收稿日期: 2023-03-21
修回日期: 2023-05-10
网络出版日期: 2023-11-06
基金资助
河北省高等学校科学技术研究项目(2021013);河北省大学生创新创业训练计划(202155)
Immunoprotection of nuclei acid vaccine dual-targeting rhoptry protein 18 and surface antigen 30 of Toxoplasma gondii in mice
Received date: 2023-03-21
Revised date: 2023-05-10
Online published: 2023-11-06
Supported by
Higher Education Science and Technology Research Project of Hebei Province(2021013);College Student Innovation and Entrepreneurship Training Program of Hebei Province(202155)
目的 研究刚地弓形虫棒状体蛋白18（ROP18）和膜表面抗原30（P30）复合核酸疫苗的免疫保护作用。 方法 PCR扩增弓形虫p30和rop18基因，构建pVAX1-p30、pVAX1-rop18-p30质粒并进行PCR鉴定、酶切鉴定、测序鉴定。用LipofectamineTM 3000转染试剂将pVAX1（2 μg）和pVAX1-rop18-p30质粒转染至HeLa细胞内，24 h后间接免疫荧光法检测目的蛋白的表达情况。75只雌性BALB/c小鼠随机分为pVAX1-rop18-p30组、pVAX1-rop18组、pVAX1-p30组、pVAX1组和PBS组，每组15只，分别于股四头肌注射100 μg（100 μl）的pVAX1-rop18-p30、pVAX1-rop18（本实验室保存）、pVAX1-p30、pVAX1质粒或PBS（100 μl），共免疫3次，间隔2周，末次免疫时质粒剂量为200 μg。每次免疫前和末次免疫后2周采集小鼠血样，ELISA检测血清IgG抗体水平。末次免疫后2周每组取3只小鼠，无菌取脾细胞，培养后ELISA检测培养上清中γ干扰素（IFN-γ）和白细胞介素2（IL-2）、IL-4、IL-10、IL-12的水平。末次免疫后2周，每组取12只小鼠，每鼠腹腔注射1 000个弓形虫RH株速殖子进行急性感染，记录小鼠存活时间，评价免疫保护性。组间比较采用单因素方差分析。 结果 弓形虫p30和rop18基因PCR扩增片段分别为789、1 665 bp。pVAX1-rop18-p30质粒经PCR、酶切鉴定均获得预期大小的条带，测序鉴定结果正确。pVAX1-rop18-p30转染HeLa细胞后，胞质内可见黄绿色荧光，pVAX1转染的HeLa细胞内无黄绿色荧光。ELISA检测结果显示，末次免疫后2周，pVAX1-rop18-p30组吸光度（A450值）为0.788 ± 0.025，高于pVAX1-rop18组（0.512 ± 0.027）、pVAX1-p30组（0.498 ± 0.027）、pVAX1组（0.122 ± 0.014）和PBS组（0.109 ± 0.011）（F = 77.6，P < 0.05），pVAX1-rop18组和pVAX1-p30组之间A450值差异无统计学意义（F = 67.80，P > 0.05）；小鼠血清IgG抗体水平随着免疫次数的增加和时间延长而升高。末次免疫后2周，pVAX1-rop18-p30组小鼠脾细胞培养上清中IFN-γ、IL-2、IL-4、IL-10和IL-12的水平分别为（678.77 ± 3.69）、（375.28 ± 2.65）、（130.82 ± 4.72）、（279.68 ± 3.67）和（579.68 ± 3.67）pg/ml，pVAX1-rop18组分别为（448.59 ± 6.52）、（256.34 ± 5.58）、（126.28 ± 7.48）、（156.58 ± 4.59）和（231.50 ± 3.21）pg/ml；pVAX1-p30组分别为（423.67 ± 4.82）、（277.92 ± 4.23）、（115.17 ± 4.37）、（137.18 ± 5.62）和（190.47 ± 4.18）pg/ml，pVAX1组分别为（48.97 ± 2.65）、（47.65 ± 2.76）、（47.14 ± 2.04）、（45.29 ± 2.31）和（46.21 ± 2.17）pg/ml；PBS组分别为（47.69 ± 3.42）、（46.77 ± 3.35）、（48.59 ± 4.75）、（44.92 ± 4.91）和（46.88 ± 3.56）pg/ml。pVAX1-rop18-p30组、pVAX1-rop18组和pVAX1-p30组的IFN-γ、IL-2、IL-4、IL-10、IL-12水平均高于pVAX1组和PBS组（F = 1 582.0、531.5、268.7、215.0、170.5，均P < 0.05）；pVAX1-rop18-p30组的IFN-γ、IL-10、IL-12水平均高于pVAX1-rop18和pVAX1-p30组（F = 1 620.8、1 208.0、728.0，均P < 0.05）；pVAX1-rop18-p30组、pVAX1-rop18组和pVAX1-p30组间的IL-2和IL-4水平差异无统计学意义（F = 53.21，P > 0.05）。弓形虫急性感染后，pVAX1-rop18-p30组、pVAX1-rop18组、pVAX1-p30组、pVAX1组和PBS组小鼠平均存活时间分别为（288 ± 2）、（196 ± 3）、（230 ± 6）、（144 ± 2）和（146 ± 11）h。pVAX1-rop18-p30组、pVAX1-rop18组和pVAX1-p30组小鼠平均存活时间长于pVAX1组和PBS组（F = 100.1，P < 0.05）；pVAX1-rop18-p30组长于pVAX1-rop18组和pVAX1-p30组（F = 38.7，P < 0.05） 结论 pVAX1-rop18-p30复合核酸疫苗能诱导小鼠产生细胞免疫和体液免疫，对弓形虫感染的免疫保护效果优于pVAX1-rop18和pVAX1-p30单基因核酸疫苗。
姜文静 , 孟雅莉 , 赵利娜 , 王春苗 , 张晓磊 . 刚地弓形虫棒状体蛋白18和膜表面抗原30复合核酸疫苗对小鼠的免疫保护作用[J]. 中国寄生虫学与寄生虫病杂志, 2023 , 41(5) : 532 -538 . DOI: 10.12140/j.issn.1000-7423.2023.05.002
