不可分型流感嗜血杆菌外膜蛋白P6基因真核表达质粒的构建及其免疫原性分析
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摘要
不可分型流感嗜血杆菌(nontypeable Haemophilus influenzae, NTHi)是一种在人群中携带率很高的病原菌，该菌能够引起急性中耳炎，鼻窦炎，结膜炎，慢性支气管炎的反复发作及恶化等严重感染性疾病，对人类尤其是婴幼儿及老年人的健康威胁极大，因此目前亟待研究一种新型、安全有效的疫苗来预防NTHi的感染。研究证明，在所有流感嗜血杆菌菌株中(含不可分型流感嗜血杆菌)高度保守的外膜蛋白P6(outer membrane protein P6, P6)可以诱导机体产生保护性的体液免疫、细胞免疫及黏膜免疫，但是天然P6蛋白含量少且提取过程极为繁琐。近年来核酸免疫的发展为我们研制疫苗提供了新的思路，本文就是通过构建真核表达重组质粒pcDNA3.1/His A-P6，并将其免疫动物观察其免疫效果，以确定P6在NTHi疫苗研制中的应用价值。
以NTHi全基因组为模板通过PCR扩增获得P6目的基因，构建真核质粒pcDNA3.1/His A-P6，通过菌落PCR、酶切、基因测序的方法确定重组质粒构建成功，并将重组质粒转染至HeLa细胞，以间接免疫荧光蛋白法检测其表达产物；动物实验中，将45只8w龄BALB/c小鼠随机分为3组：PBS组、pcDNA3.1/His A空质粒组、pcDNA3.1/His A-P6重组质粒组，每组每只小鼠分别经股四头肌注射免疫100μL PBS，100μL含100μg pcDNA3.1/His A的PBS，100μL含100μg pcDNA3.1/HisA-P6的PBS，于第0d，14d和28d共进行3次免疫。末次免疫后2w，每组取10只小鼠CCK-8法分析脾淋巴细胞增殖情况；ELISA法分析其脾淋巴细胞IFN-γ和IL-4产生水平；末次免疫后3w，每组其余5只小鼠鼻内接种1×108 CFU/只NTHi，于第3d、7d将100μL PBS灌洗鼻腔检测NTHi清除率；接种NTHi后1w处死小鼠，取其鼻粘膜组织，HE染色法分析鼻粘膜病理变化。
成功构建了真核载体pcDNA3.1/His A-P6，经PCR、酶切、测序证实插入的基因片段为P6蛋白编码基因,而且该重组质粒能够在HeLa细胞中表达目的蛋白。动物实验中，pcDNA3.1/His A-P6免疫组组小鼠特异性脾淋巴细胞刺激指数及其所产生的IFN-γ水平均高于pcDNA3.1/His A、PBS对照组(P<0.01)，而IL-4水平各组无差异；pcDNA3.1/HisA-P6组在NTHi感染后第3d、7d对其清除率分别为80%、90%，显著高于pcDNA3.1/His A、PBS对照组(P<0.01)；HE染色显示目的基因组小鼠鼻粘膜组织结构基本正常，而对照组鼻粘膜紊乱、脱落。
该研究成功构建了真核表达质粒pcDNA3.1/His A-P6，将其免疫BALB/c小鼠后可诱导小鼠产生明显的抗NTHi保护效应，提示pcDNA3.1/His A-P6核酸疫苗具有潜在的疫苗研究与开发价值。
Nontypeable Haemophilus influenzae (NTHi), which has a highprevalence rate among the population is frequently associated withmany serious diseases, such as acute otitis media (AOM), sinusitis,conjunctivitis, the repeated outbreak and acute exacerbation of chronicpulmonary diseases (COPD), and it has become a great healthy threat tohuman, especially to the infant and the elderly. Thus, it is high time toresearch a new safe and effective vaccine to prevent NTHi infections.The outer membrane protein P6, which is commonly found in all Histrains (including NTHi), could evoke protective humoral, cellular andmucosal immunity; however, the harvest of P6 is very little and trivial.In recent years, the development of nucleinic acid vaccine has offered anew approach to developing vaccines. In this study, the eukaryoticexpression plasmid for P6 named pcDNA3.1/His A-P6 was constructedand used to immunize BALB/c mice, then the immunizing effects wereobserved to ascertain the applied value of P6 in the vaccine developmentfor NTHi.
