蓝舌病病毒12型NS1蛋白单克隆抗体制备及其抗原表位鉴定
发布时间：2019-06-01 13:56
【摘要】：蓝舌病(Bluetongue disease,BT)是由蓝舌病病毒(Bluetongue virus,BTV)引起的一种虫媒传染病,可以引起绵羊等反刍动物的出血性疾病,主要通过库蠓等昆虫叮咬传播,偶尔通过精液和胎盘传播。BTV基因组的多样性和节段性增加了病毒复制和组装过程中基因重组的可能性,目前已发现27个不同的血清型,并且同一血清型内含有大量的变异毒株。此外,全气候变暖引起库蠓等分布范围的变化,导致世界范围内爆发BT的可能性增加。与环状病毒属其他成员一致,疫苗免疫是预防BT和BTV传播最有效的途径,但是BTV血清型之间具有很大的抗原差异性,彼此之间缺乏有效的免疫保护性。因此,制备具有型特异和群特异的单克隆抗体(monoclonal antibody,MAbs)为开发更好的诊断和研究试剂奠定基础,在BT防控方面具有重要意义。BTV基因组由10个分节段的双链RNA组成(Seg-1~Seg-10),其中S5基因编码的NS1蛋白在BTV各血清型之间高度保守。本研究利用实验室保存的携带BTV12 S5基因的重组质粒p EASY-NS1-BTV12为模板(Gen Bank登录号:KM485310)设计上下游引物,PCR扩增获得S5基因编码区,获得的目的基因进行酶切后分别克隆转化至原核表达载体p ET-30a和杆状病毒表达载体p Fast BacTMHT A,构建重组质粒p ET-NS1-BTV12和p FAST-NS1-BTV12。将重组质粒p ET-NS1-BTV12转化至E.coli BL21(DE3)表达感受态细胞中,利用0.5m M IPTG成功诱导表达重组BTV12 NS1蛋白。SDS-PAGE结果显示原核重组NS1蛋白(r P-NS1-BTV12)以包涵体形式表达。使用Ni2+NTA树脂对其进行纯化,Western blot(WB)验证重组NS1蛋白能与本实验室保存的BT阳性羊血清发生反应。将重组质粒p FAST-NS1-BTV12转化至E.coli DH10BacTM感受态细胞,获得重组杆粒(r Bacmid DNA)。将r Bacmid DNA转染至sf21昆虫细胞,得到高效表达S5基因的重组杆状病毒BACV-NS1。SDS-PAGE结果显示sf21昆虫细胞成功表达重组NS1蛋白(r E-NS1-BTV12),同样以包涵体形式存在。电洗脱法洗脱纯化重组NS1蛋白,所获得蛋白同样可以与BT阳性羊血清发生反应。利用免疫BALB/c小鼠的脾细胞和SP2/0骨髓瘤细胞融合技术获得杂交瘤细胞,并通过间接ELISA筛选出25株稳定分泌抗NS1蛋白的杂交瘤细胞株。WB结果显示,25株MAbs与r P-NS1-BTV12和r E-NS1-BTV12均呈阳性反应,而与只表达空载体p ET-30a和野生型杆状病毒的蛋白呈阴性反应。IFA显示25株MAbs中22株杂交瘤细胞与BTV12呈阳性反应,而3株杂交瘤细胞((2B7、3E9和4B9))与BTV12呈阴性反应。22株阳性杂交瘤细胞中6株杂交瘤细胞(1F11、2F2、2C11、3C2、3G6和4E5)与感染BTV1-24型均呈阳性反应;而1F8杂交瘤细胞株仅与BTV12反应。剩余的15株杂交瘤细胞与BTV血清型的反应图谱各有差异。为鉴定25株MAbs所识别的抗原表位,我们设计了55对覆盖整个NS1蛋白的引物,通过原核表达方法表达出55条MBP融合短肽,以此作为包被抗原通过间接ELISA方法共筛选7个线性B细胞表位,另有4株MAbs(1B2、2E8、3E9和3D10)不与任何短肽反应,推测可能为构象表位。随后,对获得的B细胞线性表位在呼肠孤病毒科环状病毒属成员之间进行氨基酸序列保守性分析。结果表明,所获得表位在不同地区BTV12中保守性高;同时,对BTV1-26以及相关病毒如AHSV、EHDV和CV氨基酸序列进行分析,表明7个线性表位在BTV各血清型之间相对保守,但是与AHSV、EHDV和CV氨基酸序列差异较大。
[Abstract]:Bluetongue disease (BT) is an insect-borne infectious disease caused by Bluetongue virus (BTV), which can cause the hemorrhagic diseases of ruminants such as sheep and other ruminants. The diversity and segmental nature of the BTV genome increases the possibility of gene recombination during viral replication and assembly, and 27 different serotypes have been found, and a large number of variant strains are present in the same serotype. In addition, global warming caused a change in the distribution range of the reservoir, resulting in an increase in the possibility of a BT in the world. In line with other members of the ring-like virus, the vaccine immunization is the most effective way to prevent BT and BTV transmission, but there is a large difference in antigen between the BTV serotypes and a lack of effective immune protection from each other. Therefore, the preparation of monoclonal antibodies (Mbs) with specific and group-specific groups lays the foundation for the development of better diagnosis and research reagents, and is of great significance in the prevention and control of BT. The BTV genome consists of 10 segments of double-stranded RNA (Seg-1-Seg-10), wherein the S5 gene-encoded NS1 protein is highly conserved among the BTV serotypes. The recombinant plasmid pEASY-NS1-BTV12 carrying the BTV12S5 gene stored in the laboratory is used as a template (Gen Bank accession number: KM485310) to design the upstream and downstream primers, and the PCR amplification obtains the S5 gene coding region, The obtained target gene was cloned into a prokaryotic expression vector pET-30a and a baculovirus expression vector pFast BacTHT A, respectively, and the recombinant plasmids pET-NS1-BTV12 and p FAST-NS1-BTV12 were constructed. The recombinant plasmid pET-NS1-BTV12 was transformed into E. coli BL21 (DE3) to express the competent cells, and the recombinant BTV12NS1 protein was successfully induced with 0.5 mM IPTG. The results of SDS-PAGE show that the prokaryotic