牛分结核枝杆菌ag85b基因重组腺病毒的构建及其实验免疫研究
发布时间：2018-08-01 16:57
【摘要】：实验目的:牛结核病(Bovine tuberculosis,bTB)一直以来在世界范围内使临床医学、社会和经济领域承受着巨大的压力。卡介苗(Bacille Calmette-Guerin,BCG)作为预防牛结核病的传统疫苗有着很好的免疫效果,而最近有研究表明该疫苗只对年幼的动物体具有免疫保护作用而对成年动物免疫保护效果很弱,因此开发新型高效bTB疫苗已经迫在眉睫。Ag85B是牛结核分枝杆菌早期分泌的主要蛋白,可以诱导机体同时产生体液免疫和细胞免疫,因此将ag85b基因作为本研究的靶基因对研究bTB基因工程疫苗具有重要的意义。人5型腺病毒作为一种外源基因的表达载体在基因治疗疫苗的研究领域一直以来有着非常广泛的应用。重组腺病毒载体去掉了基因组中早期转录区域的E1区而成为复制缺陷型的腺病毒表达载体,因此只能在含有E1区的AD293互补细胞中进行繁殖。在CMV启动子的作用下,将外源基因插入到腺病毒载体上并在互补细胞系中整合到腺病毒载体基因组中,最终重组腺病毒可以大量表达目的蛋白。因而此类疫苗目前被认为是一种安全而高效的bTB活病毒疫苗。实验方法:以质粒pGEM-85bL作为PCR模板,对目的基因Ag85b进行PCR扩增并连接到腺病毒穿梭载体pacAd5CMVK-NpA上得到重组腺病毒穿梭质粒我们命名为pacAd5CMV-ag85b,将其与腺病毒骨架质粒pacAd5 9.2-100用PacⅠ限制性内切酶线性化后在转染试剂X-tremeGENE HP的作用下共转染到AD293细胞里面进行重组腺病毒的包装。包装成功的重组腺病毒我们命名为rAd5-CMV-ag85b并对其基因组进行PCR鉴定。大量扩增rAd5-CMV-Ag85b后用腺病毒纯化试剂盒对其纯化,TCID50病毒滴度测定方法测定纯化后的rAd5-CMV-ag85b病毒滴度。间接免疫荧光试验(IFA)和反转录PCR试验(RT-PCR)进一步检测目的基因ag85b在体外的表达情况及生物学活性。最后将rAd5-CMV-ag85b、空载腺病毒rAd5-CMV-NpA、0.9%NaCl和BCG以多点肌肌肉注射的方式对6周龄雄性BALB/c小白鼠进行免疫,两周后加强免疫,再过两周利用流式细胞仪对各组免疫小鼠体内的CD3+(PE标记)、CD4+(PE标记)、CD8+(FITC标记)T淋巴细胞和IL-2(APC标记)、TNF-α(PE标记)、IFN-γ(FITC标记)细胞因子进行检测,同时利用间接酶联免疫吸附实验(ELISA)对免疫小鼠血清内的特异性免疫球蛋白IgG进行检测。实验结果:1、获得目的基因ag85b,成功构建重组腺病毒穿梭载体pacAd5CMV-ag85b,并在AD293细胞内成功包装出了重组腺病毒rAd5-CMV-ag85b;2、大量扩增rAd5-CMV-ag85b并纯化,TCID50检测病毒的滴度为1×109.22/mL。IFA和RT-PCR检测Ag85B蛋白在体外能够高效表达并有较高的生物学活性;3、血清学水平检测到rAd5-CMV-ag85b组免疫小鼠的Ig G水平低于BCG组差异不显著(P0.05),高于空载腺病毒组和0.9%NaCl组差异显著(P0.05)。流式细胞仪检测到rAd5-CMV-ag85b组小鼠体内的CD4+T淋巴细胞数高于0.9%NaCl组且差异显著(P0.05),高于rAd-CMV-NpA组低于BCG组但这三组之间差异均不显著(P0.05)。免疫小鼠体内CD8+T淋巴细胞数量BCG组高于rAd5-CMV-ag85b组差异不显著(P0.05),高于其他两组差异显著(P0.05)。CD3+T淋巴细胞数rAd5-CMV-ag85b组低于BCG组高于其他两组且各组之间的差异均不显著(P0.05)。流式细胞仪检测到分泌TNF-α的淋巴细胞数,rAd5-CMV-ag85b组高于其他三组且与BCG组相比差异不显著(P0.05),与空载腺病毒组和0.9%Nacl组相比差异显著(P0.05)。分泌IL-2的淋巴细胞数rAd5-CMV-ag85b组高于其他三组,且四组之间的差异均不显著(P0.05)。分泌IFN-γ的淋巴细胞数,rAd5-CMV-ag85b组高于BCG组但差异不显著(P0.05),高于空载腺病毒组和0.9%Nacl组且与这两组之间均差异显著(P0.05)。流式检测结果表明rAd5-CMV-ag85b对BALB/c小鼠具有一定的免疫远性,从而为牛结核病活载体疫苗的研究奠定了基础。
[Abstract]:Bovine tuberculosis (bTB) has been under great pressure in clinical medicine, social and economic fields throughout the world. Bacille Calmette-Guerin (BCG), as a traditional vaccine to prevent bovine tuberculosis, has a good immune effect, and recent studies have shown that the vaccine is only young. The immune protection effect of the object is very weak to the adult animal, so the development of the new efficient bTB vaccine has been imminent as the main protein of the early secreted.Ag85B of Mycobacterium tuberculosis. It can induce body humoral immunity and cell immunity at the same time. Therefore, the Ag85B gene is used as the target gene of this study to study bTB Gene engineering vaccine is of great significance. Human type 5 adenovirus, as an expression vector of foreign gene, has been widely used in the research field of gene therapy vaccine. Recombinant adenovirus vector has removed the E1 region of the early transcriptional region of the genome and became a replication defective adenovirus expression vector. Therefore, only the recombinant adenovirus vector has become a replication defective adenovirus expression vector. It can be propagated in the AD293 complementary cells containing the E1 region. Under the action of CMV promoter, the exogenous gene is inserted into the adenovirus vector and integrated into the adenovirus vector genome. Finally, the recombinant adenovirus can express the target protein in large amount. Live virus vaccine. Experimental methods: the plasmid pGEM-85bL was used as the PCR template, the target gene Ag85b was amplified by PCR and connected to the adenovirus shuttle vector pacAd5CMVK-NpA to get the recombinant adenovirus shuttle plasmid, which was named pacAd5CMV-ag85b, and it was linearized with the adenovirus skeleton plasmid pacAd5 9.2-100 with Pac I restriction endonuclease. The transfection reagent X-tremeGENE HP was transfected into AD293 cells to pack the recombinant adenovirus. The successful recombinant adenovirus packaging was named rAd5-CMV-ag85b and the genome was identified by PCR. After large amplification of rAd5-CMV-Ag85b, the recombinant adenovirus purification kit was purified, and the TCID50 virus titer determination method was pure. After rAd5-CMV-ag85b