ADV全基因優(yōu)化核酸疫苗的構(gòu)建及其免疫效果的初步研究
發(fā)布時(shí)間：2018-09-04 08:24
【摘要】：水貂阿留申病(Aleutian disease,AD)是由水貂阿留申病毒(Aleutian disease virus,ADV)引起的一種侵害水貂免疫系統(tǒng)的疾病,又稱為漿細(xì)胞增多癥。由于其特殊的致病機(jī)理,導(dǎo)致目前尚無(wú)有效防治水貂阿留申病的疫苗。為了更好的研制有效對(duì)抗水貂阿留申病的疫苗,本實(shí)驗(yàn)以實(shí)驗(yàn)室所分離的水貂阿留申病強(qiáng)毒株DNA序列為模板,運(yùn)用重疊延伸PCR技術(shù),去除VP2基因中編碼428-446氨基酸的核苷酸序列,并與pcDNA3.1(+)載體連接,構(gòu)建全基因突變重組核酸疫苗pcDNA3.1-ADV-428;同時(shí)在去除428-446氨基酸序列的基礎(chǔ)上截去487-501氨基酸序列,構(gòu)建全基因突變重組核酸疫苗pcDNA3.1-ADV-428-487。將構(gòu)建的重組質(zhì)粒轉(zhuǎn)染至L929細(xì)胞中,通過(guò)間接免疫熒光(IFA)實(shí)驗(yàn)檢測(cè)其表達(dá)效果。選取健康小鼠分組,經(jīng)肌肉注射進(jìn)行免疫,用間接ELISA法檢測(cè)抗體;在第42天取免疫小鼠的脾細(xì)胞用流式細(xì)胞儀檢測(cè)、CD4+、CD8+和CD3+T淋巴細(xì)胞亞群;用酶聯(lián)免疫檢測(cè)試劑盒,檢測(cè)免疫后小鼠血清中IL-4、IL-10、IL-12和IFN-γ四種細(xì)胞因子的含量。最后實(shí)驗(yàn)結(jié)果表明所構(gòu)建的重組核酸疫苗具有良好的反應(yīng)原性和免疫原性。將以上所構(gòu)建的核酸疫苗經(jīng)肌肉注射接種到水貂體內(nèi)進(jìn)行免疫。免疫完成后對(duì)水貂進(jìn)行攻毒,然后每隔兩周對(duì)水貂進(jìn)行指尖采血。應(yīng)用流式細(xì)胞術(shù)檢測(cè)全血中的CD8+淋巴細(xì)胞亞群;應(yīng)用間接ELISA法檢測(cè)血清中的抗體水平;應(yīng)用血清蛋白電泳檢測(cè)血清中γ球蛋白占總蛋白的百分比以及應(yīng)用聚乙二醇沉淀比濁法檢測(cè)水貂血清中抗原抗體復(fù)合物的含量。實(shí)驗(yàn)結(jié)果表明pcDNA3.1-ADV-428和pcDNA3.1-ADV-428-487組在水貂試驗(yàn)中γ球蛋白、CIC以及CD8+T淋巴細(xì)胞的量都要低于對(duì)照組,且pcDNA3.1-ADV-428-487的保護(hù)效果要優(yōu)于pcDNA3.1-ADV-428,所構(gòu)建的核酸疫苗展現(xiàn)了良好的疫苗潛質(zhì)。
[Abstract]:Mink Aleutian disease (Aleutian disease,AD) is a disease of mink immune system caused by mink Aleutian virus (Aleutian disease virus,ADV), also known as plasmacytosis. Due to its special pathogenic mechanism, there is no effective vaccine against Aleutian disease in mink. In order to develop an effective vaccine against Aleutian disease in mink, the DNA sequence of Aleutian virulent strain isolated in the laboratory was used as template, and the nucleotide sequence encoding 428-446 amino acid in VP2 gene was removed by overlapping extension PCR technique. The recombinant nucleic acid vaccine pcDNA3.1-ADV-428; was constructed by ligating with pcDNA3.1 () vector. The 487-501 amino acid sequence was cut off on the basis of removing 428-446 amino acid sequence, and the recombinant nucleic acid vaccine pcDNA3.1-ADV-428-487. was constructed. The recombinant plasmid was transfected into L929 cells and its expression was detected by indirect immunofluorescence (IFA) assay. Healthy mice were divided into groups and immunized by intramuscular injection, antibody was detected by indirect ELISA method, spleen cells of immunized mice were detected by flow cytometry and CD3 T lymphocyte subsets were detected by flow cytometry on the 42nd day, Elisa kit was used. The levels of four cytokines, IL-4,IL-10,IL-12 and IFN- 緯, in serum of immunized mice were determined. The results show that the recombinant nucleic acid vaccine has good reactivity and immunogenicity. The nucleic acid vaccine was injected intramuscularly into mink for immunization. After immunization, the mink is poisoned, and the mink is collected at the fingertips every two weeks. CD8 lymphocyte subsets in whole blood were detected by flow cytometry, antibody levels in serum were detected by