本文選題：藍(lán)舌病病毒 + 群特異性抗原��； 參考：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：藍(lán)舌病是由藍(lán)舌病病毒引起、侵害綿羊和牛等家畜、以高熱、白細(xì)胞減少、黏膜潰瘍性炎癥變化等為主要癥狀的反芻動物傳染病。該病是由庫蠓等昆蟲媒介傳播的非接觸性傳染病,純種細(xì)毛羊為最易感動物,平均死亡率為35%,最高可達(dá)80%。BTV血清型多達(dá)26個,且互相之間無有效保護(hù)作用。該病流行范圍廣,傳播速度快,2006年前BTV4在希臘、西班牙、法國、葡萄牙等國大面積流行;2006年后BTV8在歐洲多國大規(guī)模爆發(fā),造成了極大的經(jīng)濟(jì)損失。BTV分為10個節(jié)段,主要編碼7種結(jié)構(gòu)蛋白(VP1~VP7)和4種非結(jié)構(gòu)蛋白(NS1、NS2、NS3/NS3a和NS4)。VP7蛋白是一組特定的蛋白質(zhì)的BTV,S7基因編碼349個氨基酸,位于病毒的核衣殼表面,不同血清型的VP7蛋白超過94%保守的氨基酸之間;VP2蛋白是BTV的型特異性蛋白,由L2基因編碼,大小范圍在950到960個氨基酸之間,位于病毒外層衣殼,各血清型之間氨基酸序列變化范圍在22.7%到72.9%之間。本研究對8型及4型VP2蛋白和8型BTV的VP7蛋白進(jìn)行原核表達(dá),制備在此基礎(chǔ)上的單克抗隆體,為后期建立血清學(xué)診斷方法提供物質(zhì)基礎(chǔ),為口岸檢疫和防止新血清型流入我國提供技術(shù)儲備。根據(jù)Gen Bank中公布的BTV8 S7基因、BTV4 L2基因和BTV8 L2基因序列合成基因,設(shè)計引物將BTV4和BTV8的L2基因各分為4A、4B、4C和8A、8B、8C三段,進(jìn)行原核表達(dá)。分別將S7基因和L2分段基因克隆到原核表達(dá)載體p ET-28a(His標(biāo)簽)和p MAL-c5x(MBP標(biāo)簽)中構(gòu)建重組質(zhì)粒。對轉(zhuǎn)化到感受態(tài)細(xì)胞B21中陽性質(zhì)粒1.0mmol/L的IPTG對其進(jìn)行表達(dá)。通過檢測,重組蛋白His-VP7、His-4A、His-4B、His-4C、His-8A、His-8B、His-8C和MBP-VP7、MBP-4A、MBP-4B、MBP-4C、MBP-8A、MBP-8B、MBP-8C均已獲得表達(dá)。對帶有MBP標(biāo)簽的重組蛋白通過直鏈淀粉樹脂進(jìn)行純化,帶有His標(biāo)簽的重組蛋白用切膠純化的方法進(jìn)行純化。以帶有His標(biāo)簽的VP7和VP2蛋免白疫BALB/c小鼠,利用體細(xì)外胞合融技術(shù)將免疫后小鼠的細(xì)脾胞與SP2/0骨瘤髓細(xì)胞進(jìn)行細(xì)胞融合。以帶有MBP標(biāo)簽的重組純化后蛋白,創(chuàng)建間接ELISA方法對雜交瘤細(xì)胞進(jìn)行選擇性培養(yǎng)。共獲得了5株交雜瘤細(xì)能胞定穩(wěn)泌分抗BTV8VP7蛋單白抗體,依次為3D7、6B5、5C9、6D11和3F4;獲得3株定穩(wěn)分抗泌BTV4 VP2蛋白克單隆抗體的雜瘤交細(xì)胞1G7、8G3、5E6和2株穩(wěn)分定泌抗BTV8 VP2蛋克白單隆抗體的雜瘤交細(xì)胞2B8、3G11。Western blot證實所獲得單隆體均能與蛋白應(yīng)發(fā)重的生相組特異性反應(yīng),應(yīng)反性原較好。間接免疫熒光試驗和間接ELISA試驗結(jié)果表明,單抗3F4與BTV4發(fā)生特異性反應(yīng),與茨城病病毒、赤羽病病毒、豬輪狀病毒不發(fā)生交叉反應(yīng)。經(jīng)抗體亞類鑒定試劑盒鑒定,抗BTV8 VP7蛋白的單克隆抗體中,3D7、3F4、5C9、6D11的亞類均為Ig G1,6B5的亞類為Ig M;抗BTV4 VP2蛋白的單克隆抗體中,1G7、8G3的亞類為Ig G1,5E6的亞類為Ig G2b;對BTV8 VP2蛋白的單克隆抗體3G11,2B8是Ig G2b亞型。
[Abstract]:Bluetongue is a ruminant infectious disease caused by bluetongue virus, which affects livestock such as sheep and cattle and is characterized by high fever, leukopenia and mucosal ulcerative inflammation. The disease is a non-contact infectious disease transmitted by insect vectors such as Culicoides. Pure fine wool sheep are the most susceptible animals with an average mortality rate of 35 and a maximum of 26 serotypes of 80.BTV, and there is no effective protective effect between them. Before 2006, BTV4 was prevalent in Greece, Spain, France, Portugal and other countries. After 2006, BTV8 broke out in many countries in Europe, causing great economic loss. BTV was divided into 10 segments. VP7 mainly encodes seven structural proteins (VP1VP7) and four nonstructural proteins (NS1NS2NS3a and NS4) .VP7 protein is a specific group of proteins that encode 349 amino acids, located on the core-shell surface of the virus. VP7 proteins of different serotypes are more than 94% conserved amino acids. VP2 proteins are