virus titer, indirect immunofluorescence test (IFA) and reverse transcriptional PCR test (RT-PCR), the expression and biological activity of the target gene Ag85B in vitro were further detected. Finally, rAd5-CMV-ag85b, rAd5-CMV-NpA, 0.9%NaCl and BCG were injected into the 6 week male BALB/c mice with multiple muscle muscles. Immunization was carried out after two weeks, and after two weeks, the CD3+ (PE marker), CD4+ (PE marker), CD8+ (FITC labeled) T lymphocyte and IL-2 (APC marker), TNF- alpha (PE marker), IFN- gamma cytokine were detected by flow cytometry for two weeks, and indirect enzyme linked immunosorbent assay (indirect enzyme linked immunosorbent assay) was used to immunize small The specific immunoglobulin IgG in the rat serum was detected. 1, the target gene Ag85B was obtained, the recombinant adenovirus shuttle vector pacAd5CMV-ag85b was successfully constructed, and the recombinant adenovirus rAd5-CMV-ag85b was successfully packaged in AD293 cells; 2, large amplification of rAd5-CMV-ag85b and purification, and TCID50 detection of the virus's titer of 1 x 109.22/mL.I FA and RT-PCR detected the high expression of Ag85B protein in vitro and had high biological activity in vitro. 3, the serological level showed that the level of Ig G in the rAd5-CMV-ag85b group was lower than that of the BCG group (P0.05), which was significantly higher than that in the empty adenovirus group and the 0.9%NaCl group (P0.05). The flow cytometry detected the rAd5-CMV-ag85b group. The number of CD4+T lymphocytes in the group was higher than that in the 0.9%NaCl group (P0.05), which was higher than that in the group rAd-CMV-NpA, but the difference between the three groups was not significant (P0.05). The number of CD8+T lymphocytes in the immune mice was not significantly higher than that in the rAd5-CMV-ag85b group (P0.05), higher than the other two groups (P0.05).CD3+T lymphocyte counts. Group MV-ag85b was lower than group BCG and the difference between the other two groups was not significant (P0.05). Flow cytometry detected the number of lymphocytes secreting TNF- alpha, rAd5-CMV-ag85b group was higher than the other three groups and was not significantly different from that of the BCG group (P0.05), and the difference was significant compared with the empty adenovirus group and the 0.9%Nacl group (P0.05). The lymph thinning of IL-2 was secreted. The number of rAd5-CMV-ag85b groups was higher than that of the other three groups, and the difference between the four groups was not significant (P0.05). The number of lymphocytes secreting IFN- gamma was higher than that of the BCG group, but the difference was not significant (P0.05), which was higher than that of the empty adenovirus group and the 0.9%Nacl group (P0.05). The flow test results showed rAd5-CMV-ag85b to BALB. /c mice have a certain immunological potential, thus laying the foundation for the research of live carrier vaccine against bovine tuberculosis.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.618
本文编号：2158231
[Abstract]:Bovine tuberculosis (bTB) has been under great pressure in clinical medicine, social and economic fields throughout the world. Bacille Calmette-Guerin (BCG), as a traditional vaccine to prevent bovine tuberculosis, has a good immune effect, and recent studies have shown that the vaccine is only young. The immune protection effect of the object is very weak to the adult animal, so the development of the new efficient bTB vaccine has been imminent as the main protein of the early secreted.Ag85B of Mycobacterium tuberculosis. It can induce body humoral immunity and cell immunity at the same time. Therefore, the Ag85B gene is used as the target gene of this study to study bTB Gene engineering vaccine is of great significance. Human type 5 adenovirus, as an expression vector of foreign gene, has been widely used in the research field of gene therapy vaccine. Recombinant adenovirus vector has removed the E1 region of the early transcriptional region of the genome and became a replication defective adenovirus expression vector. Therefore, only the recombinant adenovirus vector has become a replication defective adenovirus expression