25型藍舌病病毒VP7和VP2蛋白的原核表達及其單克隆抗體的制備
發(fā)布時間：2018-08-05 16:57
【摘要】：藍舌病(Bluetongue,BT)是由藍舌病病毒(Bluetongue virus,BTV)引起的,依靠媒介昆蟲(庫蠓、伊蚊等)在反芻動物之間傳播的一種烈性非接觸性的傳染病。純種細毛羊?qū)υ摬∽蠲舾小T最早于1876年被發(fā)現(xiàn),目前在熱帶和溫帶的多個國家都已分離到BTV,并且該病的分布范圍在不斷的擴大,呈現(xiàn)全球性分布的趨勢。我國于1979年第一次發(fā)現(xiàn)該病的存在,目前在我國29個省份已檢測到感染BTV陽性的病畜。迄今為止,全世界范圍內(nèi)共分離到26種不同血清型的BTV,且不同的血清型之間無交叉保護作用。BTV基因組由10個雙鏈RNA組成,包括大片段(L1-L3)、中片段(M4-M6)和小片段(S7-S10)。其共編碼7個結(jié)構(gòu)蛋白(VP1-VP7)和4種非結(jié)構(gòu)蛋白(NS1、NS2、NS3/NS3a和NS4)。VP7是由S7基因編碼的主要結(jié)構(gòu)蛋白,由349個氨基酸組成,位于病毒核衣殼的表面,約占病毒核心蛋白總量36%。VP7是BTV的群特異性抗原,不同血清型之間的VP7蛋白的同源性高達94%。VP2由L2基因編碼,在不同血清型病毒間保守性最低。VP2蛋白主要參與病毒的吸附和進入,與病毒的毒力有關(guān)。VP2可誘導(dǎo)產(chǎn)生中和抗體,是BTV型特異性抗原主要的決定因素。25型BTV是2008年從瑞士山羊血液樣本中分離獲得。25型BTV的VP2與其他血清型病毒VP2的同源性僅為23%-79%。因感染25型BTV的牲畜無典型癥狀,故常被忽略。但是一旦爆發(fā),發(fā)病率和死亡率較高。因此建立針對25型BTV的特異性血清學檢測方法對BTV的防控有重要的意義。本研究為制備25型BTV的VP7和VP2蛋白單克隆抗體(Monoclonal antibody,Mc Ab),利用原核表達系統(tǒng)p ET-28a(+)和pGEX-6P-1表達VP7蛋白,分別命名為VP7a和VP7p。經(jīng)SDS-PAGE和Western blot分析大小依次為44 kDa和64 k Da,與預(yù)期蛋白大小一致。采用原核表達系統(tǒng)p ET-28a(+)和p MAL-c5X部分重疊表達三段VP2蛋白,分別命名為VP2-A、VP2-B、VP2-C和VP2-A1、VP2-B1、VP2-C1。經(jīng)SDS-PAGE和Western blot分析,大小依次為50k Da、48k Da、48k Da、84 kDa、82 kDa和82 k Da,與預(yù)期蛋白大小一致。分別將重組蛋白VP7a和重組VP2-A、B、C作為免疫抗原,腹腔免疫BALB/c小鼠,采用細胞融合技術(shù)將骨髓瘤細胞SP2/0與免疫后小鼠的脾細胞進行細胞融合,通過有限稀釋法進行細胞亞克隆,獲得穩(wěn)定產(chǎn)生抗體的雜交瘤細胞。分別以VP7p和VP2-A1、B1、C1蛋白作為包被抗原,利用間接ELISA方法進行Mc Ab的篩選。共獲得5株穩(wěn)定分泌抗25型BTV VP7的雜交瘤細胞株,分別命名為3H7、5F12、6E10、6G11和1C8;同時篩選出2株針對VP2蛋白的雜交瘤細胞株,分別命名為2E7和5B3。經(jīng)抗體亞類試劑盒鑒定除1C8為Ig M外,其余各株皆為Ig G。2E7和5B3經(jīng)抗體亞類試劑盒鑒定分別為Ig G1和IgG 2b。經(jīng)連續(xù)傳代并凍存復(fù)蘇后,以上7株雜交瘤細胞皆可穩(wěn)定分泌抗體,具有良好的穩(wěn)定性。間接免疫熒光鑒定結(jié)果顯示,3H7可與8型BTV發(fā)生特異性結(jié)合,其余各株皆不發(fā)生反應(yīng),說明該株單抗能夠特異性地識別8型BTV,且不與赤羽病病毒(AKAV)、茨城病毒(IBAV)、豬輪狀病毒(PRV)發(fā)生交叉反應(yīng),表明單抗具有良好的特異性。Western blot結(jié)果證明,3H7單抗能識別重組VP7蛋白;2E7和5B3可特異性識別重組VP2蛋白�？乖砦化B加試驗結(jié)果表明,五株針對VP7蛋白的單克隆抗體,3H7、6E10和1C8針對不同的抗原表位,而5F12和6G11針對相同的抗原表位;兩株針對VP2的單抗識別的是不同的抗原位點。重組VP7、VP2蛋白和相應(yīng)的單克隆抗體為25型BTV血清學檢測方法的建立及VP7和VP2蛋白的結(jié)構(gòu)與功能研究奠定了物質(zhì)基礎(chǔ)。
[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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65
本文編號：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.
【學位授予單位】：東北農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65
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相關(guān)期刊論文 前2條
1 花群義,徐自忠,周曉黎,董俊;競爭酶聯(lián)免疫吸附試驗檢測藍舌病抗體的研究[J];動物醫(yī)學進展;2002年02期
2 宋紅梅;楊濤;馬健男;王群;張維軍;徐青元;楊增岐;吳東來;;藍舌病病毒VP7基因的原核表達[J];中國預(yù)防獸醫(yī)學報;2009年12期
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