Construction and immunological evaluation of recombinant Salmonella choleraesuis expressing GAPDH and OmP26 of Glaesserella parasuis
-
摘要:目的
构建能同时表达副猪格拉瑟菌Glaesserella parasuis关键抗原GAPDH和OmP26的重组猪霍乱沙门氏菌Salmonella choleraesuis，并评估其作为二联疫苗候选株的潜力，以期为预防G. parasuis和S. choleraesuis感染提供一种新型高效的解决方法。
方法选取多种血清型的G. parasuis中广泛存在的抗原GAPDH和OmP26作为外源抗原，以S. choleraesuis C500Δasd缺失株为载体，构建同时表达GAPDH和OmP26的G. parasuis-S. choleraesuis重组菌株，并对其生物学特性和免疫效果展开研究。
结果PCR与测序结果共同表明，本研究成功构建重组菌株C501(pYA-GAPDH-OmP26)，该菌株能稳定携带GAPDH和OmP26基因片段(大小分别为1 020和798 bp)，在连续传代100次中均能稳定扩增目标片段；且生长曲线、生化特性与对照菌C501(pYA3493)一致。重组菌株C501(pYA-GAPDH-OmP26)对S. choleraesuis C78-1和G. parasuis 5型强毒株SH0165的保护率分别为62.5%和50.0%，而C501(pYA3493)对S. choleraesuis C78-1的保护率为50.0%，对G. parasuis 5型强毒株SH0165则无保护作用。
结论重组沙门氏菌C501(pYA-GAPDH-OmP26)能稳定携带异源基因，具有与亲本菌株相近的生物学特性及良好的表达特性，能够诱导机体对G. parasuis和S. choleraesuis产生联合免疫反应，为G. parasuis-S. choleraesuis二联基因工程疫苗的研发奠定了基础。
Abstract:ObjectiveThe objective of this study was to construct a recombinant Salmonella choleraesuis strain co-expressing GAPDH and OmP26, two immunogenic antigens derived from Glaesserella parasuis, evaluate its potential as a bivalent vaccine candidate, and provide a novel and efficacious solution for the prevention of both G. parasuis and S. choleraesuis infections in swine populations.
MethodGAPDH and OmP26, which are widely present in various serotypes of G. parasuis, were selected as exogenous antigens. A recombinant strain of G. parasuis-S. choleraesuis, which was capable of expressing both the GAPDH and OmP26, was constructed by using S. choleraesuis C500Δasd deletion strain as vector. The biological characteristic and immune effect of the recombinant strain were then investigated.
ResultResults of PCR and Sanger-sequencing showed that we successfully constructed the recombinant strain C501 (pYA-GAPDH-OmP26), which was able to stably harbor GAPDH and OmP26 (sizes of 1 020 and 798 bp, respectively) . The target fragments were stably amplified from the strain in 100 consecutive passages, and the recombinant strain was consistent with the parent strain C501(pYA3493) in terms of growth curves and biochemical characteristics. The recombinant strain C501 (pYA-GAPDH-OmP26) showed 62.5% and 50.0% protection rates against S. choleraesuis C78-1 and G. parasuis type 5 strong strain SH0165, respectively, while C501 (pYA3493) showed 50.0% protection rate against S. choleraesuis C78-1 and no protection effect against G. parasuis type 5 strong strain SH0165.
ConclusionThe recombinant S. choleraesuis C501 (pYA-GAPDH-OmP26) can stably carry heterologous genes. Compared with the parent strain, the recombinant strain has similar biological and good expression characteristics, can induce the organism’s combined immune response against G. parasuis and S. choleraesuis. The study lays the foundation for the development of the bivalent genetically engineered vaccine for G. parasuis and S. choleraesuis.
-
Keywords:
- Pig /
- Glaesserella parasuis /
- Salmonella choleraesuis /
- C500Δasd deletion strain /
- GAPDH /
- OmP26
-
![]()
图 1 目的基因PCR扩增片段
Figure 1. PCR amplified fragments of target genes
M: DNA Marker, 1: GAPDH, 2: OmP26.
![]()
图 2 重组质粒pYA-GAPDH和pYA-OmP26的酶切鉴定
Figure 2. Enzymatic identification of recombinant plasmids pYA-GAPDH and pYA-OmP26
M: DNA Marker, 1: pYA-GAPDH, 2: pYA-OmP26.
