Immunological Efficacy of Streptococcus agalactiae SIP Oral Vaccine on Tilapia (Oreochromis niloticus)
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摘要: 为研究无乳链球菌Streptococcus agalactiae表面免疫蛋白（surface immunogenic protein，SIP）微胶囊口服疫苗对罗非鱼Oreochromis niloticus的免疫效果，将制备的无乳链球菌SIP微胶囊疫苗口服免疫健康的罗非鱼，以注射免疫无乳链球菌灭活疫苗为阳性对照，口服包埋PBS的微胶囊颗粒为阴性对照，分别于免疫后0、7、14、28 d取脾脏组织，通过实时荧光定量PCR方法检测脾脏组织中的白细胞分化抗原CD4、白介素IL-8、IL-10、肿瘤坏死因子TNF-α、核转录因子NF-κB等5种免疫相关基因表达的变化，并于第5周进行人工感染攻毒试验，计算无乳链球菌SIP微胶囊口服疫苗对罗非鱼的相对免疫保护率。结果显示，免疫接种之后，SIP微胶囊疫苗口服免疫组（SIP疫苗组）和全菌灭活疫苗注射免疫组（灭活疫苗组）罗非鱼脾脏中的5种免疫相关基因表达均不同程度上调，且相对表达量显著高于阴性对照免疫组（阴性对照组）（P < 0.05）。在人工感染攻毒试验中，SIP疫苗组和灭活疫苗组的死亡率均显著低于阴性对照组（P < 0.05），灭活疫苗组比SIP疫苗组得到了更高的免疫保护率，但两组的死亡率差异不显著（P>0.05）。说明罗非鱼无乳链球菌SIP微胶囊口服疫苗免疫效果好，在罗非鱼无乳链球菌病的免疫防治中有良好的应用前景。Abstract: The immunological efficacy of Streptococcus agalactiae surface immunogenic protein (SIP) oral vaccine on tilapia (Oreochromis niloticus) was studied. Randomly grouped fish were injected with the inactivated vaccine as the positive control (Group Inactivated Vaccine), or orally administered with SIP vaccine (Group SIP Vaccine) for treatment or PBS as the negative control (Group Negative Control). Spleen samples from the tilapias were collected at 0, 7, 14 and 28 d after the immunization. The gene expressions of the cluster determinant 4 (CD4), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor α(TNF-α) and nuclear transcription factors κB(NF-κB) were determined by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Four weeks after the treatments, the groups were challenged by the artificial infection of S. agalactiae to observe the relative percent survivals (RPS) on the fish. The results show that the expressions on CD4, IL-8, IL-10, TNF-α and NF-κB genes of Group SIP Vaccine and Group Inactivated Vaccine increased in varying degrees, and were significantly higher (P < 0.05) than those of Group Negative Control. The death rates of fish in Group SIP Vaccine and Group Inactivated Vaccine were significantly lower (P < 0.05) than that of Group Negative Control. The RPS of Group Inactivated Vaccine was higher than that of Group SIP Vaccine, but not significantly different (P>0.05). It appeared that the SIP oral vaccine could be used with satisfaction in protecting tilapias from S. agalactiae infection.
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图 1 免疫后5个免疫相关基因在罗非鱼脾脏中的相对表达量
注：A为CD4，B为IL-8，C为IL-10，D为TNF-α，E为NF-κB。不同字母表示同一时间点不同处理组间相对表达量有显著性差异(P < 0.05)。
Figure 1. Expression of immunity-related genes in spleen of tilapia after vaccination
表 1 荧光定量PCR所用引物
Table 1. Primers used in qRT-PCR
引物名 引物序列(5′-3′) NCBI登录号 GAPDH-F CCAGGTGGTGTCCTCTGATT XM_005455438.2 GAPDH-R GTGCATGTACAGCAGCAGGT CD4-F GTTGATCTTGCACGGAGGTC XM_005455473.3 CD4-R ATGAACTGTCCCATCTGCCA IL-8-F CTGTCATGGTCTGCATCTC NM_001279704.1 IL-8-R GAGGAAGTGGTCTTCTGCT IL-10-F ATCAGTCCGTCGAAGACTCC KP645179.1 IL-10-R CAGAACACCAGCGTGGTTAG TNF-α-F GCTGGAGGCCAATAAAATCA NM_001279533.1 TNF-α-R CCTTCGTCAGTCTCCAGCTC NF-κB-F GCTGTGCAGTCTAACAGGTG XM_003454831.4 NF-κB-R ACCTTAAACTCCACCGTCGT 
