Abstract：We constructed eukaryotic expression plasmids of synthetic TAG gene of Pneumocystis p55 antigen to lay the foundation for the of vaccine research of nucleic Pneumocystis pneumonia. In a series of synthetic genes for the antigen gene TAG by double digestion, it was cloned in pIRES-hrGFP-1a eukaryotic expression vector to construct recombinant plasmids as pIRES-hrGFP-1a/TAG. After propagating in E. coli TG-1, the recombinant plasmids were transfected into HEK293 cells. After 48 h incubation, Western blot was performed to identify the expression of pIRES-hrGFP-1a/TAG antigenic gene. Results showed that construction of pIRES-hrGFP-1a/TAG recombinant plasmid was expressed by restriction enzyme digestion and sequencing, and the sequence was verifyed, successfully transfected into HEK293 cells. Western blot was used to test the effective gene expression in HKE293 cells. In this study, the recombinant plasmids of pIRES-hrGFP-1a/TAG are conducted successfully and expressed in the HEK293 cells, which provide a basis for clarification of immunologic function of pIRES-hrGFP-1a/TAG and study of DNA vaccine.
樊华, 国九英, 马素丽, 张楠, 安春丽. 人工合成肺孢子菌p55抗原串联基因真核表达载体的构建及表达[J]. 中国人兽共患病学报, 2015, 31(5): 399-402.
FAN Hua, GUO Jiu-ying, MA Su-li, ZHANG Nan, AN Chun-li. Construction and identification of eukaryotic expression plasmids of synthetic TAG gene of Pneumocystis p55 antigen. Chinese Journal of Zoonoses, 2015, 31(5): 399-402.
[1] Stringer JR, Beard CB, Miller RF, et al. A new name ( Pneumocystis jiroveci ) for Pneumocystis from humans[J]. Emerg Infect Dis, 2002, 8(9): 891-896. doi:10.3201/eid0809.020096 [2] Duchini A, Goss JA, Karpen S, et al. Vaccinations for adult solidorgan transplant recipients: current recommendations and protocols[J]. Clin Microbiol Rev, 2003, 16(3): 357-364. doi:10.1128/CMR.16.3.357-364.2003 [3] Fauci AS. HIV and AIDS: 20 years of science[J]. Nat Med, 2003, 9(7): 839-843. doi:10.1038/nm0703-839 [4] Li XL, Ma XT. Domestic past 5 years 4 153 cases analysis of Pneumocystis pneumonia [J]. J Med Forum, 2014, 35(5): 61-63. (in Chinese) 李晓亮,马希涛.国内近5年肺孢子菌肺炎4 153例分析[J]. 医药论坛杂志,2014,35(5):61-63. [5] Keely SP, Cushion MT, Stringer JR. Diversity at the locus associated with transcription of a variable surface antigen of Pneumocystis carinii as an index of population structure and dynamics in infected rats[J]. Infect Immun, 2003, 71(1): 47-60. doi:10.1128/IAI.71.I.47-60.2003 [6] Smulian AG, Theus SA, Denko N, et al. A 55 kDa antigen of Pneumocystis carinii : analysis of the cellular immune response and characterization of the gene[J]. Mol Microbio, 1993, l7: 745-753. doi:10.1111/j.1365-2958.1993.tb01165.x [7] Theus SA, Sullivan DW, Walzer PD, et al. Cellular responses to a 55-kiloDalton recombinant Pneumocystis carinii antigen[J]. Infect Immun, 1994, 62(8): 3479-3484. [8] Wang XL, Luan HZ, Liu Tong. Cloning of chimericgene of p55 antigen from Pneumocystis and analysis on the expression Product[J]. Chin J Zoonoses, 2011, 27(9): 774-778. doi:10.3969/j.issn.1002-2694.2011.09.002 (in Chinese) 王雪莲, 栾和芝, 刘彤. 肺孢子虫(菌)p55抗原嵌合基因的克隆及表达产物分析[J].中国人兽共患病学报,2011,27(9):774-778. [9] Forte M, Maartens G, Campbell F, et al. T-lymphocyte responses to Pneumocystis carinii in healthy and HIV-positive individuals[J]. J Acquir Immune Defic Syndr, 1992, 5(4): 409-416. [10] Weiss LM, Cushion MT, Didier E, et al. The 12th International Workshops on Opportunistic Protists (IWOP-12)[J]. J Eukaryot Microbiol, 2013, 60: 298-308. doi:10.1111/JEU.12034 [11] Ma L, Kutty G, Jia Q, et al. Characterization of variants of the gene encoding the p55 antigen in Pneumocystis from rats and mice[J]. Med Microbiol, 2003, (52): 955-960. doi:10.1099/jmm.0.05131-0 [12] Theus SA, Linke MJ, Andrews RP, et al. Proliferative and cytokine responses to a major surface glycoprotein of Pneumocystis carinii [J]. Infect Immun, 1993, 61: 4703-4709. [13] Theus SA, Andrews RP, Linke MJ, et al. Characteracterization of rat CD4 T cell clones specific for the major surface glycoprotein of Pneumocystis carinii [J]. Eukaryot Microbiol, 1997, 44: 96-100. doi:10.1111/j.1550-7408.1997.tb05944.x [14] Theus SA, Smulian AG, Steele P, et al. Immunization with the major surface glycoprotein of elicits a protective response[J]. Vaccine, 1998, 16: 1149-1157. doi:10.1016/S0264-410X(98)80113-8 [15] Duan YN, Yi LH, Chen JL, et al. Protective effect of DNA vaccine with the gene encoding 55 kDa antigen fragment against Pneumocystis carinii in mice[J]. Asian Pac J Trop Med, 2011, 4(5): 353-356. doi:10.1016/S1995-7645(11)60102-8 [16] Feng Y, Guo S, Jiang T, et al. Active immunization against Pneumocystis carinii with p55-v3 DNA vaccine in rats[J]. Can J Microbiol, 2011, 57(5): 375-381. doi:10.1139/W11-023
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