【摘要】： 1999年6月，天津醫(yī)科大學(xué)微生物教研室從急性呼吸道感染致死的靈長類動物狨猴肺組織中分離出一株高血凝效價病毒，經(jīng)過一系列的研究證實為一株副粘病毒，即副粘病毒天津株。經(jīng)電鏡觀察具有典型的副粘病毒特征。用交叉血凝抑制試驗對分離毒株進行鑒定，擴增HN基因測序后，用生物信息學(xué)方法與多種核酸序列進行比較，發(fā)現(xiàn)該毒株與仙臺病毒(SeV)特征最為接近。進一步研究發(fā)現(xiàn)該動物中心的工作人員都有此病毒的抗體，且在正常人群(獻血員)中抗體反應(yīng)也是陽性，陽性率高達46％(14／40)，提示此病原體與人類有密切的關(guān)系，可能是一株人和絨猴共患的呼吸道病毒。我們現(xiàn)在已經(jīng)完成了對該株病毒的全基因組測序，通過全基因組系統(tǒng)進化樹分析，其與SeV同源性較高，也證明了該株病毒很可能為SeV一個新的基因型。 為進一步研究該毒株的生物學(xué)特性及致病機制，本研究構(gòu)建了副粘病毒天津株包膜糖蛋白HN膜外區(qū)三個部分及整個M基因的原核表達質(zhì)粒pET-28a-HN1、pET-28a-HN2、pET-28a-HN3及pMal-M，誘導(dǎo)表達、純化了重組蛋白HNl(aa61-aa260)、HN2(aa253-aa452)、HN3(aa376-aa575)和MBP-M，并對其生物學(xué)和免疫學(xué)活性進行了初步研究。 我們提取病毒RNA逆轉(zhuǎn)錄為cDNA。根據(jù)前期研究全基因組測序中的HN、M的基因序列，設(shè)計并合成引物，以該株病毒的cDNA為模板擴增目的片段，雙酶切、純化回收含粘末端的目的片段和空質(zhì)粒，在T4 DNA連接酶作用下連接、轉(zhuǎn)化克隆菌E．coli TOP10。經(jīng)雙酶切、PCR和基因測序鑒定篩選構(gòu)建正確的目的基因，轉(zhuǎn)化表達菌E．coli BL21，IPTG誘導(dǎo)表達。pET-28a-HN1、pET-28a-HN2和pET-28a-HN3所選用的載體質(zhì)粒帶有6×His標簽，用Ni金屬螯合柱進行回收，而pMal-M表達的蛋白帶有MBP融合蛋白，用Amylose resin回收得到純化的蛋白。用超速離心純化的病毒和純化表達蛋白分別制備PcAb，利用ELISA、Dot Blot及Western Blot等方法對表達蛋白進行免疫學(xué)鑒定。通過血凝試驗和血凝抑制試驗對副粘病毒天津株HN1、HN2和HN3蛋白的血凝功能及重組蛋白多克隆抗體的血凝抑制功能進行了初步研究。結(jié)果顯示：HN1、HN2和HN3表現(xiàn)出來的天然抗原性明顯不同，，抗原性強弱依次為HN2＞HN3＞HN1；M蛋白的ELISA和Dot Blot方法鑒定結(jié)果也為陽性。而HN3的血凝活性明顯強于HN蛋白的其他部位，其次為HN2蛋白，HN1幾乎沒有血凝功能。另外，利用ELISA方法對副粘病毒天津株HN1、HN2和HN3蛋白與甲型、乙型流感病毒的WHO標準血清和天津CDC獲得的陽性血清的交叉免疫情況進行了研究，結(jié)果顯示：HN1、HN2和HN3三種蛋白與這些血清存在交叉免疫反應(yīng)。 綜上，本研究成功的構(gòu)建了HN1、HN2、HN3和M的原核重組表達質(zhì)粒pET-28a-HN2、pET-28a-HN3及pMa1-M，經(jīng)誘導(dǎo)、表達、純化得到了重組蛋白HN1、HN2、HN3和M，并對其進行了免疫學(xué)和生物學(xué)的初步鑒定，為進一步研究副粘病毒天津株的致病機制，制備單克隆抗體和建立該毒株特異的檢測方法奠定了基礎(chǔ)。
文內(nèi)圖片：
圖片說明：圖IM、HNI、HNZ和HN3基因RT-PCR瓊脂糖凝膠電泳
[Abstract]:In June,1999, a high-blood-haemagglutination virus was isolated from the lung tissue of the primate from the acute respiratory tract infection by the microorganism teaching and research room of Tianjin Medical University. A series of studies have been proved to be a paramyxovirus, that is, the paramyxovirus Tianjin strain. The characteristic of paramyxovirus was observed by electron microscope. The isolated strains were identified by cross-hemagglutination inhibition test. After the HN gene was amplified and compared with a variety of nucleic acid sequences, the strain was found to be closest to the Sendai virus (SeV). The antibody of this virus was found to be positive in the normal population (blood donors), and the positive rate was as high as 46% (14/40), suggesting that the pathogen had a close relationship with the human. It may be a respiratory virus that is common to a human and a velvet monkey. We have now completed the full-genome sequencing of the strain, which is highly homologous to the SeV by a full-genome phylogenetic tree analysis, which also demonstrates that the strain is likely to be a new genotype of SeV. In order to study the biological