type-specific proteins of BTV. They are encoded by L2 gene and range in size from 950 to 960 amino acids, and are located in the outer capsid of the virus. The amino acid sequences of the serotypes ranged from 22.7% to 72.9%. In this study, prokaryotic expression of VP2 protein and VP7 protein of type 8 and 4 and BTV type 8 were carried out. For port quarantine and prevention of new serotypes into China to provide technical reserves. According to the sequence synthesis genes of BTV8S7 gene and BTV8L2 gene published in GenBank, the L2 gene of BTV4 gene and BTV8 gene were divided into three segments, 4AX4BN4C and 8Am8Bm8C, respectively, for prokaryotic expression. S7 gene and L2 segment gene were cloned into prokaryotic expression vector pET-28a (his tag) and pMAL-c5x (MBP tag) respectively to construct recombinant plasmid. The positive plasmid 1.0 mmol / L was expressed by IPTG. The expression of the recombinant protein His-VP7HIS-4AnHIS-4CHIS-8CU His-8BHN His-8BH8C and MBP-VP7 MBP-4AMAMBP-4BnMBP-4CnMBP-8BMBP-8C has been detected and the results show that the recombinant protein His-4BP7A, His-4B, His-4C, His-8B, His-8B, and MBP-8B, respectively, have been expressed. The recombinant protein with MBP label was purified by amylose resin, and the recombinant protein with his label was purified by cutting gel. The BALB / c mice immunized with his label VP7 and VP2 eggs were fused with SP2 / 0 osteoma myeloma cells. Using recombinant purified protein with MBP label, indirect Elisa was established for selective culture of hybridoma cells. A total of 5 strains of hybridoma were obtained, which were stable and secreted against BTV8VP7 egg monowhite antibody. 3D7B5B5C9C9D11 and 3F4 were obtained respectively, and three hybridoma hybrids, 1G7VP2 protein monoclonal antibody, and 2 hybridoma cells stably secreting anti-BTV8 VP2 protein monoclonal antibody 2B8G11.Western blot were obtained. The specific response of the hair weight biofacies group, It is better to have antigenicity. The results of indirect immunofluorescence assay and indirect Elisa showed that McAb 3F4 reacted specifically with BTV4, but did not cross react with Tarim disease virus, red feather virus and porcine rotavirus. The antibody subclass identification kit was used to identify, Among the monoclonal antibodies against BTV8 VP7 protein, the subclasses of 3D7F4F4F4C9C9D11 are all Ig G1O6B5, of the monoclonal antibodies against BTV4VP2 protein, the subclass of 1G7 / 8G3 is Ig G2b, and that of the monoclonal antibody 3G112B8 against BTV8 VP2 protein is Ig G2b subtype, and that of anti BTV8 VP2 protein subclass is Ig G2b subtype, and the subclass of anti BTV8 VP2 protein is Ig G2b subtype. The monoclonal antibody against BTV8 VP2 protein 3G112B8 is Ig G2b subtype.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.65

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