vector. It can be propagated in the AD293 complementary cells containing the E1 region. Under the action of CMV promoter, the exogenous gene is inserted into the adenovirus vector and integrated into the adenovirus vector genome. Finally, the recombinant adenovirus can express the target protein in large amount. Live virus vaccine. Experimental methods: the plasmid pGEM-85bL was used as the PCR template, the target gene Ag85b was amplified by PCR and connected to the adenovirus shuttle vector pacAd5CMVK-NpA to get the recombinant adenovirus shuttle plasmid, which was named pacAd5CMV-ag85b, and it was linearized with the adenovirus skeleton plasmid pacAd5 9.2-100 with Pac I restriction endonuclease. The transfection reagent X-tremeGENE HP was transfected into AD293 cells to pack the recombinant adenovirus. The successful recombinant adenovirus packaging was named rAd5-CMV-ag85b and the genome was identified by PCR. After large amplification of rAd5-CMV-Ag85b, the recombinant adenovirus purification kit was purified, and the TCID50 virus titer determination method was pure. After rAd5-CMV-ag85b virus titer, indirect immunofluorescence test (IFA) and reverse transcriptional PCR test (RT-PCR), the expression and biological activity of the target gene Ag85B in vitro were further detected. Finally, rAd5-CMV-ag85b, rAd5-CMV-NpA, 0.9%NaCl and BCG were injected into the 6 week male BALB/c mice with multiple muscle muscles. Immunization was carried out after two weeks, and after two weeks, the CD3+ (PE marker), CD4+ (PE marker), CD8+ (FITC labeled) T lymphocyte and IL-2 (APC marker), TNF- alpha (PE marker), IFN- gamma cytokine were detected by flow cytometry for two weeks, and indirect enzyme linked immunosorbent assay (indirect enzyme linked immunosorbent assay) was used to immunize small The specific immunoglobulin IgG in the rat serum was detected. 1, the target gene Ag85B was obtained, the recombinant adenovirus shuttle vector pacAd5CMV-ag85b was successfully constructed, and the recombinant adenovirus rAd5-CMV-ag85b was successfully packaged in AD293 cells; 2, large amplification of rAd5-CMV-ag85b and purification, and TCID50 detection of the virus's titer of 1 x 109.22/mL.I FA and RT-PCR detected the high expression of Ag85B protein in vitro and had high biological activity in vitro. 3, the serological level showed that the level of Ig G in the rAd5-CMV-ag85b group was lower than that of the BCG group (P0.05), which was significantly higher than that in the empty adenovirus group and the 0.9%NaCl group (P0.05). The flow cytometry detected the rAd5-CMV-ag85b group. The number of CD4+T lymphocytes in the group was higher than that in the 0.9%NaCl group (P0.05), which was higher than that in the group rAd-CMV-NpA, but the difference between the three groups was not significant (P0.05). The number of CD8+T lymphocytes in the immune mice was not significantly higher than that in the rAd5-CMV-ag85b group (P0.05), higher than the other two groups (P0.05).CD3+T lymphocyte counts. Group MV-ag85b was lower than group BCG and the difference between the other two groups was not significant (P0.05). Flow cytometry detected the number of lymphocytes secreting TNF- alpha, rAd5-CMV-ag85b group was higher than the other three groups and was not significantly different from that of the BCG group (P0.05), and the difference was significant compared with the empty adenovirus group and the 0.9%Nacl group (P0.05). The lymph thinning of IL-2 was secreted. The number of rAd5-CMV-ag85b groups was higher than that of the other three groups, and the difference between the four groups was not significant (P0.05). The number of lymphocytes secreting IFN- gamma was higher than that of the BCG group, but the difference was not significant (P0.05), which was higher than that of the empty adenovirus group and the 0.9%Nacl group (P0.05). The flow test results showed rAd5-CMV-ag85b to BALB. /c mice have a certain immunological potential, thus laying the foundation for the research of live carrier vaccine against bovine tuberculosis.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.618
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