characteristics and pathogenesis of the strain, the prokaryotic expression plasmids pET-28a-HN1, pET-28a-HN2, pET-28a-HN3 and pMal-M of the whole M gene of the envelope glycoprotein HN membrane of the paramyxovirus were constructed, and the recombinant protein HNl (aa61-aa260), HN2 (aa253-aa452), HN3 (aa376-aa575) were purified. and the biological and immunological activity of the MBP-M is I've got a preliminary study. Let's get it. The method comprises the following steps of: designing and synthesizing a primer according to the gene sequence of HN and M in the whole genome sequencing in the early stage, amplifying the target fragment by using the cDNA of the strain virus as a template, double-enzyme cutting, purifying and recovering the target fragment containing the sticky end and the empty plasmid, and connecting the target fragment and the empty plasmid under the action of the T4 DNA ligase, E. coli TOP10. The correct target gene was constructed by double-enzyme digestion, PCR and gene sequencing. The expression of E. coli was transformed. The expression of pET-28a-HN1, pET-28a-HN2 and pET-28a-HN3 was carried out with a 6-fold His-tag, and the pMal-M-expressed protein had MBP fusion protein. The purified protein was recovered by esin. The PcAb was prepared using the virus and purified expression protein purified by ultracentrifugation, using ELISA, Dot Blot and Western Blot. The function of HN1, HN2 and HN3 protein of the paramyxovirus and the polyclonal antibody of the recombinant protein were tested by the hemagglutination test and the hemagglutination inhibition test. The results showed that the natural antigenicity of HN1, HN2 and HN3 was different, the antigenicity of HN2> HN3> HN1, and the ELISA and Dot B of M protein. The results of the test were positive, and the HN3 had a stronger hemagglutination activity than that of the other parts of the HN protein, followed by the HN2 egg. In addition, the cross-immunization of HN1, HN2 and HN3 proteins of the paramyxovirus and the positive sera obtained by the WHO standard serum of the influenza A and the influenza B virus and the positive serum obtained by the Tianjin CDC were carried out by the ELISA method. The results showed that: HN1, HN2 and HN3 proteins HN1, HN2, HN3 and pET-28a-HN3 and pMa1-M were successfully constructed and the recombinant proteins HN1, HN2, HN3 and HN3 and HN3 and pMa1-M were successfully constructed. M, and the preliminary identification of the immunology and biology was carried out to further study the pathogenesis of the paramyxovirus in Tianjin, and to prepare the monoclonal antibody.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R373.1

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1 袁立軍;副粘病毒天津株HN、M基因克隆與表達的研究[D];天津醫(yī)科大學(xué);2007年

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