(dá)載體連接,轉(zhuǎn)化培養(yǎng),利用IPTG在大腸桿菌E.coli BL21(DE3)中進(jìn)行誘導(dǎo)表達(dá)。對表達(dá)所得蛋白進(jìn)行純化,鑒定。3.以鼠抗人IL-12抗體包被PVDF膜,用生物素鏈霉親和素法建立起Elispot IL-12檢測技術(shù)。4.用誘導(dǎo)表達(dá)得到的PPE36、TB10.4蛋白刺激培養(yǎng)正常人及感染結(jié)核分枝桿菌患者外周血單個(gè)核細(xì)胞,對Elispot IL-12檢測技術(shù)的臨床應(yīng)用可行性進(jìn)行驗(yàn)證。結(jié)果:1.實(shí)驗(yàn)所得的結(jié)核分枝桿菌PPE36、TB10.4基因序列經(jīng)測序證明與GenBank中序列一致,并成功表達(dá)出相應(yīng)的蛋白。2.SDS-PAGE電泳結(jié)果顯示PPE36蛋白在相對分子量為30kDa處、TB10.4在相對分子量為10kDa處各有一明顯條帶,與預(yù)期大小一致。3.Elispot結(jié)果顯示,空白對照組及正常人組沒有出現(xiàn)斑點(diǎn),而感染結(jié)核分枝桿菌患者組及陽性對照組出現(xiàn)明顯的斑點(diǎn)。結(jié)論:1.利用基因工程方法體外獲取了結(jié)核分枝桿菌PPE36、TB10.4蛋白。2.經(jīng)鑒定,目的蛋白大小與文獻(xiàn)報(bào)道一致。3.初步建立了結(jié)核分枝桿菌反應(yīng)性T細(xì)胞Elispot IL-12檢測方法。
[Abstract]:Objective: to establish a new method for the detection of cellular immunity after Mycobacterium tuberculosis infection, so as to provide a new method for the specific diagnosis, treatment and monitoring of curative effect of tuberculosis. Method 1: 1. Primers were designed and synthesized for the gene fragment of Mycobacterium tuberculosis (PPE36) TB10.4. The PCR reaction was carried out by using Mycobacterium tuberculosism DNA (H37Rv) as template. The amplified product was ligated with the clone vector and transformed into E. coli E.coli DH5a culture. The plasmid was identified and preserved. The plasmid was connected with the expression vector and transformed into culture. The expression was induced by IPTG in Escherichia coli E.coli BL21 (DE3). The expressed protein was purified and identified. The PVDF membrane was coated with mouse anti-human IL-12 antibody and the Elispot IL-12 detection technique was established by biotin streptavidin method. The peripheral blood mononuclear cells (PBMC) of normal persons and patients infected with Mycobacterium tuberculosis were stimulated with PPE36 TB10.4 protein and the feasibility of clinical application of Elispot IL-12 detection technique was verified. The result is 1: 1. The sequence of Mycobacterium tuberculosis (PPE36) TB10.4 gene was confirmed by sequencing, and the corresponding protein was successfully expressed. 2. SDS-PAGE electrophoresis showed that the PPE36 protein had a distinct band at the relative molecular weight of 30kDa and the relative molecular weight of TB10.4 was 10kDa. The results of Elispot were consistent with the expected size. 3. The results of Elispot showed that there were no spots in the blank control group and the normal control group, but obvious spots appeared in the mycobacterium tuberculosis infection group and the positive control group. Conclusion 1. The gene engineering method was used to obtain the protein of Mycobacterium tuberculosis PPE36 TB10.4 in vitro. It was identified that the size of the target protein was consistent with that reported in the literature. A method for the detection of reactive T cell Elispot IL-12 of Mycobacterium tuberculosis was established.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R446.5

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