Objective To study the immunoprotective effect of a nucleic acid vaccine dual-targeting rhoptry protein 18 (ROP18) and surface antigen 30 (P30) of Toxoplasma gondii. Methods The p30 and rop18 genes of T. gondii were amplified by PCR to construct recombinant plasmids pVAX1-p30 and pVAX1-rop18-p30, which were verified by PCR, enzyme digestion, and sequencing. HeLa cells were transfected with pVAX1 plasmids (2 μg) and pVAX1-rop18-p30 plasmids using LipofectamineTM 3000 transfection reagents, and the expression of target protein was detected by indirect immunofluorescence assay after 24 hours. Seventy-five female BALB/c mice were randomly divided into five groups (pVAX1-rop18-p30 group, pVAX1-rop18 group, pVAX1-p30 group, pVAX1 group, and PBS group), with each group containing 15 mice. The plasmids [100 μg (100 μl)] and PBS (100 μl) were injected into the quadriceps femoris muscle, with a total of three injections at two-week intervals, and the last injection had a plasmid dosage of 200 μg. The levels of serum IgG were quantified by ELISA before each injection and two weeks after the final dose. The spleen cells were collected aseptically from three mice in each group two weeks after the last dose, and the levels of IFN-γ, IL-2, IL-4, IL-10, and IL-12 in the culture supernatant were measured by ELISA. Two weeks after the last immunization, 12 mice were selected from each group, and 1 000 tachyzoites of T. gondii RH strain were injected intraperitoneally into each mouse to induce acute infections. The survival time of the mice was recorded, and their immune protection was evaluated. Single-factor analysis of variance was used for inter-group comparison. Results PCR amplification fragments of p30 and rop18 genes with lengths of 789 bp and 1 665 bp. The pVAX1-rop18-p30 recombinant plasmids were successfully identified through PCR, enzyme digestion, and sequencing. HeLa cells transfected with pVAX1-rop18-p30 exhibited yellow-green fluorescence in the cytoplasm, while no such fluorescence was observed in cells transfected with pVAX1. ELISA results indicated that two weeks after the last immunization, the A450 value in the pVAX1-rop18-p30 group (0.788 ± 0.025) was significantly higher than that in the pVAX1-rop18 group (0.512 ± 0.027), pVAX1-p30 group (0.498 ± 0.027), pVAX1 group (0.122 ± 0.014), and PBS group (0.109 ± 0.011) (F = 77.6, P < 0.05). There was no statistically significant difference in A450 values between the pVAX1-rop18 group and the pVAX1-p30 group (F = 67.80, P > 0.05). The serum IgG levels increased with the frequency and duration of immunization. Two weeks after the final immunization, the levels of IFN-γ, IL-2, IL-4, IL-10, and IL-12 in the supernatant of splenocyte cultures were the highest in the pVAX1-rop18-p30 group [(678.77 ± 3.69), (375.28 ± 2.65), (130.82 ± 4.72), (279.68 ± 