牛分結(jié)核枝桿菌ag85b基因重組腺病毒的構(gòu)建及其實(shí)驗(yàn)免疫研究
發(fā)布時(shí)間：2018-08-01 16:57
【摘要】：實(shí)驗(yàn)?zāi)康?牛結(jié)核病(Bovine tuberculosis,bTB)一直以來在世界范圍內(nèi)使臨床醫(yī)學(xué)、社會和經(jīng)濟(jì)領(lǐng)域承受著巨大的壓力�？ń槊�(Bacille Calmette-Guerin,BCG)作為預(yù)防牛結(jié)核病的傳統(tǒng)疫苗有著很好的免疫效果,而最近有研究表明該疫苗只對年幼的動物體具有免疫保護(hù)作用而對成年動物免疫保護(hù)效果很弱,因此開發(fā)新型高效bTB疫苗已經(jīng)迫在眉睫。Ag85B是牛結(jié)核分枝桿菌早期分泌的主要蛋白,可以誘導(dǎo)機(jī)體同時(shí)產(chǎn)生體液免疫和細(xì)胞免疫,因此將ag85b基因作為本研究的靶基因?qū)ρ芯縝TB基因工程疫苗具有重要的意義。人5型腺病毒作為一種外源基因的表達(dá)載體在基因治療疫苗的研究領(lǐng)域一直以來有著非常廣泛的應(yīng)用。重組腺病毒載體去掉了基因組中早期轉(zhuǎn)錄區(qū)域的E1區(qū)而成為復(fù)制缺陷型的腺病毒表達(dá)載體,因此只能在含有E1區(qū)的AD293互補(bǔ)細(xì)胞中進(jìn)行繁殖。在CMV啟動子的作用下,將外源基因插入到腺病毒載體上并在互補(bǔ)細(xì)胞系中整合到腺病毒載體基因組中,最終重組腺病毒可以大量表達(dá)目的蛋白。因而此類疫苗目前被認(rèn)為是一種安全而高效的bTB活病毒疫苗。實(shí)驗(yàn)方法:以質(zhì)粒pGEM-85bL作為PCR模板,對目的基因Ag85b進(jìn)行PCR擴(kuò)增并連接到腺病毒穿梭載體pacAd5CMVK-NpA上得到重組腺病毒穿梭質(zhì)粒我們命名為pacAd5CMV-ag85b,將其與腺病毒骨架質(zhì)粒pacAd5 9.2-100用PacⅠ限制性內(nèi)切酶線性化后在轉(zhuǎn)染試劑X-tremeGENE HP的作用下共轉(zhuǎn)染到AD293細(xì)胞里面進(jìn)行重組腺病毒的包裝。包裝成功的重組腺病毒我們命名為rAd5-CMV-ag85b并對其基因組進(jìn)行PCR鑒定。大量擴(kuò)增rAd5-CMV-Ag85b后用腺病毒純化試劑盒對其純化,TCID50病毒滴度測定方法測定純化后的rAd5-CMV-ag85b病毒滴度。間接免疫熒光試驗(yàn)(IFA)和反轉(zhuǎn)錄PCR試驗(yàn)(RT-PCR)進(jìn)一步檢測目的基因ag85b在體外的表達(dá)情況及生物學(xué)活性。最后將rAd5-CMV-ag85b、空載腺病毒rAd5-CMV-NpA、0.9%NaCl和BCG以多點(diǎn)肌肌肉注射的方式對6周齡雄性BALB/c小白鼠進(jìn)行免疫,兩周后加強(qiáng)免疫,再過兩周利用流式細(xì)胞儀對各組免疫小鼠體內(nèi)的CD3+(PE標(biāo)記)、CD4+(PE標(biāo)記)、CD8+(FITC標(biāo)記)T淋巴細(xì)胞和IL-2(APC標(biāo)記)、TNF-α(PE標(biāo)記)、IFN-γ(FITC標(biāo)記)細(xì)胞因子進(jìn)行檢測,同時(shí)利用間接酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)對免疫小鼠血清內(nèi)的特異性免疫球蛋白IgG進(jìn)行檢測。實(shí)驗(yàn)結(jié)果:1、獲得目的基因ag85b,成功構(gòu)建重組腺病毒穿梭載體pacAd5CMV-ag85b,并在AD293細(xì)胞內(nèi)成功包裝出了重組腺病毒rAd5-CMV-ag85b;2、大量擴(kuò)增rAd5-CMV-ag85b并純化,TCID50檢測病毒的滴度為1×109.22/mL。IFA和RT-PCR檢測Ag85B蛋白在體外能夠高效表達(dá)并有較高的生物學(xué)活性;3、血清學(xué)水平檢測到rAd5-CMV-ag85b組免疫小鼠的Ig G水平低于BCG組差異不顯著(P0.05),高于空載腺病毒組和0.9%NaCl組差異顯著(P0.05)。流式細(xì)胞儀檢測到rAd5-CMV-ag85b組小鼠體內(nèi)的CD4+T淋巴細(xì)胞數(shù)高于0.9%NaCl組且差異顯著(P0.05),高于rAd-CMV-NpA組低于BCG組但這三組之間差異均不顯著(P0.05)。免疫小鼠體內(nèi)CD8+T淋巴細(xì)胞數(shù)量BCG組高于rAd5-CMV-ag85b組差異不顯著(P0.05),高于其他兩組差異顯著(P0.05)。CD3+T淋巴細(xì)胞數(shù)rAd5-CMV-ag85b組低于BCG組高于其他兩組且各組之間的差異均不顯著(P0.05)。流式細(xì)胞儀檢測到分泌TNF-α的淋巴細(xì)胞數(shù),rAd5-CMV-ag85b組高于其他三組且與BCG組相比差異不顯著(P0.05),與空載腺病毒組和0.9%Nacl組相比差異顯著(P0.05)。分泌IL-2的淋巴細(xì)胞數(shù)rAd5-CMV-ag85b組高于其他三組,且四組之間的差異均不顯著(P0.05)。分泌IFN-γ的淋巴細(xì)胞數(shù),rAd5-CMV-ag85b組高于BCG組但差異不顯著(P0.05),高于空載腺病毒組和0.9%Nacl組且與這兩組之間均差異顯著(P0.05)。流式檢測結(jié)果表明rAd5-CMV-ag85b對BALB/c小鼠具有一定的免疫遠(yuǎn)性,從而為牛結(jié)核病活載體疫苗的研究奠定了基礎(chǔ)。
[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.
【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】：2016
【分類號】：S852.618
本文編號：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.
【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】：2016
【分類號】：S852.618
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相關(guān)期刊論文 前3條
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