细小病毒B19-VP1U保守区外氨基酸对磷脂酶A2活性的影响

发布时间：2018-04-25 09:39

本文选题：细小病毒B19 + VP1u　；参考：《华中师范大学》2012年硕士论文

【摘要】：人细小病毒B19于1975年被Yvonne Cossart首次发现,是感染人的细小病毒之一,且能潜在抑制红细胞的生成,能引起多种疾病。B19感染具有特异性,其倾向于感染表面具有红细胞糖苷酯受体的原红细胞。B19侵入细胞除需要红细胞糖苷酯受体外还需要辅助受体a5β1整联蛋白以及具有活性的自主抗原KU80。不同年龄段的宿主感染B19则表现为不同的临床症状,比如在成年人中,关节炎、关节痛以及发热症状发生频率比较高；而在儿童中,贫血症发生频率相对较高。B19的衣壳由结构蛋白VP1和VP2构成,除了VP1的N端有额外的227个氨基酸序列外,VP1和VP2其它氨基酸序列相同。研究发现,VP1特有的227个氨基酸具有磷脂酶A2活性,其中第130-195位氨基酸为酶活性的保守区域,该保守区外的氨基酸可能对于形成蛋白质正确的三维结构以保持酶的稳定及作用底物与酶的中心充分接近非常重要。 B19-VP1U在病毒的生活周期中具有重要的功能,尤其磷脂酶A2活性对病毒的感染是必须的,若病毒缺乏VP1完整序列则病毒不具有感染性并且不能从细胞核转运到细胞质,因此,B19-VP1U的磷脂酶A2活性在病毒的致病性、诱导自主免疫反应及炎症的发生具有重要的作用。若把B19-VP1U磷脂酶A2活性部位的关键氨基酸进行突变则会导致酶活的下降或丧失,本实验主要研究保守区外氨基酸对酶活性的影响。具体方案如下： (1)根据查阅的相关文献从VP1u保守区外氨基酸的N端每隔10个或30个氨基酸进行截短突变,将截短突变的基因片段克隆到载体pMAL-c2x上,构建正确的重组载体。小规模诱导目的蛋白的表达,SDS-PAGE及Western-blot检测目的蛋白后,大规模诱导,然后利用Amylose树脂对目的蛋白进行纯化。 (2)根据相关文献及对VP1u相关序列的分析,从VP1u保守区外氨基酸的C端每隔7个或8个氨基酸进行截短突变,将截短突变的基因片段克隆到载体pMAL-c2x上,经测序得正确的重组质粒。目的蛋白经小规模诱导后,SDS-PAGE及Western-blot检测,然后进行大规模诱导,利用Amylose树脂对目的蛋白进行纯化。 (3)获得目的蛋白后,用Broadford试剂盒测定目的蛋白的浓度,保证蛋白浓度在0.5mg/ml以上,然后采用相应的试剂盒测其酶活,最后分析实验结果。通过测定磷脂酶A2的活性,结果发现当从N端截短12个AA时测的酶的活性值与完整VP1u相比降低了约35%,但截短67个AA时酶的活性就明显下降了,当截短96个AA时酶的活性几乎丧失,因此推测N端第1-96个AA之间的区域对酶的活性有显著的影响；C端截短7个AA时对酶的活性没有显著影响几乎与VPlu相同,但截短16个AA时酶的活性就显著下降了,当截短24个AA时酶的活性几乎丧失,因此推测C端第7-24个AA之间的区域对酶的活性有显著影响。推测,对保守区外氨基酸截短突变而造成的磷脂酶A2活性的改变可能与B19-VP1U蛋白的正确的三维构象有很大的关系,因为蛋白质要保持活性必须具有完整的三维结构。这些结果将为进一步研究和解释B19感染的分子生物学机制提供了一定的理论基础。
[Abstract]:Human parvovirus B19 was first discovered by Yvonne Cossart in 1975. It is one of the parvovirus infected people and can potentially inhibit the formation of red blood cells. It can cause a variety of disease.B19 infection to be specific. It tends to infection of erythrocyte.B19 invading cells with red cell glycosidic ester receptor on the surface in addition to the need of erythrocyte glycosidic ester in vitro The host infection of the A5 beta 1 integrin and the active independent antigen KU80. for different ages of the host infected B19 showed different clinical symptoms, such as in adults, arthritis, joint pain, and fever, and in children, a relatively high frequency of.B19's capsid in children had a relatively high frequency. Protein VP1 and VP2 constitute the same sequence of other amino acids in VP1 and VP2 in addition to the additional 227 amino acid sequences at the N end of VP1. The study found that the 227 amino acids specific to VP1 have the activity of phospholipase A2, and the 130-195 amino acid is the conservative region of the enzyme activity, and the amino acid outside the conservative region may be three correct for the formation of protein. It is very important to maintain the stability of the enzyme and to keep the substrate close to the center of the enzyme.
B19-VP1U plays an important role in the life cycle of the virus, especially the phospholipase A2 activity is necessary for the virus infection. If the virus lacks the complete sequence of VP1, the virus is not infective and can not be transported from the nucleus to the cytoplasm. Therefore, the phospholipase A2 activity of B19-VP1U is in the virulence of the virus, induces the autoimmune reaction and inflammation. The occurrence of the disease has an important role. If the key amino acid of the B19-VP1U phospholipase A2 active site is mutated, the enzyme activity will be reduced or lost. This experiment mainly studies the effect of the amino acid on the enzyme activity in the conservative region.
(1) according to the related literature, a short mutation was carried out from 10 or 30 amino acids from the N terminal of the VP1u conservative region. The truncated gene fragment was cloned to the carrier pMAL-c2x, and the correct recombinant vector was constructed. A small scale induced the expression of the target protein and the SDS-PAGE and Western-blot were used to detect the target protein on a large scale. The Amylose resin was used to purify the target protein.
(2) according to the related literature and the analysis of the VP1u related sequences, every 7 or 8 amino acids were truncated from the C terminal of the amino acids outside the conservative region of VP1u. The truncated gene fragment was cloned to the carrier pMAL-c2x, and the correct recombinant plasmid was sequenced. After the target protein was induced by the small norm, SDS-PAGE and Western-blot were detected and then entered. The target protein was purified by Amylose resin.
(3) after the target protein was obtained, the concentration of the target protein was measured by Broadford kit, the concentration of the protein was above 0.5mg/ml, and then the enzyme activity was measured with the corresponding kit. Finally, the experimental results were analyzed.
By measuring the activity of phospholipase A2, it was found that the activity of the enzyme was reduced by about 35% compared with the complete VP1u when the 12 AA was truncated from the N end, but the activity of the enzyme decreased obviously when the 67 AA was truncated, and the activity of the enzyme was almost lost when the 96 AA was truncated. Therefore, it was speculated that the region between the 1-96 AA of the N terminal had a significant effect on the activity of the enzyme. The activity of the enzyme had no significant effect on the activity of the enzyme at the 7 AA truncated C terminal, but the activity of the enzyme decreased significantly when the 16 AA was truncated and the activity of the enzyme was almost lost when the 24 AA was truncated. Therefore, it was speculated that the region between the 7-24 AA at the end of the C side had a significant effect on the activity of the enzyme. The changes in the activity of phospholipase A2 may have a great relationship with the correct three-dimensional conformation of the B19-VP1U protein, because the protein to maintain the activity must have a complete three-dimensional structure. These results will provide a certain theoretical basis for further research and interpretation of the molecular biological mechanism of B19 infection.

【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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