25型蓝舌病病毒VP7和VP2蛋白的原核表达及其单克隆抗体的制备
发布时间：2018-08-05 16:57
【摘要】：蓝舌病(Bluetongue,BT)是由蓝舌病病毒(Bluetongue virus,BTV)引起的,依靠媒介昆虫(库蠓、伊蚊等)在反刍动物之间传播的一种烈性非接触性的传染病。纯种细毛羊对该病最敏感。BT最早于1876年被发现,目前在热带和温带的多个国家都已分离到BTV,并且该病的分布范围在不断的扩大,呈现全球性分布的趋势。我国于1979年第一次发现该病的存在,目前在我国29个省份已检测到感染BTV阳性的病畜。迄今为止,全世界范围内共分离到26种不同血清型的BTV,且不同的血清型之间无交叉保护作用。BTV基因组由10个双链RNA组成,包括大片段(L1-L3)、中片段(M4-M6)和小片段(S7-S10)。其共编码7个结构蛋白(VP1-VP7)和4种非结构蛋白(NS1、NS2、NS3/NS3a和NS4)。VP7是由S7基因编码的主要结构蛋白,由349个氨基酸组成,位于病毒核衣壳的表面,约占病毒核心蛋白总量36%。VP7是BTV的群特异性抗原,不同血清型之间的VP7蛋白的同源性高达94%。VP2由L2基因编码,在不同血清型病毒间保守性最低。VP2蛋白主要参与病毒的吸附和进入,与病毒的毒力有关。VP2可诱导产生中和抗体,是BTV型特异性抗原主要的决定因素。25型BTV是2008年从瑞士山羊血液样本中分离获得。25型BTV的VP2与其他血清型病毒VP2的同源性仅为23%-79%。因感染25型BTV的牲畜无典型症状,故常被忽略。但是一旦爆发,发病率和死亡率较高。因此建立针对25型BTV的特异性血清学检测方法对BTV的防控有重要的意义。本研究为制备25型BTV的VP7和VP2蛋白单克隆抗体(Monoclonal antibody,Mc Ab),利用原核表达系统p ET-28a(+)和pGEX-6P-1表达VP7蛋白,分别命名为VP7a和VP7p。经SDS-PAGE和Western blot分析大小依次为44 kDa和64 k Da,与预期蛋白大小一致。采用原核表达系统p ET-28a(+)和p MAL-c5X部分重叠表达三段VP2蛋白,分别命名为VP2-A、VP2-B、VP2-C和VP2-A1、VP2-B1、VP2-C1。经SDS-PAGE和Western blot分析,大小依次为50k Da、48k Da、48k Da、84 kDa、82 kDa和82 k Da,与预期蛋白大小一致。分别将重组蛋白VP7a和重组VP2-A、B、C作为免疫抗原,腹腔免疫BALB/c小鼠,采用细胞融合技术将骨髓瘤细胞SP2/0与免疫后小鼠的脾细胞进行细胞融合,通过有限稀释法进行细胞亚克隆,获得稳定产生抗体的杂交瘤细胞。分别以VP7p和VP2-A1、B1、C1蛋白作为包被抗原,利用间接ELISA方法进行Mc Ab的筛选。共获得5株稳定分泌抗25型BTV VP7的杂交瘤细胞株,分别命名为3H7、5F12、6E10、6G11和1C8;同时筛选出2株针对VP2蛋白的杂交瘤细胞株,分别命名为2E7和5B3。经抗体亚类试剂盒鉴定除1C8为Ig M外,其余各株皆为Ig G。2E7和5B3经抗体亚类试剂盒鉴定分别为Ig G1和IgG 2b。经连续传代并冻存复苏后,以上7株杂交瘤细胞皆可稳定分泌抗体,具有良好的稳定性。间接免疫荧光鉴定结果显示,3H7可与8型BTV发生特异性结合,其余各株皆不发生反应,说明该株单抗能够特异性地识别8型BTV,且不与赤羽病病毒(AKAV)、茨城病毒(IBAV)、猪轮状病毒(PRV)发生交叉反应,表明单抗具有良好的特异性。Western blot结果证明,3H7单抗能识别重组VP7蛋白;2E7和5B3可特异性识别重组VP2蛋白。抗原表位叠加试验结果表明,五株针对VP7蛋白的单克隆抗体,3H7、6E10和1C8针对不同的抗原表位,而5F12和6G11针对相同的抗原表位;两株针对VP2的单抗识别的是不同的抗原位点。重组VP7、VP2蛋白和相应的单克隆抗体为25型BTV血清学检测方法的建立及VP7和VP2蛋白的结构与功能研究奠定了物质基础。
[Abstract]:Bluetongue (BT) is a strong non contagious disease transmitted by vector (Bluetongue virus, BTV) in ruminant animals. The most sensitive.BT of pure breed fine wool sheep is first found in 1876, and is now separated to BTV in many tropical and temperate countries. And the distribution of the disease is constantly expanding, showing a global distribution trend. In 1979, China first discovered the existence of the disease. At present, 29 provinces in China have detected BTV positive infected animals. So far, 26 different serum types of BTV are isolated and no serotypes are intersected between different serotypes. The protective.BTV genome consists of 10 double stranded RNA, including a large fragment (L1-L3), a medium fragment (M4-M6) and a small fragment (S7-S10). Its co encoding 7 structural proteins (VP1-VP7) and 4 non structural proteins (NS1, NS2, NS3/NS3a and NS4) are the main structure proteins encoded by the S7 genes, which are composed of 349 amino acids and are located on the surface of the virus nucleocapsid. The total 36%.VP7 of the core protein of the virus is a group specific antigen of BTV. The homology of VP7 protein between different serotypes is high as 94%.VP2 is encoded by the L2 gene. The lowest conserved.VP2 protein among different serotype viruses is mainly involved in the adsorption and entry of the virus, and the.VP2 can induce neutralizing antibody with the virulence of the virus, which is the specificity of the BTV type. The main determinant of antigen.25 BTV is that the VP2 of type.25 BTV isolated from the blood samples of the Swiss goat in 2008 and other serotype virus VP2 is homologous only to the typical symptoms of 23%-79%. infected cattle with type 25 BTV, so it is often ignored. However, once the outbreak, the incidence and death rate are high. Therefore, the specificity of the type 25 BTV is established. The method