recombinant NS1 protein (r P-NS1-BTV12) is expressed in the form of inclusion bodies. It was purified by using Ni2 + NTA resin, and Western blot (WB) was used to verify that the recombinant NS1 protein can react with the BT-positive sheep serum stored in this lab. The recombinant plasmid pFAST-NS1-BTV12 was transformed into E. coli DH10BTM competent cells to obtain a recombinant rod particle (r Bacmid DNA). The recombinant NS1 protein (r E-NS1-BTV12) was successfully expressed by the recombinant baculovirus BACV-NS1. SDS-PAGE showed that the recombinant NS1 protein (r E-NS1-BTV12) was successfully expressed by the sf21 insect cells. The purified recombinant NS1 protein is eluted and purified by the electroelution method, and the obtained protein can also react with the serum of the BT-positive sheep serum. The hybridoma cells were obtained by the fusion of the spleen cells of the BALB/ c mice and the SP2/0 myeloma cell fusion technique, and the hybridoma cell lines stably secreting the anti-NS1 protein were screened by indirect ELISA. The results of WB showed that 25 strains of MAbs were positive with r P-NS1-BTV12 and r E-NS1-BTV12, and were negative to the proteins expressing only the empty vector pET-30a and the wild-type baculovirus. The two hybridoma cells (1F11, 2F2, 2C11, 3C2, 3G6 and 4E5) of the 22 positive hybridoma cells were positive with BTV12. And the 1F8 hybridoma cell line only reacts with the BTV12. The reaction profiles of the remaining 15 hybridoma cells and the BTV serotype were different. In order to identify the antigenic epitopes identified by 25 MAbs,55 pairs of primers covering the whole NS1 protein were designed, and 55 MBP fusion short peptides were expressed by a prokaryotic expression method, and 7 linear B cell epitopes were screened by an indirect ELISA method as the envelope antigen. Four other MAbs (1B2, 2E8, 3E9, and 3D10) do not react with any short peptides, and may be presumed to be a conformational epitope. Subsequently, an amino acid sequence conservative analysis was performed on the obtained B-cell linear table position between members of the reovirus family. The results showed that the obtained epitope was highly conserved in the BTV12 in different regions; at the same time, the BTV1-26 and the related viruses such as AHSV, EHDV and CV amino acid sequences were analyzed, indicating that the 7 linear table bits were relatively conservative among the various serotypes of the BTV, but the difference in the amino acid sequence with the AHSV, EHDV and CV was large.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
本文编号：2490326
[Abstract]:Bluetongue disease (BT) is an insect-borne infectious disease caused by Bluetongue virus (BTV), which can cause the hemorrhagic diseases of ruminants such as sheep and other ruminants. The diversity and segmental nature of the BTV genome increases the possibility of gene recombination during viral replication and assembly, and 27 different serotypes have been found, and a large number of variant strains are present in the same serotype. In addition, global warming caused a change in the distribution range of the reservoir, resulting in an increase in the possibility of a BT in the world. In line with other members of the ring-like virus, the vaccine immunization is the most effective way to prevent BT and BTV transmission, but there is a large difference in antigen between the BTV serotypes and a lack of effective immune protection from each other. Therefore, the preparation of monoclonal antibodies (Mbs) with specific and group-specific groups lays the foundation for the development of better diagnosis and research reagents, and is of great significance in the prevention