The P6 gene was amplified from the NTHi chromosomal DNA byPCR；Then the eukaryotic plasmid named pcDNA3.1/His A-P6 wasconstructed and verified by PCR, enzyme digestion analysis and DNAsequencing. The pcDNA3.1/His A-P6 plasmid was also transfected intoHeLa cells to detect its expression by indirect immunofluores cenceassay. A total of 45 8-week-old mice were separated into three cohorts of 15 animals each at random. The PBS group, pcDNA3.1/His A groupand pcDNA3.1/His A-P6 group were respectively immunized byintramuscular injection into the left thigh quadriceps muscle with 100μLPBS, 100μL PBS containing 100μg pcDNA3.1/His A plasmid and100μL PBS containing 100μg pcDNA3.1/His A-P6 plasmids three timeson days 0, 14, 28. 2 weeks after the last immunization, 10 mice fromeach group were killed, the proliferation of spleen lymphocyte wasanalysed by CCK-8 counting; IFN-γand IL-4 in supernatant ofspleenocytes cultured with NTHi in vitro was detected byenzyme-linked immunosorbent assay; 3 weeks after the lastimmunization, live NTHi strains at a dose of 1×108 cfu/head wasintranasally challenged to 15 mice (5 from each group), nasopharyngealwashings (NPWs) were obtained by meticulously washing with 100μLsterile PBS on days 3, 7 after the challenge to determine the clearance ofNTHi; 1 week after NTHi challenge, nasal mucosa histopathology wasanalysed by hematoxylin and eosin staining.
The eukaryotic expression plasmid pcDNA3.1/His A-P6 was successfulllyconstructed, and the sequence of the inserted gene was verified tobe P6 by PCR, enzyme digestion analysis and DNA sequencing; Inaddition, the recombinant plasmids could express target protein P6 inHeLa cells. In the animal experiment, the mice spleen lymphocyteimmunized with pcDNA3.1/His A-P6 evoked a higher production ofIFN-γand stimulation index than those of controls which wereimmunized by pcDNA3.1/His A or PBS (P<0.01), while there was nodifference in IL-4 level among them; The nasopharyngeal clearance rateof NTHi immunized with pcDNA3.1/His A-P6 was also apparentlyhigher than those of controls (P<0.01); The H&E staining showed thenasal mucosa immunized with pcDNA3.1/His A-P6 was nearly normal,while the nasal mucosa of controls disordered and fell off.
This study has successfully constructed the eukaryotic expression plasmid pcDNA3.1/His A-P6 and this eukaryotic plasmid for P6 inducesobviously protective effects against NTHi in the BALB/c mice model,which displays the recombinant vector for P6 has a potential value forNTHi vaccine research and development .
以NTHi全基因组为模板通过PCR扩增获得P6目的基因，构建真核质粒pcDNA3.1/His A-P6，通过菌落PCR、酶切、基因测序的方法确定重组质粒构建成功，并将重组质粒转染至HeLa细胞，以间接免疫荧光蛋白法检测其表达产物；动物实验中，将45只8w龄BALB/c小鼠随机分为3组：PBS组、pcDNA3.1/His A空质粒组、pcDNA3.1/His A-P6重组质粒组，每组每只小鼠分别经股四头肌注射免疫100μL PBS，100μL含100μg pcDNA3.1/His A的PBS，100μL含100μg pcDNA3.1/HisA-P6的PBS，于第0d，14d和28d共进行3次免疫。末次免疫后2w，每组取10只小鼠CCK-8法分析脾淋巴细胞增殖情况；ELISA法分析其脾淋巴细胞IFN-γ和IL-4产生水平；末次免疫后3w，每组其余5只小鼠鼻内接种1×108 CFU/只NTHi，于第3d、7d将100μL PBS灌洗鼻腔检测NTHi清除率；接种NTHi后1w处死小鼠，取其鼻粘膜组织，HE染色法分析鼻粘膜病理变化。
成功构建了真核载体pcDNA3.1/His A-P6，经PCR、酶切、测序证实插入的基因片段为P6蛋白编码基因,而且该重组质粒能够在HeLa细胞中表达目的蛋白。动物实验中，pcDNA3.1/His A-P6免疫组组小鼠特异性脾淋巴细胞刺激指数及其所产生的IFN-γ水平均高于pcDNA3.1/His A、PBS对照组(P<0.01)，而IL-4水平各组无差异；pcDNA3.1/HisA-P6组在NTHi感染后第3d、7d对其清除率分别为80%、90%，显著高于pcDNA3.1/His A、PBS对照组(P<0.01)；HE染色显示目的基因组小鼠鼻粘膜组织结构基本正常，而对照组鼻粘膜紊乱、脱落。
该研究成功构建了真核表达质粒pcDNA3.1/His A-P6，将其免疫BALB/c小鼠后可诱导小鼠产生明显的抗NTHi保护效应，提示pcDNA3.1/His A-P6核酸疫苗具有潜在的疫苗研究与开发价值。
Nontypeable Haemophilus influenzae (NTHi), which has a highprevalence rate among the population is frequently associated withmany serious diseases, such as acute otitis media (AOM), sinusitis,conjunctivitis, the repeated outbreak and acute exacerbation of chronicpulmonary diseases (COPD), and it has become a great healthy threat tohuman, especially to the infant and the elderly. Thus, it is high time toresearch a new safe and effective vaccine to prevent NTHi infections.The outer membrane protein P6, which is commonly found in all Histrains (including NTHi), could evoke protective humoral, cellular andmucosal immunity; however, the harvest of P6 is very little and trivial.In recent years, the development of nucleinic acid vaccine has offered anew approach to developing vaccines. In this study, the eukaryoticexpression plasmid for P6 named pcDNA3.1/His A-P6 was constructedand used to immunize BALB/c mice, then the immunizing effects wereobserved to ascertain the applied value of P6 in the vaccine developmentfor NTHi.
