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  本文選題：犬細小病毒　切入點：VP2蛋白　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：犬細小病毒病是由犬細小病毒(Canine parvovirus, CPV)引起的傳染犬科動物的,高度接觸性、致死性疾病,以出血性腸炎和亞急性心肌炎為特征。犬細小病毒病主要通過糞-口途徑傳播,幼犬最易感染。目前,犬細小病毒病已經(jīng)在我國廣泛流行,嚴重威脅著我國的養(yǎng)犬業(yè)和皮毛動物養(yǎng)殖業(yè)。疫苗接種是防控犬細小病毒病的最有效方法。犬細小病毒病滅活疫苗只能誘導(dǎo)一個短期的免疫反應(yīng)；由于新的抗原變異毒株的出現(xiàn),弱毒疫苗接種有時會出現(xiàn)免疫失敗。隨著反向遺傳技術(shù)的發(fā)展和成熟,單股負鏈RNA病毒成為了疫苗載體的研究熱點。犬瘟熱(Canine distemper, CD)是由犬瘟熱病毒(Canine distemper virus, CDV)引發(fā)的感染多種動物的急性致死性傳染病,給我國的犬養(yǎng)殖業(yè)和毛皮動物業(yè)造成了嚴重的損失。目前,全世界應(yīng)用最為廣泛的犬瘟熱疫苗是弱毒疫苗。利用犬瘟熱弱毒疫苗為載體,研制犬細小病毒病、犬瘟熱二聯(lián)活載體疫苗,可實現(xiàn)一苗兩防,降低疫苗生產(chǎn)成本,也可針對新出現(xiàn)的抗原變異亞型毒株快速地進行疫苗更新。按照哺乳動物的密碼子偏好性對外源抗原蛋白基因進行密碼子優(yōu)化,能夠提高該蛋白在哺乳動物體內(nèi)或細胞內(nèi)的表達量,為提高疫苗的免疫效力提供了新的方法。本研究,利用本實驗已建立的CD弱毒疫苗株CDV/R-20/8的反向遺傳技術(shù)平臺,構(gòu)建了表達密碼子優(yōu)化CPV VP2蛋白的重組犬瘟熱病毒r20/8-CPVVP2opti。免疫熒光實驗和Western blot實驗表明,重組病毒r20/8-CPVVP2opti感染的BHK-21細胞中,CPV VP2蛋白得到了正確表達。重組病毒r20/8-CPVVP2opti保持了親本毒株r20/8在Vero細胞上的生長特性,經(jīng)實驗測定具有生長滴度高的特點,其最高生長滴度可達到106.75 TCID50/ml。Western blot證實,密碼子優(yōu)化可顯著提高CPVVP2蛋白在重組犬瘟熱病毒r20/8-CPVVP2opti感染細胞中的表達量。以表達密碼子優(yōu)化CPVVP2蛋白重組犬瘟熱病毒r20/8-CPVVP2opti表達野生型CPVVP2蛋白重組犬瘟熱病毒r20/8-CPVVP2和親本毒株r20/8分別按104.5TCID50/100μl/只的劑量以肌肉注射的方式,兩次免疫接種小鼠。r20/8-CPVVP2opti、r20/8-CPVVP2r20/8免疫小鼠,均誘導(dǎo)低水平的CDV中和抗體反應(yīng),且3組免疫小鼠血清中CDV中和抗體滴度無顯著性差異；然而,r20/8-CPVVP2opti、r20/8-CPVVP2免疫小鼠,均可誘導(dǎo)顯著的CPV血凝抑制(HI)抗體反應(yīng),加強免疫后1周,CPV HI抗體反應(yīng)顯著增強,且r20/8-CPVVP2opti免疫小鼠血清中CPV HI抗體滴度顯著地高于r20/8-CPVVP2免疫小鼠免疫小鼠血清中CPV HI抗體滴度。結(jié)果表明,利用密碼子優(yōu)化CPV VP2蛋白基因構(gòu)建CPV、CDV活載體疫苗,能增強在免疫動物體內(nèi)誘導(dǎo)的CPV HI抗體反應(yīng),有助于提高該疫苗抗CPV感染的免疫效力。本研究結(jié)果表明,表達密碼子優(yōu)化CPV VP2蛋白重組CDVr20/8-CPVVP2opti具有良好的免疫原性,具有作為預(yù)防犬細小病毒病和犬瘟熱二聯(lián)活載體疫苗的潛力。
[Abstract]:Canine parvovirus disease (CPV) is a highly contagious and fatal disease of Canine parvovirus (CPV), characterized by hemorrhagic enteritis and subacute myocarditis. Puppies are most susceptible to infection. At present, canine parvovirus disease has been widespread in China. Vaccination is the most effective method to prevent and control canine parvovirus disease. Inactivated canine parvovirus vaccine can only induce a short-term immune response. As a result of the emergence of new antigenic variant strains, attenuated vaccines sometimes fail. As reverse genetic technology develops and matures, Single strand negative strand RNA virus has become a hot topic in vaccine vector. Canine distemper (CDV) is an acute fatal infectious disease caused by canine distemper virus (CDV). At present, the most widely used canine distemper vaccine in the world is attenuated virus vaccine. Using canine distemper attenuated vaccine as carrier, canine parvovirus disease is developed. Canine