肺炎嗜衣原體MOMP核酸疫苗的構(gòu)建及其免疫效果的初步研究
發(fā)布時間：2019-01-30 16:09
【摘要】： 目的:構(gòu)建肺炎嗜衣原體(Chlamydophila pneumoniae, Cpn)重組單基因疫苗和雙基因融合疫苗,建立動物感染模型,比較兩種核酸疫苗在抗Cpn感染中的免疫效果。為研制高效、新型的Cpn核酸疫苗奠定實驗基礎(chǔ)。 方法:用Prime軟件分析Cpn外膜蛋白MOMP和人IL-2基因序列,設(shè)計相應(yīng)特異性引物,分別用PCR和RT-PCR擴增MOMP全基因及人IL-2基因,同時以編碼6個氨基酸的18個寡核苷酸作為接頭,通過重組PCR法將MOMP與IL-2基因融合,PCR產(chǎn)物經(jīng)酶切純化后克隆至pcDNA3.1(+)真核表達載體中;陽性克隆經(jīng)雙酶切及測序鑒定后,大量制備純化后作為核酸疫苗,分別將重組質(zhì)粒pcDNA3.1(+)-MOMP、pcDNA3.1(+)-IL-2、pcDNA3.1(+)-MOMP-IL-2及對照空質(zhì)粒pcDNA3.1(+)分組注射于BALB/c小鼠后腿股四頭肌,每次劑量為100μg,末次免疫后2w,免疫熒光組化法檢測肺炎嗜衣原體MOMP基因在小鼠組織內(nèi)的表達情況,無菌分離小鼠脾細胞,經(jīng)特異性重組蛋白抗原刺激后, ELISA雙抗體夾心法測定培養(yǎng)上清中IFN-γ水平,ELISA間接法測定BALB/c小鼠血清中抗MOMP和抗MOMP-IL-2特異性抗體水平;MTT法、淋巴細胞轉(zhuǎn)化試驗測定脾淋巴細胞特異性增殖反應(yīng)。 結(jié)果: 1.成功構(gòu)建了MOMP、IL-2以及MOMP-IL-2融合基因的原核表達載體和真核表達載體。 2.成功構(gòu)建pcDNA3.1(+)-MOMP、pcDNA3.1(+)-MOMP-IL-2融合基因核酸疫苗,其均能在組織細胞內(nèi)有效表達融合靶蛋白。 3.小鼠接種疫苗后,能產(chǎn)生特異性抗體,6w后抗體的最高滴度均達1:1280 4. MOMP單基因核酸疫苗組和MOMP-IL-2融合基因核酸疫苗組的鼠脾淋巴細胞經(jīng)相應(yīng)特異性抗原刺激后,培養(yǎng)上清中IFN-γ均顯著升高(分別升至586±42.3pg/mL、695±43.7pg/mL)。 5. MOMP單基因核酸疫苗組和MOMP-IL-2融合基因核酸疫苗組小鼠脾淋巴細胞經(jīng)相應(yīng)特異性抗原刺激后,其刺激指數(shù)分別為(1.508±0.010、1.573±0.012)。 6.在小鼠肌組織中用PCR可檢測到MOMP基因。 結(jié)論: 1. Cpn MOMP單基因核酸疫苗和MOMP-IL-2融合基因核酸疫苗均能刺激機體產(chǎn)生較強細胞免疫應(yīng)答和體液免疫應(yīng)答。 2.雙基因核酸疫苗誘生特異性抗體的效率與單基因核酸疫苗有明顯差異;且能誘導(dǎo)更強,更持久的細胞免疫應(yīng)答。
[Abstract]:Aim: to construct recombinant single gene vaccine and double gene fusion vaccine of Chlamydia pneumoniae (Chlamydophila pneumoniae, Cpn) and to establish animal infection model and compare the immune effect of two nucleic acid vaccines against Cpn infection. For the development of high-efficiency, new Cpn nucleic acid vaccine to lay the experimental foundation. Methods: the sequences of Cpn outer membrane protein MOMP and human IL-2 gene were analyzed by Prime software. Specific primers were designed to amplify the whole MOMP gene and human IL-2 gene by PCR and RT-PCR, respectively. At the same time, 18 oligonucleotides encoding 6 amino acids were used as the junction, the MOMP gene was fused with IL-2 gene by recombinant PCR method, and the PCR product was purified by enzyme digestion and cloned into pcDNA3.1 () eukaryotic expression vector. The positive clones were digested by double enzyme and sequenced, and then purified as nucleic acid vaccine. The recombinant plasmids pcDNA3.1 ()-MOMP,pcDNA3.1 ()-IL-2, were prepared respectively. PcDNA3.1 ()-MOMP-IL-2 and control plasmid pcDNA3.1 () were injected into the quadriceps femoris of the hind leg of BALB/c mice at a dose of 100 渭 g each time for 2 weeks after the last immunization. The expression of chlamydia pneumoniae MOMP gene in mouse tissues was detected by immunofluorescence histochemistry. Mouse spleen cells were isolated without bacteria. After stimulation with specific recombinant protein antigen, the level of IFN- 緯 in culture supernatant was measured by ELISA double antibody sandwich method. The levels of anti MOMP and anti MOMP-IL-2 specific antibodies in serum of BALB/c mice were determined by ELISA indirect method. MTT assay and lymphocyte transformation test were used to determine the specific proliferation of splenic lymphocytes. Results: 1. The prokaryotic expression vector and eukaryotic expression vector of MOMP,IL-2 and MOMP-IL-2 fusion gene were successfully constructed. 2. PcDNA3.1 ()-MOMP,pcDNA3.1 ()-MOMP-IL-2 fusion gene nucleic acid vaccine was successfully constructed. 3. After inoculation, specific antibody was produced in mice, and the highest titer of antibody was 1: 12804 after 6 weeks. The splenic lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group were stimulated by specific antigen, and the IFN- 緯 in the culture supernatant was significantly increased (up to 586 鹵42.3 PG / mL 695 鹵43.7pg/mL, respectively). 