The P6 gene was amplified from the NTHi chromosomal DNA byPCR；Then the eukaryotic plasmid named pcDNA3.1/His A-P6 wasconstructed and verified by PCR, enzyme digestion analysis and DNAsequencing. The pcDNA3.1/His A-P6 plasmid was also transfected intoHeLa cells to detect its expression by indirect immunofluores cenceassay. A total of 45 8-week-old mice were separated into three cohorts of 15 animals each at random. The PBS group, pcDNA3.1/His A groupand pcDNA3.1/His A-P6 group were respectively immunized byintramuscular injection into the left thigh quadriceps muscle with 100μLPBS, 100μL PBS containing 100μg pcDNA3.1/His A plasmid and100μL PBS containing 100μg pcDNA3.1/His A-P6 plasmids three timeson days 0, 14, 28. 2 weeks after the last immunization, 10 mice fromeach group were killed, the proliferation of spleen lymphocyte wasanalysed by CCK-8 counting; IFN-γand IL-4 in supernatant ofspleenocytes cultured with NTHi in vitro was detected byenzyme-linked immunosorbent assay; 3 weeks after the lastimmunization, live NTHi strains at a dose of 1×108 cfu/head wasintranasally challenged to 15 mice (5 from each group), nasopharyngealwashings (NPWs) were obtained by meticulously washing with 100μLsterile PBS on days 3, 7 after the challenge to determine the clearance ofNTHi; 1 week after NTHi challenge, nasal mucosa histopathology wasanalysed by hematoxylin and eosin staining.
The eukaryotic expression plasmid pcDNA3.1/His A-P6 was successfulllyconstructed, and the sequence of the inserted gene was verified tobe P6 by PCR, enzyme digestion analysis and DNA sequencing; Inaddition, the recombinant plasmids could express target protein P6 inHeLa cells. In the animal experiment, the mice spleen lymphocyteimmunized with pcDNA3.1/His A-P6 evoked a higher production ofIFN-γand stimulation index than those of controls which wereimmunized by pcDNA3.1/His A or PBS (P<0.01), while there was nodifference in IL-4 level among them; The nasopharyngeal clearance rateof NTHi immunized with pcDNA3.1/His A-P6 was also apparentlyhigher than those of controls (P<0.01); The H&E staining showed thenasal mucosa immunized with pcDNA3.1/His A-P6 was nearly normal,while the nasal mucosa of controls disordered and fell off.
This study has successfully constructed the eukaryotic expression plasmid pcDNA3.1/His A-P6 and this eukaryotic plasmid for P6 inducesobviously protective effects against NTHi in the BALB/c mice model,which displays the recombinant vector for P6 has a potential value forNTHi vaccine research and development .
引文
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18 Kodama S, Hirano T, Noda K, et al. A single nasal dose of fms-like tyrosinekinase receptor-3 ligand, but not peritoneal application,enhances nontypeableHaemophilus influenzae-specific long-term mucosal immune responses in thenasopharynx [J]. Vaccine, 2010, 28(13):2510-2516.19 Sabirov A, Kodama S, Hirano T, et al. Intranasal immunization enhances clearanceof nontypeable Haemophilus influenzae and reduces stimulation of tumor necrosisfactor alpha production in the murine model of otitis media [J]. Infect Immun, 2001,69(5): 2964- 2971.
20 Sabirov A, Kodama S, Sabirova N, et al. Intranasal immunization with outermembrane protein P6 and cholera toxin induces specific sinus mucosal immunityand enhances sinus clearance of nontypeable Haemophilus influenzae [J]. Vaccine,2004, 22(23-24): 3112-3121.