3.67), (579.68 ± 3.67) pg/ml], followed by the pVAX1-rop18 group [(448.59 ± 6.52), (256.34 ± 5.58), (126.28 ± 7.48), (156.58 ± 4.59), (231.50 ± 3.21) pg/ml] and pVAX1-p30 group [(423.67 ± 4.82), (277.92 ± 4.23), (115.17 ± 4.37), (137.18 ± 5.62), (190.47 ± 4.18) pg/ml], while the pVAX1 group [(48.97 ± 2.65), (47.65 ± 2.76), (47.14 ± 2.04), (45.29 ± 2.31), (46.21 ± 2.17) pg/ml] and PBS group [(47.69 ± 3.42), (46.77 ± 3.35), (48.59 ± 4.75), (44.92 ± 4.91), (46.88 ± 3.56) pg/ml] showed significantly lower levels (F = 1 582.0, 531.5, 268.7, 215.0, 170.5; all P < 0.05). The levels of IFN-γ, IL-10, IL-12 were elevated in the pVAX1-rop18-p30 group compared with the pVAX1-rop18 group and the pVAX1-p30 group (F = 1 620.8, 1 208.0, 728.0; all P < 0.05), the levels of IL-2 and IL-4 didn’t show significant changes between the pVAX1-rop18-p30 group, the pVAX1-rop18 group and the pVAX1-p30 group (F = 53.21, P > 0.05). After acute infection, the mice mean survival time in the pVAX1-rop18-p30 group, the pVAX1-rop18 group, the pVAX1-p30 group, the pVAX1 group and the PBS group were (288 ± 2), (196 ± 3), (230 ± 6), (144 ± 2), (146 ± 11) h, the mean survival time of mice in the pVAX1-rop18-p30 group was significantly longer than that in the pVAX1 group and PBS group (F = 100.1, P < 0.05), as well as the pVAX1-rop18 and pVAX1-p30 groups (F = 38.7, P < 0.05). Conclusion The pVAX1-rop18-p30 dual-targeting nucleic acid vaccine could induce cellular and humoral immunity in mice; its immunprotectiv effect against T. gondii infection is better than that of the vaccine mono-targeting either pVAX1-rop18 or pVAX1-p30.
| [1] | Mutka T, Seyfang A, Yoo JY, et al. Adverse pregnancy outcomes in Toxoplasma gondii seropositive Hispanic women[J]. J Obstet Gynaecol Res, 2023, 49(3): 893-903. |
| [2] | Wang JL, Zhang NZ, Li TT, et al. Advances in the development of anti-Toxoplasma gondii vaccines: challenges, opportunities, and perspectives[J]. Trends Parasitol, 2019, 35(3): 239-253. |
| [3] | El Hajj H, Lebrun M, Fourmaux MN, et al. Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane[J]. Cell Microbiol, 2007, 9(1): 54-64. |
| [4] | Anuradha B, Preethi C. Seroprevalence of Toxoplasma IgG antibodies in HIV positive patients in and around Khammam, Telangana State[J]. J Clin Diagn Res, 2014, 8(9): DL01-DL02. |
| [5] | Lopes AP, Granada S, Oliveira AC, et al. Toxoplasmosis in dogs: first report of Toxoplasma gondii infection in any animal species in Angola[J]. Pathog Glob Health, 2014, 108(7): 344-346. |
| [6] | Luo HQ, Li K, Zhang H, et al. Seroprevalence of Toxoplasma gondii infection in zoo and domestic animals in Jiangxi Province, China[J]. Parasite, 2017, 24: 7. |
| [7] | Nzelu IN, Kwaga JKP, Kabir J, et al. Detection and genetic characterisation of Toxoplasma gondii circulating in free-range chickens, pigs and seropositive pregnant women in Benue State, Nigeria[J]. PLoS Negl Trop Dis, 2021, 15(6): e0009458. |