5. The stimulation index of spleen lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group was (1.508 鹵0.010 10) 1.53 鹵0.012 (P < 0.05). 6. MOMP gene could be detected by PCR in mouse muscle tissue. Conclusion: 1. Both Cpn MOMP single gene nucleic acid vaccine and MOMP-IL-2 fusion gene nucleic acid vaccine can stimulate strong cellular and humoral immune responses. 2. The efficiency of inducing specific antibody by double gene nucleic acid vaccine was significantly different from that of single gene nucleic acid vaccine, and it could induce a stronger and more lasting cellular immune response.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392.1
[Abstract]:Aim: to construct recombinant single gene vaccine and double gene fusion vaccine of Chlamydia pneumoniae (Chlamydophila pneumoniae, Cpn) and to establish animal infection model and compare the immune effect of two nucleic acid vaccines against Cpn infection. For the development of high-efficiency, new Cpn nucleic acid vaccine to lay the experimental foundation. Methods: the sequences of Cpn outer membrane protein MOMP and human IL-2 gene were analyzed by Prime software. Specific primers were designed to amplify the whole MOMP gene and human IL-2 gene by PCR and RT-PCR, respectively. At the same time, 18 oligonucleotides encoding 6 amino acids were used as the junction, the MOMP gene was fused with IL-2 gene by recombinant PCR method, and the PCR product was purified by enzyme digestion and cloned into pcDNA3.1 () eukaryotic expression vector. The positive clones were digested by double enzyme and sequenced, and then purified as nucleic acid vaccine. The recombinant plasmids pcDNA3.1 ()-MOMP,pcDNA3.1 ()-IL-2, were prepared respectively. PcDNA3.1 ()-MOMP-IL-2 and control plasmid pcDNA3.1 () were injected into the quadriceps femoris of the hind leg of BALB/c mice at a dose of 100 渭 g each time for 2 weeks after the last immunization. The expression of chlamydia pneumoniae MOMP gene in mouse tissues was detected by immunofluorescence histochemistry. Mouse spleen cells were isolated without bacteria. After stimulation with specific recombinant protein antigen, the level of IFN- 緯 in culture supernatant was measured by ELISA double antibody sandwich method. The levels of anti MOMP and anti MOMP-IL-2 specific antibodies in serum of BALB/c mice were determined by ELISA indirect method. MTT assay and lymphocyte transformation test were used to determine the specific proliferation of splenic lymphocytes. Results: 1. The prokaryotic expression vector and eukaryotic expression vector of MOMP,IL-2 and MOMP-IL-2 fusion gene were successfully constructed. 2. PcDNA3.1 ()-MOMP,pcDNA3.1 ()-MOMP-IL-2 fusion gene nucleic acid vaccine was successfully constructed. 3. After inoculation, specific antibody was produced in mice, and the highest titer of antibody was 1: 12804 after 6 weeks. The splenic lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group were stimulated by specific antigen, and the IFN- 緯 in the culture supernatant was significantly increased (up to 586 鹵42.3 PG / mL 695 鹵43.7pg/mL, respectively). 5. The stimulation index of spleen lymphocytes in MOMP single gene vaccine group and MOMP-IL-2 fusion gene nucleic acid vaccine group was (1.508 鹵0.010 10) 1.53 鹵0.012 (P < 0.05). 6. MOMP gene could be detected by PCR in mouse muscle tissue. Conclusion: 1. Both Cpn MOMP single gene nucleic acid vaccine and MOMP-IL-2 fusion gene nucleic acid vaccine can stimulate strong cellular and humoral immune responses. 2. The efficiency of inducing specific antibody by double gene nucleic acid vaccine was significantly different from that of single gene nucleic acid vaccine, and it could induce a stronger and more lasting cellular immune response.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392.1
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