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：2018-06-22 16:46

本文选题：蓝舌病病毒 + 群特异性抗原　；参考：《东北农业大学》2015年硕士论文

【摘要】：蓝舌病是由蓝舌病病毒引起、侵害绵羊和牛等家畜、以高热、白细胞减少、黏膜溃疡性炎症变化等为主要症状的反刍动物传染病。该病是由库蠓等昆虫媒介传播的非接触性传染病,纯种细毛羊为最易感动物,平均死亡率为35%,最高可达80%。BTV血清型多达26个,且互相之间无有效保护作用。该病流行范围广,传播速度快,2006年前BTV4在希腊、西班牙、法国、葡萄牙等国大面积流行;2006年后BTV8在欧洲多国大规模爆发,造成了极大的经济损失。BTV分为10个节段,主要编码7种结构蛋白(VP1~VP7)和4种非结构蛋白(NS1、NS2、NS3/NS3a和NS4)。VP7蛋白是一组特定的蛋白质的BTV,S7基因编码349个氨基酸,位于病毒的核衣壳表面,不同血清型的VP7蛋白超过94%保守的氨基酸之间;VP2蛋白是BTV的型特异性蛋白,由L2基因编码,大小范围在950到960个氨基酸之间,位于病毒外层衣壳,各血清型之间氨基酸序列变化范围在22.7%到72.9%之间。本研究对8型及4型VP2蛋白和8型BTV的VP7蛋白进行原核表达,制备在此基础上的单克抗隆体,为后期建立血清学诊断方法提供物质基础,为口岸检疫和防止新血清型流入我国提供技术储备。根据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,设计引物将BTV4和BTV8的L2基因各分为4A、4B、4C和8A、8B、8C三段,进行原核表达。分别将S7基因和L2分段基因克隆到原核表达载体p ET-28a(His标签)和p MAL-c5x(MBP标签)中构建重组质粒。对转化到感受态细胞B21中阳性质粒1.0mmol/L的IPTG对其进行表达。通过检测,重组蛋白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均已获得表达。对带有MBP标签的重组蛋白通过直链淀粉树脂进行纯化,带有His标签的重组蛋白用切胶纯化的方法进行纯化。以带有His标签的VP7和VP2蛋免白疫BALB/c小鼠,利用体细外胞合融技术将免疫后小鼠的细脾胞与SP2/0骨瘤髓细胞进行细胞融合。以带有MBP标签的重组纯化后蛋白,创建间接ELISA方法对杂交瘤细胞进行选择性培养。共获得了5株交杂瘤细能胞定稳泌分抗BTV8VP7蛋单白抗体,依次为3D7、6B5、5C9、6D11和3F4;获得3株定稳分抗泌BTV4 VP2蛋白克单隆抗体的杂瘤交细胞1G7、8G3、5E6和2株稳分定泌抗BTV8 VP2蛋克白单隆抗体的杂瘤交细胞2B8、3G11。Western blot证实所获得单隆体均能与蛋白应发重的生相组特异性反应,应反性原较好。间接免疫荧光试验和间接ELISA试验结果表明,单抗3F4与BTV4发生特异性反应,与茨城病病毒、赤羽病病毒、猪轮状病毒不发生交叉反应。经抗体亚类鉴定试剂盒鉴定,抗BTV8 VP7蛋白的单克隆抗体中,3D7、3F4、5C9、6D11的亚类均为Ig G1,6B5的亚类为Ig M;抗BTV4 VP2蛋白的单克隆抗体中,1G7、8G3的亚类为Ig G1,5E6的亚类为Ig G2b;对BTV8 VP2蛋白的单克隆抗体3G11,2B8是Ig G2b亚型。
[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.
【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】