犬瘟熱病毒H基因與犬IL-6融合基因的克隆與原核表達(dá)
發(fā)布時間：2018-11-10 23:25
【摘要】：犬瘟熱(Canine distemper)是由犬瘟熱病毒(CDV)引起的一種高度烈性傳染病,呈現(xiàn)典型的雙相熱型,主要侵害呼吸系統(tǒng)、消化系統(tǒng)和神經(jīng)系統(tǒng),表現(xiàn)為眼、鼻分泌物轉(zhuǎn)化為粘液性或者膿性,肺部聽診呼吸音粗歷等卡他性炎癥,以及非化膿性腦膜-脊髓炎,并且其癥狀幾乎為90%以上都會有,死亡率也高達(dá)6 0%-8 0%,并且該病易和其他病毒、細(xì)菌混合感染,造成大量死亡,因此常被稱為“毀滅性傳染病”。目前本病遍布世界各地,造成巨大的經(jīng)濟(jì)損失。近年來,隨著生態(tài)環(huán)境發(fā)生改變、病毒也變異進(jìn)化,導(dǎo)致CDV可感染犬科、鼬科、浣熊科、貓科、大熊貓科(貓除外)等多種動物,是當(dāng)前危害養(yǎng)殖業(yè)和野生動物保護(hù)業(yè)的最大疫病之一。CDV基因組編碼核衣殼蛋白(N)、基質(zhì)膜蛋白(M)、磷蛋白(P)、大蛋白(L)、融合蛋白(F)和血凝蛋白(H)6個蛋白,其中,H蛋白基因由1947個核糖核甘酸組成,含有一個開放性閱讀框架,其編碼的H蛋白為CDV的膜表面主要糖蛋白之一,可以誘導(dǎo)機(jī)體產(chǎn)生中和抗體,在免疫中起著重要的作用,并且能吸附和識別細(xì)胞表面受體,并能協(xié)助F蛋白使CDV的囊膜與細(xì)胞膜發(fā)生融合,進(jìn)而侵入宿主細(xì)胞,此外,H蛋白含有多個細(xì)胞毒性T淋巴細(xì)胞(CTL)表位,可產(chǎn)生特異的CTL活性,可作為預(yù)防CD基因工程疫苗中的靶基因。白細(xì)胞介素-6(IL-6)是一種多功能的細(xì)胞因子,能激活靶基因,還可以作為造血源細(xì)胞、T細(xì)胞、B細(xì)胞、內(nèi)皮細(xì)胞、破骨細(xì)胞等的分化和生長因子,增強(qiáng)自然殺傷細(xì)胞(NK)的殺傷活性等,在機(jī)體的免疫中具有重要作用,是一類天然的免疫增強(qiáng)劑。在醫(yī)學(xué)領(lǐng)域商品化的人IL-6之已經(jīng)用于治療過敏性疾病、感染性疾病和腫瘤疾病等的治療。由于IL-6不會產(chǎn)生免疫原性,不會發(fā)生自身免疫性疾病,克服了傳統(tǒng)免疫佐劑一大缺點(diǎn)。所以可以將IL-6基因克隆到載體中,做為一種高效、安全、經(jīng)濟(jì)的新型免疫佐劑,其前景非常樂觀。根據(jù)發(fā)表的犬瘟熱病毒的基因序列設(shè)計(jì)一對引物,用犬瘟熱病毒株感染Vero細(xì)胞收集病毒,用逆轉(zhuǎn)錄PCR(RT-PCR)方法擴(kuò)增出H基因序列大小為1125bp的基因片段。擴(kuò)增產(chǎn)物連接到pMD18-T載體中,轉(zhuǎn)化,提取質(zhì)粒酶切鑒定、PCR分析,重組質(zhì)粒進(jìn)行測序,結(jié)果顯示獲得基因序列與GenBanK中登錄的H基因相似性為100%。再從健康犬血提取淋巴細(xì)胞,分離犬IL-6RNA,構(gòu)建重組質(zhì)粒pMD-IL-6,酶切鑒定,PCR分析,重組質(zhì)粒測序,結(jié)果顯示重組質(zhì)粒與GenBanK中登錄的IL-6基因相似性高達(dá)98%。將重組質(zhì)粒pMD-H和pMD-IL-6同時酶切后,構(gòu)建重組表達(dá)質(zhì)粒pMD-H-IL-6,酶切鑒定,PCR分析,重組質(zhì)粒測序分析結(jié)果為1758bp,將重組質(zhì)粒轉(zhuǎn)化進(jìn)宿主菌,收集菌液進(jìn)行SDS-PAGE和Western-blot等分析檢測,結(jié)果表明CDV H基因可以高效表達(dá),最終表達(dá)目的蛋白分子量約為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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65
本文編號：2323867
[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.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65
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