牙龈卟啉单胞菌HA2基因和白细胞介素IL-15基因重组质粒的构建
|
摘要
构建抗牙龈卟啉单胞菌的牙周炎基因疫苗p VAX1-HA2、pVAX1-HA2/IL-15,体外检测其在293T细胞的表达。以HA2基因(牙龈卟啉单胞菌牙龈素—血凝素基因编码区的核心功能区)为目的基因与IL-15基因为免疫佐剂构建真核表达质粒,用Lip2000介导瞬时转染293T细胞,RT-PCR检测目的基因mRNA水平及酶联免疫吸附试验检测IL-15蛋白表达水平。重组质粒p VAX1-HA2、pVAX1-HA2/IL-15经酶切及DNA测序鉴定构建正确,转染的293T细胞能够检测到目的基因的表达,也可以检测到IL-15蛋白的表达。说明我们成功构建了真核共表达质粒pVAX1-HA2和p VAX1-HA2/IL-15,为下一步研制抗牙龈卟啉单胞菌DNA疫苗奠定了基础。
The periodontitis gene vaccines, pVAX1-HA2 and p VAX1-HA2/IL-15 against Porphyromonas gingivalis were constructed and their expression in 293 T cells was detected in vitro. Eukaryotic expression plasmid was constructed with HA2 gene(core functional region of gingipain-hemagglutinin gene coding region of Porphyromonas gingivalis) as target gene and IL-15 gene as immune adjuvants, Lip2000 mediated transient transfection of 293 T cells, RT-PCR was used to detect the mRNA level of target gene and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of IL-15 protein. The recombinant plasmid pVAX1-HA2, pVAX1-HA2/IL-15 was constructed correctly by enzyme digestion and DNA sequencing, the transfected 293 T cells were able to detect the expression of the target gene, and the expression of IL-15 protein was also detected. These results indicated that eukaryotic co-expression plasmids pVAX1-HA2 and pVAX1-HA2/IL-15 were successfully constructed, which would lay the foundation for the further development of DNA vaccine against Porphyromonas gingivalis.
The periodontitis gene vaccines, pVAX1-HA2 and p VAX1-HA2/IL-15 against Porphyromonas gingivalis were constructed and their expression in 293 T cells was detected in vitro. Eukaryotic expression plasmid was constructed with HA2 gene(core functional region of gingipain-hemagglutinin gene coding region of Porphyromonas gingivalis) as target gene and IL-15 gene as immune adjuvants, Lip2000 mediated transient transfection of 293 T cells, RT-PCR was used to detect the mRNA level of target gene and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of IL-15 protein. The recombinant plasmid pVAX1-HA2, pVAX1-HA2/IL-15 was constructed correctly by enzyme digestion and DNA sequencing, the transfected 293 T cells were able to detect the expression of the target gene, and the expression of IL-15 protein was also detected. These results indicated that eukaryotic co-expression plasmids pVAX1-HA2 and pVAX1-HA2/IL-15 were successfully constructed, which would lay the foundation for the further development of DNA vaccine against Porphyromonas gingivalis.