21 Hotomi M, Yamanaka N, Shimada J, et al. Intranasal immunization withrecombinant outer membrane protein P6 induces specific immune responsesagainst nontypeable Haemophilus influenzae [J]. Int J Pediatr Otorhinolaryngol,2002, 65(2):109-116.
22 Bertot GM, Becker PD, Guzman CA, et al. Intranasal vaccination withrecombinant P6 protein and adamantylamide dipeptide as mucosal adjuvant confersefficient protection against otitis media and lung infection by nontypeableHaemophilus influenzae [J]. Infect Dis, 2004, 189(7):1304-1312.
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12 Sabirov A, Casey JR, Murphy TF, et al. Breastfeeding is associated with a reducedfrequency of acute otitis media and high serum antibody levels against NTHi andouter membrane protein vaccine antigen candidate P6 [J]. Pediatr Res, 2009, 65(5):565-570.
13 Yamanaka N, Hotomi M, Billal DS. Clinical bacteriology and immunology in acuteotitis media in children [J]. Infect Chemother, 2008, 14(3): 180-187.
14 Pichichero ME, Kaur R, Casey JR, et al. Antibody response to Haemophilusinfluenzae outer membrane protein D, P6, and OMP26 after nasopharyngealcolonization and acute otitis media in children [J]. Vaccine, 2010, 28(44):7184-7192.
15 Noda K, Kadama S, Umemoto S, et al. Nasal vaccination with P6 outer membraneprotein and alpha-galactosylceramide induces nontypeable Haemophilusinfluenzae-specific protective immunity associated with NKT cell activation anddendritic cell expansion in nasopharynx [J].Vaccine, 2010, 28(31): 5068-5074.
16 Berenson CS, Murphy TF, Wrona CT, et al. Outer membrane protein P6 ofnontypeable Haemophilus influenzae is a potent and selective inducer of humanmacrophage proinflammatory cytokines [J]. Infect Immun, 2005, 73(5):2728-2735.
17 Kodama H, Faden H. Cellular immunity to the P6 outer membrane protein ofnontypeable Haemophilus influenzae [J]. Infect Immun, 1995, 63(7): 2467-2472.
18 Kodama S, Hirano T, Noda K, et al. A single nasal dose of fms-like tyrosinekinase receptor-3 ligand, but not peritoneal application,enhances nontypeableHaemophilus influenzae-specific long-term mucosal immune responses in thenasopharynx [J]. Vaccine, 2010, 28(13):2510-2516.19 Sabirov A, Kodama S, Hirano T, et al. Intranasal immunization enhances clearanceof nontypeable Haemophilus influenzae and reduces stimulation of tumor necrosisfactor alpha production in the murine model of otitis media [J]. Infect Immun, 2001,69(5): 2964- 2971.
20 Sabirov A, Kodama S, Sabirova N, et al. Intranasal immunization with outermembrane protein P6 and cholera toxin induces specific sinus mucosal immunityand enhances sinus clearance of nontypeable Haemophilus influenzae [J]. Vaccine,2004, 22(23-24): 3112-3121.
21 Hotomi M, Yamanaka N, Shimada J, et al. Intranasal immunization withrecombinant outer membrane protein P6 induces specific immune responsesagainst nontypeable Haemophilus influenzae [J]. Int J Pediatr Otorhinolaryngol,2002, 65(2):109-116.
22 Bertot GM, Becker PD, Guzman CA, et al. Intranasal vaccination withrecombinant P6 protein and adamantylamide dipeptide as mucosal adjuvant confersefficient protection against otitis media and lung infection by nontypeableHaemophilus influenzae [J]. Infect Dis, 2004, 189(7):1304-1312.
23 van Ketel RJ, de Wever B, van Alphen L. Detection of Haemophilus influenzae incerebrospinal fluids by polymerase chain reaction DNA amplification [J]. MedMicrobiol, 1990, 33(4): 271-276.
24 Sakamoto N, Kurono Y, Ueyama T, et al. Detection of Haemophilus influenzae inadenoids and nasopharyngeal secretions by polymerase chain reaction [J]. ActaOtolaryngol Suppl, 1996, 523: 145-146.
25 Murphy TF, Brauer AL, Sethi S, et al. Haemophilus haemolyticus: a humanrespiratory tract commensal to be distinguished from Haemophilus influenzae [J].Infect Dis, 2007, 195(1): 81-89.
26 Yao F, Li WY, Kuang Y, et al. Expression and biological activity identification ofrecombinant Hap protein of NTHi [J]. Nan Fang Yi Ke Da Xue Xue Bao, 2010,30(5): 953-956.