![]()
图 3 重组菌株遗传稳定性
M：DNA Marker；1~4：C501(pYA-GAPDH) 25、50、75、100代；6~9：C501(pYA-OmP26) 25、50、75、100代；11~14：C501(pYA-GAPDH-OmP26) 25、50、75、100代；5、10：pYA3493。
Figure 3. Genetic stability of recombinant strains
M: DNA Marker; 1−4: C501(pYA-GAPDH) 25, 50, 75, 100 generations; 6−9: C501(pYA-OmP26) 25, 50, 75, 100 generations; 11−14: C501(pYA-GAPDH-OmP26) 25, 50, 75, 100 generations; 5, 10: pYA3493.
![]()
图 4 重组菌株C501(pYA-GAPDH-OmP26)与对照菌株C501(pYA3493)的生长曲线
Figure 4. Growth curves of recombinant strain C501 (pYA-GAPDH-OmP26) and control strain C501 (pYA3493)
![]()
图 5 重组菌株Western-Blot分析
Mr：相对分子质量，M：Marker，1：C501(pYA3493)，2：C501(pYA-GAPDH)，3：C501(pYA-OmP26)。
Figure 5. Western-Blot analysis of recombinant strains
Mr: Relative molecular mass, M: Marker, 1: C501(pYA3493), 2: C501(pYA-GAPDH), 3: C501(pYA-OmP26).
表 1 重组沙门氏菌对不同碳源的利用情况1)
Table 1 Utilization of different carbon sources by recombinant Salmonella spp.
菌株
Strain阿拉伯糖Arabinose 乳糖Lactose 棉子糖Raffinose 山梨醇Sorbitol 淀粉Starch 半乳糖Galactose 葡萄糖Glucose 甘露醇Mannitol 果糖Fructose 鼠李糖Rhamnose 麦芽糖Maltose C501(pYA3493) − − − − − − + + + + + C501(pYA-GAPDH) − − − − − − + + + + + C501(pYA-OmP26) − − − − − − + + + + + C501(pYA-GAPDH-OmP26) − − − − − − + + + + + 1) −：阴性；+：阳性。
1) −: Negative; +: Positive.
下载: 导出CSV
-
[1] 赵谦. 猪链球菌病-副猪嗜血杆菌病二联亚单位疫苗的研制[D]. 北京: 中国农业科学院, 2019. [2] OLVERA À, CALSAMIGLIA M, ARAGON V. Genotypic diversity of Haemophilus parasuis field strains[J]. Applied and Environmental Microbiology, 2006, 72(6): 3984-3992. doi: 10.1128/AEM.02834-05
[3] 薛国聪, 任涛. 副猪嗜血杆菌病流行病学及致病因子的研究进展[J]. 中国畜牧兽医, 2009, 36(5): 168-171. [4] 蒋征, 李军星, 姜平, 等. 副猪嗜血杆菌病分离与基因分型鉴定[J]. 畜牧与兽医, 2008, 40(6): 47-49. [5] ZHANG P, ZHANG C Y, ARAGON V, et al. Investigation of Haemophilus parasuis from healthy pigs in China[J]. Veterinary Microbiology, 2019, 231: 40-44. doi: 10.1016/j.vetmic.2019.02.034
[6] ZHAO Y D, GUO L L, LI J, et al. Characterization of antimicrobial resistance genes in Haemophilus parasuis isolated from pigs in China[J]. PeerJ, 2018, 6: e4613. doi: 10.7717/peerj.4613
[7] 董婉玉, 王馨雨, 张聚民, 等. 2020年~2021年我国副猪嗜血杆菌和链球菌的血清型、耐药基因型流行情况分析[J]. 中国预防兽医学报, 2022, 44(10): 1045-1051. [8] 穆永才, 罗天瑶, 徐铭. 副猪嗜血杆菌病多价油乳剂灭活疫苗的研制[J]. 黑龙江畜牧兽医, 2015(6): 108-109. [9] 王金合, 石冬梅, 郭素琴, 等. 副猪嗜血杆菌的分离鉴定及自家灭活苗的研制[J]. 中国畜牧兽医, 2009, 36(8): 144-147. [10] 张昆丽, 李春玲. 副猪嗜血杆菌病研究进展[J]. 广东农业科学, 2020, 47(12): 166-174. [11] 孔里程, 王兆飞, 孙建和. 猪链球菌保护性抗原表位串联亚单位疫苗设计及小鼠免疫保护效力分析[J]. 上海交通大学学报(农业科学版), 2019, 37(3): 1-8. [12] FU S L, ZHANG M M, OU J W, et al. Construction and immune effect of Haemophilus parasuis DNA vaccine encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mice[J]. Vaccine, 2012, 30(48): 6839-6844. doi: 10.1016/j.vaccine.2012.09.014
[13] MINIATS O P, SMART N L, ROSENDAL S. Cross protection among Haemophilus parasuis strains in immunized gnotobiotic pigs[J]. Canadian Journal of Veterinary Research, 1991, 55(1): 37-41.