引文
Anaya-Bergman C.,Rosato A.,and Lewis J.P.,2015,Iron-and hemin-dependent gene expression of Porphyromonas gingivalis,Mol.Oral Microbiol.,30(1):39-61
Geng X.Y.,Sun L.,and Yang Q.B.,2013,Analysis of hemin-binding sites of Porphyromonas gingivalis hemagglutinin-2,Zhongguo Weishengtaixue Zazhi(Chinese Journal of Microecology),25(5):558-560(耿效宇,孙璐,杨秋波,2013,牙龈卟啉单胞菌血凝素2与氯化血红素结合位点分析,中国微生态学杂志,25(5):558-560)
Ghosh A.K.,Sinha D.,Mukherjee S.,Biswas R.,and Biswas T.,2 016,IL-15 temporally reorients IL-10 biased B-1a cells toward IL-12 expression,Cellular and Molecular Immunology,13:229-239
Hiroi T.,Yanagita M.,Ohta N.,Sakaue G.,and Kiyono H.,2000,IL-15 and IL-15 receptor selectively regulate differentiation of common mucosal immune system-independent B-1 cells for Ig A responses,J.Immunol.,165(8):4329-4337
Inagaki S.,Ishihara K.,Yasaki Y.,Yamada S.,and Okuda K.,2003,Antibody responses of periodontitis patients to gingipains of Porphyromonas gingivalis,J.Periodontol.,74(10):1432-1439
Li N.,and Collyer C.A.,2011,Ginpipains from Porphyromonas gingivalis-complex domaim structures confer diverse functions,Eur J Microbiol Immunol(Bp),1(1):41-58
Lin M.,Yang Q.B.,and Yang S.H.,2009,Construction of the hemagglutinin-2-deficient mutant of Porphyromonas gingi-%valis,Zhonghua Kouqiang Yixue Zazhi(Chinese Journal of Stomatology),44(11):672-676(林梅,杨秋波,杨圣辉,2009,牙龈卟啉单胞菌的血凝素2基因缺陷型突变株的构建,中华口腔医学杂志,44(11):672-676)
Liu R.J.,Geng X.Y.,Liu Y.,and Yang Q.B.,2014,The growth inhibition of hemin-binding polypeptide fragments derived from Porphyromonas gingivalis hemagglutinin-2 on Porphyromonas gingivalis,Zhon gguo Weishegntaixue Zazhi(Chinese Journal of Microecology),26(4):425-428(刘瑞杰,耿效宇,刘颖李金陆,杨秋波,2014,牙龈卟啉单胞菌血凝素2氯化血红素结合位点多肽对牙龈卟啉单胞菌生长抑制作用,中国微生态学杂志,26(4):425-428)
Rangarajan M.,Aduse-Opoku J.,Hashim A.,McPhail G.,Luklinska Z.,Florencia Haurat M.,Feldman M.F.,and Curtis M.A.,2017,LptO(PG0027)is required for Lipid A 1-Phosphatase activity in Porphyromonas gingivalis W50,J.of Bacteriol.,99(11):e00751-16
Ryoma N.,Shogo T.,Saori K.,Minoru Y.,Haruo W.,Makoto O.,and Hidenobu S.,2014,Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses,Microbes Infect,16(1):6-16
Yang Q.B.,Yu F.Y.,Sun L.,Zhang Q.X.,and Lin M.,2015,I-dentification of Amino Acid Residues Involved in Hemin Binding in Porphyromonas gingivalis Hemagglutinin 2,Mol.Oral Microbiol.,30(5):337-346
Zhang Y.,Ai L.S.,and Lin L.,2015,Research prowess in thallus surface virulence factors and pathogenesis of Porphyrom-%onas gingivalis,Zhongguo Shiyong Kouqiangke Zazhi(Chinese Journal of Practical Stomatology),(1):43-47(张宇,艾丽思,林莉,2015,牙龈卟啉单胞菌菌体表面毒力因子及其致病性研究进展,中国实用口腔科杂志,(1):43-47)
Zhao Q.,Geng X.Y.,Liu Y.,Zhang Z.L.,and Yang Q.B.,2015,Inhibition of amino acid sequence of hemin-binding sites of Porphyromonas gingivalis hemagglutinin-2 against infection and alveolar bone absorption in mice,Zhongguo Weishengtaixue Zazhi(Chinese Journal of Microecology),(11):1284-1299(赵茜,耿效宇,刘颖,张子路,杨秋波,2015,多肽免疫抑制牙龈卟啉单胞菌引起的小鼠感染及牙槽骨吸收作用的研究,中国微生态学杂志,(11):1284-1299)