indirect ELISA method. Serum protein electrophoresis was used to detect the percentage of 緯 globulin to total protein and polyethylene glycol precipitation turbidimetry was used to detect the content of antigen-antibody complex in mink serum. The results showed that the levels of 緯 globulin CIC and CD8 T lymphocytes in pcDNA3.1-ADV-428 and pcDNA3.1-ADV-428-487 groups were lower than those in the control group, and the protective effect of pcDNA3.1-ADV-428-487 was better than that of nucleic acid vaccine constructed by pcDNA3.1-ADV-428,.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.92
本文編號(hào)：2221538
[Abstract]:Mink Aleutian disease (Aleutian disease,AD) is a disease of mink immune system caused by mink Aleutian virus (Aleutian disease virus,ADV), also known as plasmacytosis. Due to its special pathogenic mechanism, there is no effective vaccine against Aleutian disease in mink. In order to develop an effective vaccine against Aleutian disease in mink, the DNA sequence of Aleutian virulent strain isolated in the laboratory was used as template, and the nucleotide sequence encoding 428-446 amino acid in VP2 gene was removed by overlapping extension PCR technique. The recombinant nucleic acid vaccine pcDNA3.1-ADV-428; was constructed by ligating with pcDNA3.1 () vector. The 487-501 amino acid sequence was cut off on the basis of removing 428-446 amino acid sequence, and the recombinant nucleic acid vaccine pcDNA3.1-ADV-428-487. was constructed. The recombinant plasmid was transfected into L929 cells and its expression was detected by indirect immunofluorescence (IFA) assay. Healthy mice were divided into groups and immunized by intramuscular injection, antibody was detected by indirect ELISA method, spleen cells of immunized mice were detected by flow cytometry and CD3 T lymphocyte subsets were detected by flow cytometry on the 42nd day, Elisa kit was used. The levels of four cytokines, IL-4,IL-10,IL-12 and IFN- 緯, in serum of immunized mice were determined. The results show that the recombinant nucleic acid vaccine has good reactivity and immunogenicity. The nucleic acid vaccine was injected intramuscularly into mink for immunization. After immunization, the mink is poisoned, and the mink is collected at the fingertips every two weeks. CD8 lymphocyte subsets in whole blood were detected by flow cytometry, antibody levels in serum were detected by indirect ELISA method. Serum protein electrophoresis was used to detect the percentage of 緯 globulin to total protein and polyethylene glycol precipitation turbidimetry was used to detect the content of antigen-antibody complex in mink serum. The results showed that the levels of 緯 globulin CIC and CD8 T lymphocytes in pcDNA3.1-ADV-428 and pcDNA3.1-ADV-428-487 groups were lower than those in the control group, and the protective effect of pcDNA3.1-ADV-428-487 was better than that of nucleic acid vaccine constructed by pcDNA3.1-ADV-428,.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.92
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