副粘病毒天津株HN、M基因克隆与表达的研究
发布时间：2019-07-04 20:27
【摘要】： 1999年6月，天津医科大学微生物教研室从急性呼吸道感染致死的灵长类动物狨猴肺组织中分离出一株高血凝效价病毒，经过一系列的研究证实为一株副粘病毒，即副粘病毒天津株。经电镜观察具有典型的副粘病毒特征。用交叉血凝抑制试验对分离毒株进行鉴定，扩增HN基因测序后，用生物信息学方法与多种核酸序列进行比较，发现该毒株与仙台病毒(SeV)特征最为接近。进一步研究发现该动物中心的工作人员都有此病毒的抗体，且在正常人群(献血员)中抗体反应也是阳性，阳性率高达46％(14／40)，提示此病原体与人类有密切的关系，可能是一株人和绒猴共患的呼吸道病毒。我们现在已经完成了对该株病毒的全基因组测序，通过全基因组系统进化树分析，其与SeV同源性较高，也证明了该株病毒很可能为SeV一个新的基因型。 为进一步研究该毒株的生物学特性及致病机制，本研究构建了副粘病毒天津株包膜糖蛋白HN膜外区三个部分及整个M基因的原核表达质粒pET-28a-HN1、pET-28a-HN2、pET-28a-HN3及pMal-M，诱导表达、纯化了重组蛋白HNl(aa61-aa260)、HN2(aa253-aa452)、HN3(aa376-aa575)和MBP-M，并对其生物学和免疫学活性进行了初步研究。 我们提取病毒RNA逆转录为cDNA。根据前期研究全基因组测序中的HN、M的基因序列，设计并合成引物，以该株病毒的cDNA为模板扩增目的片段，双酶切、纯化回收含粘末端的目的片段和空质粒，在T4 DNA连接酶作用下连接、转化克隆菌E．coli TOP10。经双酶切、PCR和基因测序鉴定筛选构建正确的目的基因，转化表达菌E．coli BL21，IPTG诱导表达。pET-28a-HN1、pET-28a-HN2和pET-28a-HN3所选用的载体质粒带有6×His标签，用Ni金属螯合柱进行回收，而pMal-M表达的蛋白带有MBP融合蛋白，用Amylose resin回收得到纯化的蛋白。用超速离心纯化的病毒和纯化表达蛋白分别制备PcAb，利用ELISA、Dot Blot及Western Blot等方法对表达蛋白进行免疫学鉴定。通过血凝试验和血凝抑制试验对副粘病毒天津株HN1、HN2和HN3蛋白的血凝功能及重组蛋白多克隆抗体的血凝抑制功能进行了初步研究。结果显示：HN1、HN2和HN3表现出来的天然抗原性明显不同，，抗原性强弱依次为HN2＞HN3＞HN1；M蛋白的ELISA和Dot Blot方法鉴定结果也为阳性。而HN3的血凝活性明显强于HN蛋白的其他部位，其次为HN2蛋白，HN1几乎没有血凝功能。另外，利用ELISA方法对副粘病毒天津株HN1、HN2和HN3蛋白与甲型、乙型流感病毒的WHO标准血清和天津CDC获得的阳性血清的交叉免疫情况进行了研究，结果显示：HN1、HN2和HN3三种蛋白与这些血清存在交叉免疫反应。 综上，本研究成功的构建了HN1、HN2、HN3和M的原核重组表达质粒pET-28a-HN2、pET-28a-HN3及pMa1-M，经诱导、表达、纯化得到了重组蛋白HN1、HN2、HN3和M，并对其进行了免疫学和生物学的初步鉴定，为进一步研究副粘病毒天津株的致病机制，制备单克隆抗体和建立该毒株特异的检测方法奠定了基础。
文内图片：
图片说明：图IM、HNI、HNZ和HN3基因RT-PCR琼脂糖凝胶电泳
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373.1
本文编号：2510209
文内图片：
图片说明：图IM、HNI、HNZ和HN3基因RT-PCR琼脂糖凝胶电泳
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373.1
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1 袁立军;副粘病毒天津株HN、M基因克隆与表达的研究[D];天津医科大学;2007年
本文编号：2510209
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