| [8] | Kim K, Weiss LM. Toxoplasma: the next 100 years[J]. Microbes Infect, 2008, 10(9): 978-984. |
| [9] | Boothroyd JC, Dubremetz JF. Kiss and spit: the dual roles of Toxoplasma rhoptries[J]. Nat Rev Microbiol, 2008, 6(1): 79-88. |
| [10] | Zhang X, Gan XF, Cheng ZY, et al. Advances in the study of rhoptry proteins 2 of Toxoplasma gondii[J]. J Pathog Biol, 2015, 10(6): 574-576, 579. (in Chinese) |
| (张贤, 甘小凤, 程正阳, 等. 弓形虫棒状体蛋白2家族成员研究进展[J]. 中国病原生物学杂志, 2015, 10(6): 574-576, 579.) | |
| [11] | Fentress SJ, Sibley LD. The secreted kinase ROP18 defends Toxoplasma’s border[J]. Bioessays, 2011, 33(9): 693-700. |
| [12] | El Hajj H, Demey E, Poncet J, et al. The ROP2 family of Toxoplasma gondii rhoptry proteins: proteomic and genomic characterization and molecular modeling[J]. Proteomics, 2006, 6(21): 5773-5784. |
| [13] | Yuan ZG, Zhang XX, Lin RQ, et al. Protective effect against toxoplasmosis in mice induced by DNA immunization with gene encoding Toxoplasma gondii ROP18[J]. Vaccine, 2011, 29(38): 6614-6619. |
| [14] | Xu TJ, Li YK, Lin XL, et al. Effect of molecular adjuvant Ov-ASP-1 on SAG1 nucleic acid vaccine of Toxoplasma gondii[J]. Heilongjiang Anim Sci Vet Med, 2019(5): 118-122. (in Chinese) |
| (徐焘杰, 李元凯, 林霞琳, 等. 分子佐剂Ov-ASP-1对弓形虫SAG1核酸疫苗的影响[J]. 黑龙江畜牧兽医, 2019(5): 118-122.) | |
| [15] | Debard N, Buzoni-Gatel D, Bout D. Intranasal immunization with SAG1 protein of Toxoplasma gondii in association with cholera toxin dramatically reduces development of cerebral cysts after oral infection[J]. Infect Immun, 1996, 64(6): 2158-2166. |
| [16] | Nielsen HV, Lauem?ller SL, Christiansen L, et al. Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene[J]. Infect Immun, 1999, 67(12): 6358-6363. |
| [17] | Qi NS, Sun MF, Liao SQ, et al. Invasion-associated factors of apicomplexa[J]. Chin J Anim Vet Sci, 2012, 43(2): 167-174. (in Chinese) |
| (戚南山, 孙铭飞, 廖申权, 等. 顶复门原虫入侵相关因子的研究进展[J]. 畜牧兽医学报, 2012, 43(2): 167-174.) | |
| [18] | Li B, Wei F, Liu Q, et al. Construction and identification of Toxoplasma gondii ROP18 in transgenic Leishmania tarentolae[J]. Prog Vet Med, 2012, 33(2): 1-4. (in Chinese) |
| (李博, 魏峰, 刘全, 等. 弓形虫ROP18基因重组蜥蜴利什曼原虫的构建及鉴定[J]. 动物医学进展, 2012, 33(2): 1-4.) | |
| [19] | Garcia JL. Vaccination concepts against Toxoplasma gondii[J]. Expert Rev Vaccines, 2009, 8(2): 215-225. |
| [20] | Chen J, Li ZY, Petersen E, et al. DNA vaccination with genes encoding Toxoplasma gondii antigens ROP5 and GRA15 induces protective immunity against toxoplasmosis in Kunming mice[J]. Expert Rev Vaccines, 2015, 14(4): 617-624. |
| [21] | Guo LL, Zhang XL, Zhang JS, et al. Construction of eukaryotic expression vector containing rop18-rop12 of Toxoplasma gondii RH strain[J]. Chin J Parasitol Parasit Dis, 2015, 33(3): 161-166. (in Chinese) |
