藍舌病病毒12型NS1蛋白單克隆抗體制備及其抗原表位鑒定
發(fā)布時間：2019-06-01 13:56
【摘要】：藍舌病(Bluetongue disease,BT)是由藍舌病病毒(Bluetongue virus,BTV)引起的一種蟲媒傳染病,可以引起綿羊等反芻動物的出血性疾病,主要通過庫蠓等昆蟲叮咬傳播,偶爾通過精液和胎盤傳播。BTV基因組的多樣性和節(jié)段性增加了病毒復(fù)制和組裝過程中基因重組的可能性,目前已發(fā)現(xiàn)27個不同的血清型,并且同一血清型內(nèi)含有大量的變異毒株。此外,全氣候變暖引起庫蠓等分布范圍的變化,導(dǎo)致世界范圍內(nèi)爆發(fā)BT的可能性增加。與環(huán)狀病毒屬其他成員一致,疫苗免疫是預(yù)防BT和BTV傳播最有效的途徑,但是BTV血清型之間具有很大的抗原差異性,彼此之間缺乏有效的免疫保護性。因此,制備具有型特異和群特異的單克隆抗體(monoclonal antibody,MAbs)為開發(fā)更好的診斷和研究試劑奠定基礎(chǔ),在BT防控方面具有重要意義。BTV基因組由10個分節(jié)段的雙鏈RNA組成(Seg-1~Seg-10),其中S5基因編碼的NS1蛋白在BTV各血清型之間高度保守。本研究利用實驗室保存的攜帶BTV12 S5基因的重組質(zhì)粒p EASY-NS1-BTV12為模板(Gen Bank登錄號:KM485310)設(shè)計上下游引物,PCR擴增獲得S5基因編碼區(qū),獲得的目的基因進行酶切后分別克隆轉(zhuǎn)化至原核表達載體p ET-30a和桿狀病毒表達載體p Fast BacTMHT A,構(gòu)建重組質(zhì)粒p ET-NS1-BTV12和p FAST-NS1-BTV12。將重組質(zhì)粒p ET-NS1-BTV12轉(zhuǎn)化至E.coli BL21(DE3)表達感受態(tài)細胞中,利用0.5m M IPTG成功誘導(dǎo)表達重組BTV12 NS1蛋白。SDS-PAGE結(jié)果顯示原核重組NS1蛋白(r P-NS1-BTV12)以包涵體形式表達。使用Ni2+NTA樹脂對其進行純化,Western blot(WB)驗證重組NS1蛋白能與本實驗室保存的BT陽性羊血清發(fā)生反應(yīng)。將重組質(zhì)粒p FAST-NS1-BTV12轉(zhuǎn)化至E.coli DH10BacTM感受態(tài)細胞,獲得重組桿粒(r Bacmid DNA)。將r Bacmid DNA轉(zhuǎn)染至sf21昆蟲細胞,得到高效表達S5基因的重組桿狀病毒BACV-NS1。SDS-PAGE結(jié)果顯示sf21昆蟲細胞成功表達重組NS1蛋白(r E-NS1-BTV12),同樣以包涵體形式存在。電洗脫法洗脫純化重組NS1蛋白,所獲得蛋白同樣可以與BT陽性羊血清發(fā)生反應(yīng)。利用免疫BALB/c小鼠的脾細胞和SP2/0骨髓瘤細胞融合技術(shù)獲得雜交瘤細胞,并通過間接ELISA篩選出25株穩(wěn)定分泌抗NS1蛋白的雜交瘤細胞株。WB結(jié)果顯示,25株MAbs與r P-NS1-BTV12和r E-NS1-BTV12均呈陽性反應(yīng),而與只表達空載體p ET-30a和野生型桿狀病毒的蛋白呈陰性反應(yīng)。IFA顯示25株MAbs中22株雜交瘤細胞與BTV12呈陽性反應(yīng),而3株雜交瘤細胞((2B7、3E9和4B9))與BTV12呈陰性反應(yīng)。22株陽性雜交瘤細胞中6株雜交瘤細胞(1F11、2F2、2C11、3C2、3G6和4E5)與感染BTV1-24型均呈陽性反應(yīng);而1F8雜交瘤細胞株僅與BTV12反應(yīng)。剩余的15株雜交瘤細胞與BTV血清型的反應(yīng)圖譜各有差異。為鑒定25株MAbs所識別的抗原表位,我們設(shè)計了55對覆蓋整個NS1蛋白的引物,通過原核表達方法表達出55條MBP融合短肽,以此作為包被抗原通過間接ELISA方法共篩選7個線性B細胞表位,另有4株MAbs(1B2、2E8、3E9和3D10)不與任何短肽反應(yīng),推測可能為構(gòu)象表位。隨后,對獲得的B細胞線性表位在呼腸孤病毒科環(huán)狀病毒屬成員之間進行氨基酸序列保守性分析。結(jié)果表明,所獲得表位在不同地區(qū)BTV12中保守性高;同時,對BTV1-26以及相關(guān)病毒如AHSV、EHDV和CV氨基酸序列進行分析,表明7個線性表位在BTV各血清型之間相對保守,但是與AHSV、EHDV和CV氨基酸序列差異較大。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
本文編號：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S855.3
【參考文獻】
相關(guān)期刊論文 前2條
1 張yN峰;;反芻動物藍舌病及其防治策略初探[J];當代畜牧;2014年14期
2 林漢亮;冉多良;王文;;藍舌病研究進展[J];新疆畜牧業(yè);2008年01期
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