distemper binomial live vector vaccine can realize one vaccine and two prevention, and reduce the cost of vaccine production. It is also possible to quickly update the vaccine against the emerging antigen variant subtype strain, and to optimize the codon of the foreign antigen protein gene according to the codon preference of the mammalian codon. It can increase the expression of the protein in mammalian or in cells, and provide a new method to improve the immune potency of the vaccine. In this study, the reverse genetic technology platform of CD attenuated vaccine strain CDV/R-20/8 has been established in this study. Recombinant canine distemper virus r20 / 8-CPVVP2opti.Immunofluorescence and Western blot experiments showed that the recombinant canine distemper virus r20 / 8-CPVP2optivirus was constructed to express codon to optimize CPV VP2 protein. The recombinant virus r20/8-CPVVP2opti maintained the growth characteristics of the parent strain R20 / 8 on Vero cells and had the characteristics of high growth titer. The highest growth titer can reach 106.75 TCID50/ml.Western blot. Codon optimization can significantly increase the expression of CPVVP2 protein in recombinant canine distemper virus r20/8-CPVVP2opti infected cells. The expression codon optimizes the expression of CPVVP2 protein recombinant canine distemper virus r20/8-CPVVP2opti expression wild-type CPVVP2 protein recombinant canine distemper virus r20/8-CPVVP2 and parent. Strain R20 / 8 was injected intramuscularly at the dose of 104.5TCID50/100 渭 l / mouse, The mice inoculated twice with. R20 / 8-CPVP2optir 20 / 8-CPVP2P2r20 / 8 induced a low level of CDV neutralizing antibody reaction, and there was no significant difference in the titer of CDV neutralizing antibodies in the serum of the three groups of immunized mice; however, R208-CPVP2optir208-CPVP2optir20 / 8-CPVP2VP2 immunized mice. CPV hemagglutination inhibited the anti-HI antibody response significantly, and the HI antibody response was significantly increased at 1 week after enhanced immunization. The titer of CPV HI antibody in the serum of r20/8-CPVVP2opti immunized mice was significantly higher than that of r20/8-CPVVP2 immunized mice. The results showed that the codon optimized CPV VP2 protein gene was used to construct the live vector vaccine. The results showed that the expression codon optimized CPV VP2 protein recombinant CDVr20/8-CPVVP2opti had good immunogenicity. It has the potential as a bivector vaccine against canine parvovirus and canine distemper.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S858.292

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相關(guān)期刊論文 前1條

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本文編號：1665061