犬瘟热病毒H基因与犬IL-6融合基因的克隆与原核表达

发布时间：2018-11-10 23:25

【摘要】：犬瘟热(Canine distemper)是由犬瘟热病毒(CDV)引起的一种高度烈性传染病,呈现典型的双相热型,主要侵害呼吸系统、消化系统和神经系统,表现为眼、鼻分泌物转化为粘液性或者脓性,肺部听诊呼吸音粗历等卡他性炎症,以及非化脓性脑膜-脊髓炎,并且其症状几乎为90%以上都会有,死亡率也高达6 0%-8 0%,并且该病易和其他病毒、细菌混合感染,造成大量死亡,因此常被称为“毁灭性传染病”。目前本病遍布世界各地,造成巨大的经济损失。近年来,随着生态环境发生改变、病毒也变异进化,导致CDV可感染犬科、鼬科、浣熊科、猫科、大熊猫科(猫除外)等多种动物,是当前危害养殖业和野生动物保护业的最大疫病之一。CDV基因组编码核衣壳蛋白(N)、基质膜蛋白(M)、磷蛋白(P)、大蛋白(L)、融合蛋白(F)和血凝蛋白(H)6个蛋白,其中,H蛋白基因由1947个核糖核甘酸组成,含有一个开放性阅读框架,其编码的H蛋白为CDV的膜表面主要糖蛋白之一,可以诱导机体产生中和抗体,在免疫中起着重要的作用,并且能吸附和识别细胞表面受体,并能协助F蛋白使CDV的囊膜与细胞膜发生融合,进而侵入宿主细胞,此外,H蛋白含有多个细胞毒性T淋巴细胞(CTL)表位,可产生特异的CTL活性,可作为预防CD基因工程疫苗中的靶基因。白细胞介素-6(IL-6)是一种多功能的细胞因子,能激活靶基因,还可以作为造血源细胞、T细胞、B细胞、内皮细胞、破骨细胞等的分化和生长因子,增强自然杀伤细胞(NK)的杀伤活性等,在机体的免疫中具有重要作用,是一类天然的免疫增强剂。在医学领域商品化的人IL-6之已经用于治疗过敏性疾病、感染性疾病和肿瘤疾病等的治疗。由于IL-6不会产生免疫原性,不会发生自身免疫性疾病,克服了传统免疫佐剂一大缺点。所以可以将IL-6基因克隆到载体中,做为一种高效、安全、经济的新型免疫佐剂,其前景非常乐观。根据发表的犬瘟热病毒的基因序列设计一对引物,用犬瘟热病毒株感染Vero细胞收集病毒,用逆转录PCR(RT-PCR)方法扩增出H基因序列大小为1125bp的基因片段。扩增产物连接到pMD18-T载体中,转化,提取质粒酶切鉴定、PCR分析,重组质粒进行测序,结果显示获得基因序列与GenBanK中登录的H基因相似性为100%。再从健康犬血提取淋巴细胞,分离犬IL-6RNA,构建重组质粒pMD-IL-6,酶切鉴定,PCR分析,重组质粒测序,结果显示重组质粒与GenBanK中登录的IL-6基因相似性高达98%。将重组质粒pMD-H和pMD-IL-6同时酶切后,构建重组表达质粒pMD-H-IL-6,酶切鉴定,PCR分析,重组质粒测序分析结果为1758bp,将重组质粒转化进宿主菌,收集菌液进行SDS-PAGE和Western-blot等分析检测,结果表明CDV H基因可以高效表达,最终表达目的蛋白分子量约为69.5KD。
[Abstract]:Canine distemper (Canine distemper) is a highly infectious disease caused by canine distemper virus (CDV). Nasal secretions are converted into mucous or purulent, lung auscultation, and other cataracts, as well as non-suppurative meningeomyelitis. The symptoms are almost 90% or more, and the mortality rate is as high as 60% -8.0%. And the disease is easily mixed with other viruses and bacteria, resulting in mass deaths, and is often referred to as a "devastating infectious disease." At present, the disease is spread all over the world, causing huge economic losses. In recent years, as the ecological environment has changed, the virus has mutated and evolved, causing CDV to infect a variety of animals, such as the canine family, the ferret family, the raccoon family, the cat family, and the giant panda family (except cats). CDV genome encodes nucleocapsid protein (N), matrix membrane protein (M), phosphorous protein (P), large protein (L), The fusion protein (F) and hemagglutinin (H) are 6 proteins, of which H protein gene is composed of 1 447 ribose ribonucleic acid and contains an open reading frame. The H protein encoded by the fusion protein is one of the main glycoproteins on the membrane surface of CDV. It can induce the body to produce neutralizing antibodies, play an important role in immunity, and can adsorb and recognize cell surface receptors, and can assist F protein to fuse the envelope of CDV with cell membrane and then invade host cells. The H protein contains multiple cytotoxic T lymphocyte (CTL) epitopes, which can produce specific CTL activity and can be used as a target gene for the prevention of CD gene engineering vaccine. Interleukin-6 (IL-6) is a multifunctional cytokine that activates target genes and can also be used as a differentiation and growth factor in hematopoietic cells, T cells, B cells, endothelial cells, osteoclasts, etc. Enhancing the killing activity of natural killer cell (NK), which plays an important role in the body's immunity, is a kind of natural immune enhancer. Commercialized human IL-6 in medicine has been used to treat allergic, infectious and tumor diseases. Because IL-6 does not produce immunogenicity and autoimmune disease, it overcomes a big shortcoming of traditional immune adjuvant. Therefore, IL-6 gene can be cloned into the vector as a new immune adjuvant with high efficiency, safety and economy. According to the published gene sequence of canine distemper virus, a pair of primers were designed. The virus was collected from Vero cells infected with canine distemper virus strain. The H gene fragment was amplified by reverse transcription PCR (RT-PCR). The amplified product was ligated into pMD18-T vector, transformed, digested and identified by extracting plasmid, analyzed by PCR, sequenced the recombinant plasmid. The result showed that the similarity between the obtained gene sequence and H gene registered in GenBanK was 100. Then the lymphocytes were extracted from healthy dog blood, the recombinant plasmid pMD-IL-6, was digested and identified by PCR analysis, and the recombinant plasmid was sequenced. The results showed that the similarity between the recombinant plasmid and the IL-6 gene registered in GenBanK was as high as 98%. After the recombinant plasmid pMD-H and pMD-IL-6 were digested at the same time, the recombinant expression plasmid pMD-H-IL-6, was digested and identified. The result of PCR analysis and the sequencing analysis of the recombinant plasmid was 1758bp. the recombinant plasmid was transformed into the host bacteria. The results of SDS-PAGE and Western-blot analysis showed that the CDV H gene could be expressed efficiently, and the molecular weight of the target protein was about 69.5 KD.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65

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