Geng X.Y.,Sun L.,and Yang Q.B.,2013,Analysis of hemin-binding sites of Porphyromonas gingivalis hemagglutinin-2,Zhongguo Weishengtaixue Zazhi(Chinese Journal of Microecology),25(5):558-560(耿效宇,孙璐,杨秋波,2013,牙龈卟啉单胞菌血凝素2与氯化血红素结合位点分析,中国微生态学杂志,25(5):558-560)
Ghosh A.K.,Sinha D.,Mukherjee S.,Biswas R.,and Biswas T.,2 016,IL-15 temporally reorients IL-10 biased B-1a cells toward IL-12 expression,Cellular and Molecular Immunology,13:229-239
Hiroi T.,Yanagita M.,Ohta N.,Sakaue G.,and Kiyono H.,2000,IL-15 and IL-15 receptor selectively regulate differentiation of common mucosal immune system-independent B-1 cells for Ig A responses,J.Immunol.,165(8):4329-4337
Inagaki S.,Ishihara K.,Yasaki Y.,Yamada S.,and Okuda K.,2003,Antibody responses of periodontitis patients to gingipains of Porphyromonas gingivalis,J.Periodontol.,74(10):1432-1439
Li N.,and Collyer C.A.,2011,Ginpipains from Porphyromonas gingivalis-complex domaim structures confer diverse functions,Eur J Microbiol Immunol(Bp),1(1):41-58
Lin M.,Yang Q.B.,and Yang S.H.,2009,Construction of the hemagglutinin-2-deficient mutant of Porphyromonas gingi-%valis,Zhonghua Kouqiang Yixue Zazhi(Chinese Journal of Stomatology),44(11):672-676(林梅,杨秋波,杨圣辉,2009,牙龈卟啉单胞菌的血凝素2基因缺陷型突变株的构建,中华口腔医学杂志,44(11):672-676)
Liu R.J.,Geng X.Y.,Liu Y.,and Yang Q.B.,2014,The growth inhibition of hemin-binding polypeptide fragments derived from Porphyromonas gingivalis hemagglutinin-2 on Porphyromonas gingivalis,Zhon gguo Weishegntaixue Zazhi(Chinese Journal of Microecology),26(4):425-428(刘瑞杰,耿效宇,刘颖李金陆,杨秋波,2014,牙龈卟啉单胞菌血凝素2氯化血红素结合位点多肽对牙龈卟啉单胞菌生长抑制作用,中国微生态学杂志,26(4):425-428)
Rangarajan M.,Aduse-Opoku J.,Hashim A.,McPhail G.,Luklinska Z.,Florencia Haurat M.,Feldman M.F.,and Curtis M.A.,2017,LptO(PG0027)is required for Lipid A 1-Phosphatase activity in Porphyromonas gingivalis W50,J.of Bacteriol.,99(11):e00751-16
Ryoma N.,Shogo T.,Saori K.,Minoru Y.,Haruo W.,Makoto O.,and Hidenobu S.,2014,Effect of Porphyromonas gingivalis outer membrane vesicles on gingipain-mediated detachment of cultured oral epithelial cells and immune responses,Microbes Infect,16(1):6-16
Yang Q.B.,Yu F.Y.,Sun L.,Zhang Q.X.,and Lin M.,2015,I-dentification of Amino Acid Residues Involved in Hemin Binding in Porphyromonas gingivalis Hemagglutinin 2,Mol.Oral Microbiol.,30(5):337-346
Zhang Y.,Ai L.S.,and Lin L.,2015,Research prowess in thallus surface virulence factors and pathogenesis of Porphyrom-%onas gingivalis,Zhongguo Shiyong Kouqiangke Zazhi(Chinese Journal of Practical Stomatology),(1):43-47(张宇,艾丽思,林莉,2015,牙龈卟啉单胞菌菌体表面毒力因子及其致病性研究进展,中国实用口腔科杂志,(1):43-47)
Zhao Q.,Geng X.Y.,Liu Y.,Zhang Z.L.,and Yang Q.B.,2015,Inhibition of amino acid sequence of hemin-binding sites of Porphyromonas gingivalis hemagglutinin-2 against infection and alveolar bone absorption in mice,Zhongguo Weishengtaixue Zazhi(Chinese Journal of Microecology),(11):1284-1299(赵茜,耿效宇,刘颖,张子路,杨秋波,2015,多肽免疫抑制牙龈卟啉单胞菌引起的小鼠感染及牙槽骨吸收作用的研究,中国微生态学杂志,(11):1284-1299)