發(fā)布時間：2018-06-05 23:21

  本文選題：結(jié)核分枝桿菌 + Rvc�。� 參考：《中國病原生物學雜志》2017年04期

【摘要】：目的克隆、表達、純化結(jié)核分枝桿菌潛伏感染相關(guān)蛋白Rv1813c,分析其免疫活性。方法應用生物信息學方法分析Rv1813c蛋白序列,根據(jù)H37Rv基因組序列合成引物,PCR擴增Rv1813c基因,克隆至pGEM-T載體;挑取陽性克隆測序,將正確編碼基因克隆到pET30a載體上,在大腸埃希菌BL21菌株中以IPTG誘導表達重組蛋白。以金屬螯合層析分離純化重組蛋白,采用ELISA法分析其免疫反應性。結(jié)果 Rv1813c蛋白第34位氨基酸殘基之前含有跨膜區(qū)及信號肽部分,可能是一個胞外蛋白。對重組質(zhì)粒pET30a-Rv1813c測序,與設(shè)計的序列相同。重組蛋白在大腸埃希菌BL21(DE3)細胞內(nèi)以包涵體形式表達,IPTG誘導3h-4h重組蛋白在大腸埃希菌中的表達量較高,表達量約占細菌總蛋白的40%以上。純化的重組Rv1813c蛋白純度95%。ELISA分析重組Rv1813c蛋白與結(jié)核患者血清有較強的反應性,A450值為0.50±0.34,顯著高于健康組A450值0.23±0.18及非結(jié)核呼吸疾病組A450值0.30±0.27(P0.05)。結(jié)論成功構(gòu)建的結(jié)核分枝桿菌重組質(zhì)粒能高效表達Rv1813c蛋白,該蛋白具有良好的免疫反應性,為結(jié)核病的免疫機制研究奠定了基礎(chǔ)。
[Abstract]:Objective to clone, express and purify Mycobacterium tuberculosis latent infection related protein RV 1813 c and analyze its immune activity. Methods the Rv1813c protein sequence was analyzed by bioinformatics, and the Rv1813c gene was amplified by PCR according to the H37Rv genomic sequence and cloned into the pGEM-T vector, and the correct coding gene was cloned into the pET30a vector. The recombinant protein was induced by IPTG in Escherichia coli BL21 strain. The recombinant protein was purified by metal chelate chromatography and its immunoreactivity was analyzed by ELISA method. Results the 34th amino acid residues of Rv1813c protein contained transmembrane region and signal peptide, which may be an extracellular protein. The recombinant plasmid pET30a-Rv1813c was sequenced in the same sequence as the designed one. The recombinant protein was expressed as inclusion body in Escherichia coli BL21DDE3) cells. The expression of recombinant 3h-4h protein in Escherichia coli was higher than that in Escherichia coli cells, accounting for more than 40% of the total bacterial protein. 95%.ELISA analysis of purified recombinant Rv1813c protein the reactivity of recombinant Rv1813c protein to serum of tuberculosis patients was 0.50 鹵0.34, which was significantly higher than that of healthy group (0.23 鹵0.18) and non-tuberculous respiratory disease group (0.30 鹵0.27) (P 0.05). Conclusion the recombinant plasmid of Mycobacterium tuberculosis can express Rv1813c protein efficiently, and the protein has good immunoreactivity, which lays a foundation for the study of the immune mechanism of tuberculosis.
【作者單位】： 解放軍第三0九醫(yī)院全軍結(jié)核病研究所全軍結(jié)核病防治重點實驗室結(jié)核病診療新技術(shù)北京市重點實驗室;
【基金】：“十二五”國家重大科技專項(No.2013ZX10003003-005) 軍隊醫(yī)學科技“十二五”重點項目(No.BWS11J050) 北京市科技創(chuàng)新基地培育與發(fā)展工程專項(No.Z141107004414021) 解放軍第309醫(yī)院重點課題(No.2015ZD-004)
【分類號】：R378.911
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本文編號：1983850