[14] LI M, LI C L, SONG S, et al. Development and antigenic characterization of three recombinant proteins with potential for Glässer’s disease prevention[J]. Vaccine, 2016, 34(19): 2251-2258. doi: 10.1016/j.vaccine.2016.03.014
[15] ZHANG B, TANG C, YANG F L, et al. Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis[J]. Veterinary Microbiology, 2009, 136(3/4): 408-410.
[16] 刘栋, 薛少华, 龙凡利, 等. 副猪嗜血杆菌OMP5抗原免疫刺激复合物的制备及其对小鼠免疫功能的影响[J]. 黑龙江畜牧兽医, 2023(9): 67-72. [17] MARTÍNEZ-MARTÍNEZ S, FRANDOLOSO R, FERRI E F R, et al. Immunoproteomic analysis of the protective response obtained with subunit and commercial vaccines against Glässer’s disease in pigs[J]. Veterinary Immunology and Immunopathology, 2013, 151(3/4): 235-247.
[18] 赵战勤, 徐引弟, 吴斌, 等. 猪霍乱沙门氏菌ΔasdC500株的生物学特性及作为活疫苗表达载体的应用[J]. 生物工程学报, 2009, 25(1): 29-36. [19] 徐引弟, 郭爱珍, 刘维红, 等. 绿荧光蛋白基因在猪霍乱沙门氏菌C500株asd−缺失株平衡致死载体系统中的表达[J]. 中国兽医学报, 2007, 27(4): 493-496. [20] 赵战勤, 王臣, 丁轲, 等. 表达T+ Pm保护性抗原的重组猪霍乱沙门氏菌C500株的构建及其生物学特性[J]. 微生物学报, 2010, 50(1): 91-97. [21] TAKAHASHI K, NAGA S, YAGIHASHI T, et al. A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions[J]. Journal of Veterinary Medical Science, 2001, 63(5): 487-491. doi: 10.1292/jvms.63.487
[22] PANG M N, TU T, WANG Y, et al. Design of a multi-epitope vaccine against Haemophilus parasuis based on pan-genome and immunoinformatics approaches[J]. Frontiers in Veterinary Science, 2022, 9: 1053198. doi: 10.3389/fvets.2022.1053198.
[23] 房晓文, 李扬陇, 黄昌炳, 等. 仔猪副伤寒弱毒菌苗的研究[J]. 畜牧兽医学报, 1981, 12(2): 29-34. [24] 韩国全, 郭万柱, 林华, 等. 减毒猪霍乱沙门菌C500作为基因疫苗运载体的研究现状[J]. 猪业科学, 2009, 26(6): 80-83. [25] 胡娇. 猪霍乱沙门菌C500Δasd缺失株的构建及其作为疫苗活载体的初步应用[D]. 扬州: 扬州大学, 2010. [26] XU Y D, GUO A Z, LIU W H, et al. Construction and characterization of delta crp delta asd mutant host-vector balanced lethal system of Salmonella choleraesuis C500 strain[J]. Chinese Journal of Biotechnology, 2006, 22(3): 366-372.
[27] FRANDOLOSO R, MARTÍNEZ-MARTÍNEZ S, RODRÍGUEZ-FERRI E F, et al. Haemophilus parasuis subunit vaccines based on native proteins with affinity to porcine transferrin prevent the expression of proinflammatory chemokines and cytokines in pigs[J]. Clinical & Developmental Immunology, 2013, 2013: 132432. doi: 10.1155/2013/132432.
