人博卡病毒結構蛋白VP1u，非結構蛋白ORFx和NP1的原核表達與抗體制備
發(fā)布時間：2018-12-11 20:28
【摘要】：瑞典科學家Tobias Allander等于2005年10月從下呼吸道感染嬰幼兒的分泌物中發(fā)現(xiàn)了一種新的細小病毒,即人博卡病毒(HumanBocavirus, HBoV), HBoV是一種DNA病毒,單鏈線狀,病毒粒子無囊膜,基因組全長5299 nucleotide (HBoV isolate st2)屬于細小病毒科中細小病毒亞科里面的博卡病毒屬。 HBoV和所有的其他已知的細小病毒屬的病毒一樣,含有至少兩個衣殼蛋白(VP2和VP1)和一個編碼非結構蛋白(NS1)及中間開放讀碼框架,在病毒感染細胞時非結構蛋白NS1最先被轉錄和翻譯,通過與病毒復制原點結合起來從而啟動病毒DNA的復制。對病毒的序列分析發(fā)現(xiàn)一個未知蛋白ORFx,它在細小病毒科中并無同源基因。到目前為止NP1蛋白功并不清楚,采用Cratch Protein Predictor預測的NP1蛋白并用ProtFun分析后的結果顯示NP1蛋白不是具有催化功能的酶蛋白,但可能與病毒基因的翻譯過程有關[3]。為了對非結構蛋白基因ORFx, NP1和結構蛋白基因VPlu的功能更好研究,本實驗分別對它們進行克隆,原核表達及多克隆抗體的制備。 本實驗根據(jù)我們實驗室克隆得到的中間大片段人博卡病毒序列(該序列已經(jīng)提交GeneBank,登錄號為：GU 139423)設計ORFx,VP1u和NP1引物,通過PCR技術擴增目的基因。并將它們克隆到原核表達載體pMAL-c2X中,利用IPTG誘導融合蛋白的產(chǎn)生,并用Amylose親和層析柱純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,并讓融合蛋白MBP-VP1u和經(jīng)過切割MBP-ORFx蛋白免疫新西蘭大白兔,利用切割的MBP-NP1蛋白免疫小白鼠,制備出多克隆抗體,從而為進一步研究該病毒結構蛋白基因中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.
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
本文編號：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.
【學位授予單位】：華中師范大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392
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