猪整合素αvβ3真核表达载体构建及介导PEDV感染作用的初步验证

发布时间：2018-01-21 16:16

本文关键词： PEDV 猪整合素αvβ3 多克隆抗体真核表达载体　出处：《黑龙江八一农垦大学》2016年硕士论文　论文类型：学位论文

【摘要】：猪流行性腹泻(PED)是一种发病率和死亡率极高的肠道传染病,给养猪业带来巨大的经济损失。目前已经证明猪氨基肽酶N(pAPN)是PEDV的一个细胞受体,但是仅依赖于pAPN蛋白仍不能解释PEDV感染中存在的相关理论问题。因此,对PEDV受体的研究具有重要的科学意义,为抗病毒药物的研究提供依据。近年来,越来越多的研究发现整合素可以作为许多病毒的受体,具有成为共性受体蛋白的趋势。在本研究中,根据GenBank发表的猪整合素αv和猪整合素β3氨基酸序列,分别进行疏水性分析,截取具有亲水性的胞外区氨基酸序列,然后针对大肠杆菌具有偏嗜性的密码子进行优化,人工合成两段基因。将两段基因分别连接至pGEX-6p-1原核表达载体,以IPTG诱导表达。将成功表达的重组蛋白进行纯化,然后按照50?g/只的剂量免疫8周龄的BALB/c雌性小白鼠,第3次免疫3日后进行眼球采血。用间接ELISA方法检测抗体效价,然后利用Western blot方法鉴定制备的两种多克隆抗体是否能识别ST细胞、IEC细胞、PK15细胞及仔猪小肠组织中的整合素蛋白。将人工合成的猪整合素αv和猪整合素β3全基因序列连接至pCAGGS-HA真核表达载体上,再将重组质粒pCAGGS-pIαv和pCAGGS-pIβ3转染至CHO细胞,确定重组蛋白表达后接种PEDV,在接种病毒后的不同时间点对细胞感染率进行统计。结果成功获得了人工合成基因,通过原核表达系统获得了重组蛋白pGEX-6p-1-αv和重组蛋白pGEX-6p-1-β3,Western blot显示截取的猪整合素αv第37~557位氨基酸具有较好的抗原性,猪整合素β3第30~714位氨基酸具有较好的抗原性。制备的猪整合素αv和猪整合素β3多克隆抗体效价分别为1:6 400和1:12 800,能识别ST细胞、IEC细胞、PK15细胞及仔猪小肠组织中的整合素蛋白。构建了pCAGGS-pIαv和pCAGGS-pIβ3真核表达载体,共转染组的整合素表达量高于单转染组,转染后24 h~36 h的整合素αvβ3蛋白在CHO细胞内表达量最高,在该时间段接种PEDV后,过表达整合素αvβ3蛋白的CHO细胞PEDV感染率显著高于对照组。本研究获得了猪整合素αv和猪整合素β3多克隆抗体,能够识别多种细胞及组织内的天然整合素蛋白,过表达整合素αvβ3的CHO细胞使PEDV感染率提高,为PEDV感染机制及病毒与受体间相互作用的研究奠定了基础。
[Abstract]:Porcine epidemic diarrhea (PED) is an extremely high incidence and mortality of intestinal infectious diseases. It has been proved that porcine aminopeptidase (NPAPN) is a cell receptor of PEDV. However, relying on pAPN protein alone can not explain the related theoretical problems in PEDV infection. Therefore, the study of PEDV receptor has important scientific significance. In recent years, more and more studies have found that integrin can be used as the receptor of many viruses and has the tendency to become a common receptor protein. According to the porcine integrin 伪 v and porcine integrin 尾 3 amino acid sequences published by GenBank, hydrophobic analysis was carried out, and the hydrophilic extracellular amino acid sequences were intercepted. Then the codon of Escherichia coli with bias was optimized and two segments of gene were synthesized. The two genes were linked to the prokaryotic expression vector of pGEX-6p-1 respectively. Expression was induced by IPTG. The recombinant protein was purified and then purified according to 50? BALB/c female mice aged 8 weeks were immunized with g / g for 3 days after the third immunization. The antibody titers were detected by indirect ELISA method. Then Western blot was used to identify whether the two polyclonal antibodies could recognize St cells. The synthetic porcine integrin 伪 v and porcine integrin 尾 3 gene sequences were ligated to the pCAGGS-HA eukaryotic expression vector in PK15 cells and small intestine tissues of piglets. The recombinant plasmids pCAGGS-pI 伪 v and pCAGGS-pI 尾 3 were transfected into CHO cells, and then the recombinant protein was expressed and inoculated with PEDV. The cell infection rate was counted at different time points after inoculation. The recombinant protein pGEX-6p-1- 伪 v and pGEX-6p-1- 尾 3 were obtained by prokaryotic expression system. Western blot showed that the amino acid at position 37557 of porcine integrin 伪 v had good antigenicity. The antibody titers of porcine integrin 伪 v and porcine integrin 尾 3 polyclonal antibody were 1: 6 400 and 1:12 800, respectively. PCAGGS-pI 伪 v and pCAGGS-pI 尾 3 eukaryotic expression vectors were constructed, which could recognize integrin protein in PK15 cells and small intestine tissues of piglets. The expression of integrin in cotransfection group was higher than that in single transfection group. The expression of integrin 伪 v 尾 3 protein in CHO cells was the highest at 24 h and 36 h after transfection. After inoculation with PEDV at that time, the expression of integrin 伪 v 尾 3 protein in CHO cells was the highest. The PEDV infection rate of CHO cells overexpressing integrin 伪 v 尾 3 protein was significantly higher than that of control group. In this study, porcine integrin 伪 v and porcine integrin 尾 3 polyclonal antibodies were obtained. The CHO cells which can recognize the natural integrin protein in many kinds of cells and tissues and overexpression integrin 伪 v 尾 3 increase the infection rate of PEDV. It lays a foundation for the study of the mechanism of PEDV infection and the interaction between virus and receptor.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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