of serological detection is of great significance for the prevention and control of BTV. The study is to prepare the VP7 and VP2 protein monoclonal antibodies (Monoclonal antibody, Mc Ab) of type 25 BTV, and to express VP7 proteins by the P ET-28a (+) and pGEX-6P-1 of the prokaryotic expression system. The expected protein size was the same. Three segments of VP2 protein were expressed as VP2-A, VP2-B, VP2-C and VP2-A1, VP2-B1, VP2-B1, VP2-C1. via SDS-PAGE and P MAL-c5X, respectively, using the prokaryotic expression system p ET-28a (+) and P MAL-c5X. Protein VP7a and recombinant VP2-A, B, C were used as immune antigens, BALB/c mice were immunized intraperitoneally, and cell fusion technique was used to fuse myeloma cells SP2/0 and spleen cells of mice immunized after immunization. Cell subclones were carried out by finite dilution method, and the clones that produced antibodies were obtained. VP7p and VP2-A1, B1, C1 protein were used as packets respectively. Mc Ab was screened by indirect ELISA method. A total of 5 hybridoma cells secreting stable BTV VP7 were obtained, respectively named 3H7,5F12,6E10,6G11 and 1C8, and 2 hybridoma cell lines for VP2 protein were selected, and the other strains were named 2E7 and 5B3. by antibody subclass kit, except 1C8 Ig. Ig G.2E7 and 5B3 were identified as Ig G1 and IgG 2b. after continuous passage and cryopreservation. The above 7 hybridoma cells were stable to secrete antibodies and had good stability. The results of indirect immunofluorescence identification showed that 3H7 could be combined with type 8 BTV, and the other strains did not react, indicating the strain of the strain. McAbs can specifically identify type 8 BTV, and do not cross reacted with Chek feather virus (AKAV), Ibaraki virus (IBAV) and porcine rotavirus (PRV). It shows that McAbs have good specific.Western blot results, and 3H7 McAbs can identify recombinant VP7 protein; 2E7 and 5B3 can specifically identify recombinant VP2 protein. The result table of epitope superposition test is the result of 2E7 and 5B3 Five monoclonal antibodies against VP7 protein, 3H7,6E10 and 1C8 against different antigen epitopes, and 5F12 and 6G11 against the same epitopes, and the identification of the two monoclonal antibodies against VP2 are different antigenic sites. The recombinant VP7, VP2 protein and corresponding monoclonal antibodies are the establishment of the 25 type BTV serological detection method and the VP7 and VP2 protein. The study of structure and function lays a material foundation.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
本文编号：2166409
[Abstract]:Bluetongue (BT) is a strong non contagious disease transmitted by vector (Bluetongue virus, BTV) in ruminant animals. The most sensitive.BT of pure breed fine wool sheep is first found in 1876, and is now separated to BTV in many tropical and temperate countries. And the distribution of the disease is constantly expanding, showing a global distribution trend. In 1979, China first discovered the existence of the disease. At present, 29 provinces in China have detected BTV positive infected animals. So far, 26 different serum types of BTV are isolated and no serotypes are intersected between different serotypes. The protective.BTV genome consists of 10 double stranded RNA, including a large fragment (L1-L3), a medium fragment (M4-M6) and a small fragment (S7-S10). Its co encoding 7 structural proteins (VP1-VP7) and 4 non structural proteins (NS1, NS2, NS3/NS3a and NS4) are the main structure proteins encoded by the S7 genes, which are composed of 349 amino acids and are located on the surface of the virus