人博卡病毒结构蛋白VP1u，非结构蛋白ORFx和NP1的原核表达与抗体制备
发布时间：2018-12-11 20:28
【摘要】：瑞典科学家Tobias Allander等于2005年10月从下呼吸道感染婴幼儿的分泌物中发现了一种新的细小病毒,即人博卡病毒(HumanBocavirus, HBoV), HBoV是一种DNA病毒,单链线状,病毒粒子无囊膜,基因组全长5299 nucleotide (HBoV isolate st2)属于细小病毒科中细小病毒亚科里面的博卡病毒属。 HBoV和所有的其他已知的细小病毒属的病毒一样,含有至少两个衣壳蛋白(VP2和VP1)和一个编码非结构蛋白(NS1)及中间开放读码框架,在病毒感染细胞时非结构蛋白NS1最先被转录和翻译,通过与病毒复制原点结合起来从而启动病毒DNA的复制。对病毒的序列分析发现一个未知蛋白ORFx,它在细小病毒科中并无同源基因。到目前为止NP1蛋白功并不清楚,采用Cratch Protein Predictor预测的NP1蛋白并用ProtFun分析后的结果显示NP1蛋白不是具有催化功能的酶蛋白,但可能与病毒基因的翻译过程有关[3]。为了对非结构蛋白基因ORFx, NP1和结构蛋白基因VPlu的功能更好研究,本实验分别对它们进行克隆,原核表达及多克隆抗体的制备。 本实验根据我们实验室克隆得到的中间大片段人博卡病毒序列(该序列已经提交GeneBank,登录号为：GU 139423)设计ORFx,VP1u和NP1引物,通过PCR技术扩增目的基因。并将它们克隆到原核表达载体pMAL-c2X中,利用IPTG诱导融合蛋白的产生,并用Amylose亲和层析柱纯化含目的蛋白的融合蛋白,然后用Factor Xa切割融合蛋白MBP-ORFx和MBP-NP1,并让融合蛋白MBP-VP1u和经过切割MBP-ORFx蛋白免疫新西兰大白兔,利用切割的MBP-NP1蛋白免疫小白鼠,制备出多克隆抗体,从而为进一步研究该病毒结构蛋白基因中ORFx,VP1u和NP1的功能提供可靠的工具。
[Abstract]:Swedish scientist Tobias Allander equaled to the discovery in October 2005 of a new parvovirus from the secretions of infants infected with lower respiratory tract infections: human Boca virus (HumanBocavirus, HBoV), a single-stranded, single-stranded virus that has no envelope. The genome length of 5299 nucleotide (HBoV isolate st2 belongs to the genus Boca virus in the subfamily Parvoviridae. HBoV, like all other known parvoviruses, contains at least two capsid proteins (VP2 and VP1) and a coded non-structural protein (NS1) and an intermediate open reading frame. The non-structural protein NS1 is first transcribed and translated when the virus infects the cells, and the replication of virus DNA is initiated by combining with the origin of virus replication. Sequence analysis of the virus revealed an unknown protein, ORFx, which had no homologous gene in the parvoviridae. Up to now, the NP1 protein function is not clear. The NP1 protein predicted by Cratch Protein Predictor and analyzed by ProtFun showed that NP1 protein is not a catalytic enzyme protein, but may be related to the translation process of virus gene [3]. In order to study the function of nonstructural protein gene (ORFx, NP1) and structural protein gene (VPlu), we cloned them, expressed them in prokaryotic and prepared polyclonal antibody. In this study, we designed ORFx,VP1u and NP1 primers according to the sequence of human Boca virus cloned in our laboratory (which has been submitted to GeneBank, accession number: GU 139423), and amplified the target gene by PCR technique. They were cloned into prokaryotic expression vector pMAL-c2X, and the fusion protein was induced by IPTG. The fusion protein containing the target protein was purified by Amylose affinity chromatography. Then the fusion proteins MBP-ORFx and MBP-NP1, were cut by Factor Xa. The fusion protein MBP-VP1u and the cut MBP-ORFx protein were used to immunize the New Zealand white rabbits, and the mice were immunized with the cut MBP-NP1 protein. The polyclonal antibodies were prepared, so as to further study the ORFx, in the structural protein gene of the virus. The functions of VP1u and NP1 provide reliable tools.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2373174
[Abstract]:Swedish scientist Tobias Allander equaled to the discovery in October 2005 of a new parvovirus from the secretions of infants infected with lower respiratory tract infections: human Boca virus (HumanBocavirus, HBoV), a single-stranded, single-stranded virus that has no envelope. The genome length of 5299 nucleotide (HBoV isolate st2 belongs to the genus Boca virus in the subfamily Parvoviridae. HBoV, like all other known parvoviruses, contains at least two capsid proteins (VP2 and VP1) and a coded non-structural protein (NS1) and an intermediate open reading frame. The non-structural protein NS1 is first transcribed and translated when the virus infects the cells, and the replication of virus DNA is initiated by combining with the origin of virus replication. Sequence analysis of the virus revealed an unknown protein, ORFx, which had no homologous gene in the parvoviridae. Up to now, the NP1 protein function is not clear. The NP1 protein predicted by Cratch Protein Predictor and analyzed by ProtFun showed that NP1 protein is not a catalytic enzyme protein, but may be related to the translation process of virus gene [3]. In order to study the function of nonstructural protein gene (ORFx, NP1) and structural protein gene (VPlu), we cloned them, expressed them in prokaryotic and prepared polyclonal antibody. In this study, we designed ORFx,VP1u and NP1 primers according to the sequence of human Boca virus cloned in our laboratory (which has been submitted to GeneBank, accession number: GU 139423), and amplified the target gene by PCR technique. They were cloned into prokaryotic expression vector pMAL-c2X, and the fusion protein was induced by IPTG. The fusion protein containing the target protein was purified by Amylose affinity chromatography. Then the fusion proteins MBP-ORFx and MBP-NP1, were cut by Factor Xa. The fusion protein MBP-VP1u and the cut MBP-ORFx protein were used to immunize the New Zealand white rabbits, and the mice were immunized with the cut MBP-NP1 protein. The polyclonal antibodies were prepared, so as to further study the ORFx, in the structural protein gene of the virus. The functions of VP1u and NP1 provide reliable tools.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
相关期刊论文 前6条
1 王永杰,陈克平,姚勤;浓核病毒基因组研究进展[J];安徽农业科学;2005年09期
2 瞿小旺;漆正宇;段招军;刘劲松;刘巧突;刘文培;黄灿平;谢志萍;彭夫望;高寒春;郑丽舒;侯云德;;儿童急性呼吸道博卡病毒感染[J];病毒学报;2006年02期
3 郑丽舒;段招军;;人博卡病毒——呼吸道感染病新病原[J];传染病信息;2007年02期
4 林峰;曾爱平;杨恩;林海燕;郑昌华;陈弘;李桦;李旭阳;郁明素;杨宁敏;金大智;余光创;伯晓晨;文思远;王升启;;WLL-1株博卡病毒(Bocavirus)基因组测序及生物信息学分析[J];浙江检验医学;2007年01期
5 林峰;曾爱平;杨宁敏;林海燕;郑昌华;李桦;陈弘;李旭阳;郁明素;杨宝顺;金大智;;小儿人博卡病毒感染临床与实验研究[J];医学研究杂志;2006年09期
6 傅希;伍严安;;博卡病毒的研究进展[J];医学综述;2007年15期
，本文编号：2373174
本文链接：https://www.wllwen.com/xiyixuelunwen/2373174.html
最近更新
教材专著