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表 2 各实验组罗非鱼的免疫保护率
Table 2. RPS of tilapia in 3 groups
序号 分组 免疫途径 试验鱼数/尾 死亡数/尾 死亡率/% 相对免疫保护率/% 1 SIP疫苗 口服 30 10 33.3a 57a 2 阴性对照 口服 30 23 76.7b 3 灭活疫苗 注射 30 6 20.0a 74a 注：同列数据后相同字母表示差异不显著(P > 0.05)，不同字母表示差异显著(P < 0.05)。
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[1] 张新艳, 樊海平, 钟全福, 等.罗非鱼无乳链球菌的分离、鉴定及致病性研究[J].水产学报, 2008, 32(5):772-779. http://www.cnki.com.cn/Article/CJFDTOTAL-SCKX200805015.htm [2] 卢迈新, 黎炯, 叶星, 等.广东与海南养殖罗非鱼无乳链球菌的分离、鉴定与特性分析[J].微生物学通报, 2010, 37(5):766-774. http://journals.im.ac.cn/wswxtbcn/ch/reader/create_pdf.aspx?file_no=tb10050766 [3] 陈贺, 吴灶和, 王蓓, 等.无乳链球菌灭活疫苗对罗非鱼的免疫效果[J].广东海洋大学学报, 2012, 32(6):50-56. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zjhydxxb201206011 [4] 高铭蔚, 田园园, 卢迈新, 等.罗非鱼无乳链球菌PLGA微球口服疫苗免疫效果的研究[J].免疫学杂志, 2015, 31(2):105-110. http://www.cnki.com.cn/Article/CJFDTotal-MYXZ201502004.htm [5] 全琛宇, 杨磊, 王凯, 等.口服无乳链球菌弱毒疫苗对罗非鱼肠道影响的组织学观察[J].动物医学进展, 2014(10):79-84. doi: 10.3969/j.issn.1007-5038.2014.10.018 [6] 吴斌, 樊海平, 张新艳, 等.罗非鱼无乳链球菌微胶囊口服疫苗的研制及其免疫效果[J].水产学报, 2016, 40(8):1258-1264. http://www.cnki.com.cn/Article/CJFDTotal-MYXZ201502004.htm [7] 付天增, 郝金婷, 李宁求, 等.迟缓爱德华菌菌蜕疫苗对罗非鱼注射和口服免疫的效果分析[J].水生生物学报, 2016, 40(6):1158-1165. doi: 10.7541/2016.150 [8] 张生, 曾忠良, 王凡, 等.海豚链球菌灭活疫苗对罗非鱼免疫效果的研究[J].西南师范大学学报(自然科学版), 2007, 32(5):65-70. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=dsyy201106009 [9] 徐增辉. 罗非鱼海豚链球菌疫苗的研制及免疫效果的初步研究[D]. 南宁: 广西大学, 2008: 93. [10] 邢婧, 王洋, 宋晓青, 等.牙鲆(Paralichthys olivaceus)浸泡免疫鳗弧菌(Vibrio anguillarum)灭活疫苗后11种免疫相关基因的表达变化[J].海洋与湖沼, 2015, 46(5):1024-1033. [11] 王庆, 吴光辉, 巩华, 等.斜带石斑鱼浸泡免疫哈维氏弧菌灭活疫苗后MHC-Ⅱ类分子的表达变化[J].广东农业科学, 2010, 37(12):136-139. doi: 10.3969/j.issn.1004-874X.2010.12.050 [12] BRODEUR B R, BOYER M, CHARLEBOIS I, et al. Identification of group B streptococcal Sip protein, which elicits cross-protective immunity[J]. Infect Immun, 2000, 68(10):5610-5618. doi: 10.1128/IAI.68.10.5610-5618.2000 [13] 宋晓青, 邢婧, 战文斌.牙鲆经注射和浸泡免疫鳗弧菌灭活疫苗后7种免疫相关基因表达的变化[J].中国水产科学, 2014, 21(4):747-758. http://industry.wanfangdata.com.cn/dl/Detail/Periodical?id=Periodical_zgsckx201404013 [14] 庞碧剑. 罗非鱼免疫记忆及体外淋巴细胞增殖的研究[D]. 湛江: 广东海洋大学, 2012: 68. [15] KATO G, GOTO K, AKUNE I, et al. CD4 and CD8 homologues in Japanese flounder, Paralichthys olivaceus:Differences in the expressions and localizations of CD4-1, CD4-2, CD8alpha and CD8beta[J]. Dev Comp Immunol, 2013, 39(3):293-301. doi: 10.1016/j.dci.2012.09.004 [16] 肖周婷. 斜带石斑鱼IL-10和IL-16的原核表达及其在鱼体内组织特异性表达研究[D]. 湛江: 广东海洋大学, 2015: 66. [17] 杨先乐, 陈远新.鱼用疫苗的现状及其发展趋势[J].水产学报, 1996, 20(2):159-167. http://www.oalib.com/paper/5093158 -
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