nucleocapsid. The total 36%.VP7 of the core protein of the virus is a group specific antigen of BTV. The homology of VP7 protein between different serotypes is high as 94%.VP2 is encoded by the L2 gene. The lowest conserved.VP2 protein among different serotype viruses is mainly involved in the adsorption and entry of the virus, and the.VP2 can induce neutralizing antibody with the virulence of the virus, which is the specificity of the BTV type. The main determinant of antigen.25 BTV is that the VP2 of type.25 BTV isolated from the blood samples of the Swiss goat in 2008 and other serotype virus VP2 is homologous only to the typical symptoms of 23%-79%. infected cattle with type 25 BTV, so it is often ignored. However, once the outbreak, the incidence and death rate are high. Therefore, the specificity of the type 25 BTV is established. The method of serological detection is of great significance for the prevention and control of BTV. The study is to prepare the VP7 and VP2 protein monoclonal antibodies (Monoclonal antibody, Mc Ab) of type 25 BTV, and to express VP7 proteins by the P ET-28a (+) and pGEX-6P-1 of the prokaryotic expression system. The expected protein size was the same. Three segments of VP2 protein were expressed as VP2-A, VP2-B, VP2-C and VP2-A1, VP2-B1, VP2-B1, VP2-C1. via SDS-PAGE and P MAL-c5X, respectively, using the prokaryotic expression system p ET-28a (+) and P MAL-c5X. Protein VP7a and recombinant VP2-A, B, C were used as immune antigens, BALB/c mice were immunized intraperitoneally, and cell fusion technique was used to fuse myeloma cells SP2/0 and spleen cells of mice immunized after immunization. Cell subclones were carried out by finite dilution method, and the clones that produced antibodies were obtained. VP7p and VP2-A1, B1, C1 protein were used as packets respectively. Mc Ab was screened by indirect ELISA method. A total of 5 hybridoma cells secreting stable BTV VP7 were obtained, respectively named 3H7,5F12,6E10,6G11 and 1C8, and 2 hybridoma cell lines for VP2 protein were selected, and the other strains were named 2E7 and 5B3. by antibody subclass kit, except 1C8 Ig. Ig G.2E7 and 5B3 were identified as Ig G1 and IgG 2b. after continuous passage and cryopreservation. The above 7 hybridoma cells were stable to secrete antibodies and had good stability. The results of indirect immunofluorescence identification showed that 3H7 could be combined with type 8 BTV, and the other strains did not react, indicating the strain of the strain. McAbs can specifically identify type 8 BTV, and do not cross reacted with Chek feather virus (AKAV), Ibaraki virus (IBAV) and porcine rotavirus (PRV). It shows that McAbs have good specific.Western blot results, and 3H7 McAbs can identify recombinant VP7 protein; 2E7 and 5B3 can specifically identify recombinant VP2 protein. The result table of epitope superposition test is the result of 2E7 and 5B3 Five monoclonal antibodies against VP7 protein, 3H7,6E10 and 1C8 against different antigen epitopes, and 5F12 and 6G11 against the same epitopes, and the identification of the two monoclonal antibodies against VP2 are different antigenic sites. The recombinant VP7, VP2 protein and corresponding monoclonal antibodies are the establishment of the 25 type BTV serological detection method and the VP7 and VP2 protein. The study of structure and function lays a material foundation.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
【参考文献】
相关期刊论文 前2条
1 花群义,徐自忠,周晓黎,董俊;竞争酶联免疫吸附试验检测蓝舌病抗体的研究[J];动物医学进展;2002年02期
2 宋红梅;杨涛;马健男;王群;张维军;徐青元;杨增岐;吴东来;;蓝舌病病毒VP7基因的原核表达[J];中国预防兽医学报;2009年12期
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