and control of BT. The BTV genome consists of 10 segments of double-stranded RNA (Seg-1-Seg-10), wherein the S5 gene-encoded NS1 protein is highly conserved among the BTV serotypes. The recombinant plasmid pEASY-NS1-BTV12 carrying the BTV12S5 gene stored in the laboratory is used as a template (Gen Bank accession number: KM485310) to design the upstream and downstream primers, and the PCR amplification obtains the S5 gene coding region, The obtained target gene was cloned into a prokaryotic expression vector pET-30a and a baculovirus expression vector pFast BacTHT A, respectively, and the recombinant plasmids pET-NS1-BTV12 and p FAST-NS1-BTV12 were constructed. The recombinant plasmid pET-NS1-BTV12 was transformed into E. coli BL21 (DE3) to express the competent cells, and the recombinant BTV12NS1 protein was successfully induced with 0.5 mM IPTG. The results of SDS-PAGE show that the prokaryotic recombinant NS1 protein (r P-NS1-BTV12) is expressed in the form of inclusion bodies. It was purified by using Ni2 + NTA resin, and Western blot (WB) was used to verify that the recombinant NS1 protein can react with the BT-positive sheep serum stored in this lab. The recombinant plasmid pFAST-NS1-BTV12 was transformed into E. coli DH10BTM competent cells to obtain a recombinant rod particle (r Bacmid DNA). The recombinant NS1 protein (r E-NS1-BTV12) was successfully expressed by the recombinant baculovirus BACV-NS1. SDS-PAGE showed that the recombinant NS1 protein (r E-NS1-BTV12) was successfully expressed by the sf21 insect cells. The purified recombinant NS1 protein is eluted and purified by the electroelution method, and the obtained protein can also react with the serum of the BT-positive sheep serum. The hybridoma cells were obtained by the fusion of the spleen cells of the BALB/ c mice and the SP2/0 myeloma cell fusion technique, and the hybridoma cell lines stably secreting the anti-NS1 protein were screened by indirect ELISA. The results of WB showed that 25 strains of MAbs were positive with r P-NS1-BTV12 and r E-NS1-BTV12, and were negative to the proteins expressing only the empty vector pET-30a and the wild-type baculovirus. The two hybridoma cells (1F11, 2F2, 2C11, 3C2, 3G6 and 4E5) of the 22 positive hybridoma cells were positive with BTV12. And the 1F8 hybridoma cell line only reacts with the BTV12. The reaction profiles of the remaining 15 hybridoma cells and the BTV serotype were different. In order to identify the antigenic epitopes identified by 25 MAbs,55 pairs of primers covering the whole NS1 protein were designed, and 55 MBP fusion short peptides were expressed by a prokaryotic expression method, and 7 linear B cell epitopes were screened by an indirect ELISA method as the envelope antigen. Four other MAbs (1B2, 2E8, 3E9, and 3D10) do not react with any short peptides, and may be presumed to be a conformational epitope. Subsequently, an amino acid sequence conservative analysis was performed on the obtained B-cell linear table position between members of the reovirus family. The results showed that the obtained epitope was highly conserved in the BTV12 in different regions; at the same time, the BTV1-26 and the related viruses such as AHSV, EHDV and CV amino acid sequences were analyzed, indicating that the 7 linear table bits were relatively conservative among the various serotypes of the BTV, but the difference in the amino acid sequence with the AHSV, EHDV and CV was large.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S855.3
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相关期刊论文 前2条
1 张yN峰;;反刍动物蓝舌病及其防治策略初探[J];当代畜牧;2014年14期
2 林汉亮;冉多良;王文;;蓝舌病研究进展[J];新疆畜牧业;2008年01期
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