| (郭玲玲, 张晓磊, 张进顺, 等. 刚地弓形虫RH株rop18-rop12复合基因真核表达载体的构建[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(3): 161-166.) | |
| [22] | Liu RR, Zhang XL, Zhang JS, et al. Construction and identification of a dual promoter eukaryotic expression vector for rop10 and rop18 of Toxoplasma gondii[J]. J Pathog Biol, 2018, 13(6): 612-616. (in Chinese) |
| (刘荣荣, 张晓磊, 张进顺, 等. 刚地弓形虫rop10-rop18真核表达载体的构建与鉴定[J]. 中国病原生物学杂志, 2018, 13(6): 612-616.) | |
| [23] | Zhao HZ, Li LH, Song ZX, et al. Research progress of genetic engineering vaccine for Toxoplasma gondii[J]. Hubei J Anim Vet Sci, 2019, 40(4): 10-12. (in Chinese) |
| (赵海忠, 李良华, 宋忠旭, 等. 弓形虫基因工程疫苗研究进展[J]. 湖北畜牧兽医, 2019, 40(4): 10-12.) | |
| [24] | Kalef DA. Leishmania mexicana recombinant filamentous acid phosphatase as carrier for Toxoplasma gondii surface antigen 1 expression in Leishmania tarentolae[J]. J Parasi Dis, 2021, 45(4): 1135-1144. |
| [25] | Zhan GQ, Wu ST, Li GG, et al. Study on the cellular immune responses induced by p30 DNA vaccine of Toxoplasma gondii in mice[J]. Chin J Parasit Dis Contr, 2001(4): 257-259. (in Chinese) |
| (占国清, 吴少庭, 李国光, 等. 弓形虫表面抗原p30 DNA疫苗免疫小鼠诱导细胞免疫应答的研究[J]. 中国寄生虫病防治杂志, 2001(4): 257-259.) | |
| [26] | Angus CW, Klivington-Evans D, Dubey JP, et al. Immunization with a DNA plasmid encoding the SAG1 (P30) protein of Toxoplasma gondii is immunogenic and protective in rodents[J]. J Infect Dis, 2000, 181(1): 317-324. |
| [27] | Zhuang HH, Pan LT, Yao CQ, et al. Preparation of Toxoplasma gondii SAG1 recombinant antibody and identification of its epitope[J]. Chin J Vet Sci, 2021, 41(6): 1080-1085, 1104. (in Chinese) |
| (庄浩瀚, 潘灵韬, 姚晨倩, 等. 弓形虫SAG1重组抗体制备及抗原表位鉴定[J]. 中国兽医学报, 2021, 41(6): 1080-1085, 1104.) | |
| [28] | Zhao HG, Fan ZG, Huang FY, et al. Immune response in mice induced by complex gene vaccine pcSAG1-ROP5 of Toxoplasma gondii[J]. Chin J Parasitol Parasit Dis, 2013, 31(1): 1-5. (in Chinese) |
| (赵焕阁, 范志刚, 黄风迎, 等. 弓形虫复合基因疫苗pcSAG1-ROP5诱导小鼠免疫应答的研究[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(1): 1-5.) | |
| [29] | Qi TN, Shi C, Wang ZY, et al. Construction and expression of eukaryotic expression recombinant plasmid of SAG1-GRA1 gene of Toxoplasma gondii[J]. J Guizhou Med Univ, 2020, 45(3): 286-291. (in Chinese) |
| (綦廷娜, 石畅, 汪政勇, 等. 弓形虫SAG1-GRA1复合基因真核表达重组质粒的构建与表达[J]. 贵州医科大学学报, 2020, 45(3): 286-291.) | |
| [30] | Wu LM, Yang HJ, Wang JL, et al. A novel combined DNA vaccine encoding Toxoplasma gondii SAG1 and ROP18 provokes protective immunity against a lethal challenge in mice[J]. Acta Parasitol, 2021, 66(4): 1387-1395. |
| [31] | Ni XT. Construction of recombinant Lactobacillus plantarum expressing ROP18 and SAG1 and their protective efficacy against Toxoplasma gondii infection[D]. Lanzhou: Gansu Agricultural University, 2016: 30-36. (in Chinese) |
| (倪晓婷. 弓形虫ROP18和SAG1乳酸杆菌活载体疫苗株的构建及其免疫效果评价[D]. 兰州: 甘肃农业大学, 2016: 30-36.) |
/
| 〈 |
|
〉 |