文献类型：期刊文献

中文题名：无乳链球菌Sip-GAPDH嵌合核酸疫苗的制备及免疫效果评价

英文题名：Chimeric DNA vaccine development and protective immunity analysis of Sip-GAPDH of Streptococcus agalactiae isolated from tilapia(Oreochromis niloticus)

作者：王蓓[1,2,3];李桂欢[1,2,3];鲁义善[1,2,3];汤菊芬[1,2,3];吴灶和[2,3,4];简纪常[1,2,3]

机构：[1]广东海洋大学水产学院,广东湛江524088;[2]广东省水产经济动物病原生物学及流行病学重点实验室,广东湛江524088;[3]广东省水产经济动物病害控制重点实验室,广东湛江524088;[4]仲恺农业工程学院,广东广州510225

年份：2014

卷号：38

期号：11

起止页码：1926

中文期刊名：水产学报

外文期刊名：Journal of Fisheries of China

收录：CSTPCD、、北大核心2011、Scopus、北大核心、CSCD、CSCD2013_2014

基金：国家自然科学基金青年基金(31302226);国家科技支撑计划(2012BAD17B02;2012BAD17B03);广东省科技计划农业攻关项目(2012B020308009);广东省2012年鱼病防治专项资金(2130108)

语种：中文

中文关键词：无乳链球菌;罗非鱼;Sip-GAPDH;嵌合基因;核酸疫苗;免疫保护率

外文关键词：Streptococcus agalactiae;;Oreochromis niloticus;;Sip-GAPDH;;chimeric gene;;DNA vaccine;;relative percentage survival

中文摘要：为了研究无乳链球菌Sip-GAPDH嵌合核酸疫苗对罗非鱼链球菌病的免疫保护作用,本研究通过PCR和重叠延伸拼接技术(SOEing)获得融合基因Sip-GAPDH,连接至真核表达载体pcDNA3.1(+)制备DNA疫苗,通过背鳍肌肉注射方式免疫吉富罗非鱼,免疫后第7、28天取样,采用PCR及RT-PCR方法检测pcDNA-Sip-GAPDH在各个组织(包括肌肉、脑、头肾、脾脏、鳃和肝脏)中的表达情况。采用ELISA、Real-time PCR法和体外攻毒法分别分析各实验组不同时间血清抗体效价、免疫基因(IgM、IL-1β和CD8)表达变化规律和相对保护率,研究该嵌合性疫苗对吉富罗非鱼的保护效果。结果显示,实验组在免疫接种第7和第28天时,注射点周围的肌肉、脑、头肾、脾脏、鳃和肝脏均能检测到嵌合性质粒,实验组罗非鱼血清抗体效价均高于对照组并于21 d时达到峰值(1∶4 096);qPCR数据表明,实验组鱼体胸腺、头肾和脾脏的IgM、IL-1β和CD8基因的mRNA表达量均出现了上调,并且在胸腺、头肾和脾脏中的表达量分别在免疫后第12和第48 h达到峰值;免疫后42 d进行人工攻毒后计算免疫保护率为93.3%。以上研究结果表明,本研究构建的无乳链球菌Sip-GAPDH嵌合核酸疫苗在罗非鱼链球菌病防治中具有潜在的应用价值。

外文摘要：In order to study the immunogenic and protective effects of Sip-GAPDH from Streptococcus agalactiae recombined chimeric DNA vaccine for Oreochromis niloticus,we cloned Sip and GAPDH gene by PCR,and then obtained the Sip-GAPDH fusion gene using the splicing overlap extension technology(SOEing).The Sip-GAPDH fusion gene was inserted into pcDNA3.1(+) vector and confirmed by restriction endonuclease digestion,PCR amplification and sequencing,and O.niloticus were immunized with the recombined plasmid(designated as pcDNA-Sip-GAPDH) by intramuscular injection.The pcDNA-SipGAPDH expression in tissues(including muscle,brain,head kidney,spleen,gill and liver) was analyzed by PCR and RT-PCR at 7 and 28 d after immunization.To reveal the serum antibody titer,expression of immunogenes(IgM,IL-1β and CD8) and relative percentage survival(RPS),we used the enzyme-linked immunosorbent assay(ELISA),real-time PCR and bacterial challenge in vitro to demonstrate the DNA vaccine is a potential candidate for vaccine development of O.niloticus.The results indicated that pcDNASip expression could be found in all studied tissues and antibody titer rose to peak(1∶ 4 096) at 21 d after immunization.Quantitative real-time PCR(qRT-PCR) analysis showed that IgM,IL-1β and CD8 had relatively high expression levels in the head kidney,thymus,spleen.After S.agalactiae infection,transcripts of IgM,IL-1β and CD8 increased and reached its peak 12 h in thymus and head kidney,and 48 h in spleen,respectively.Additionally,the relative percentage survival(RPS) value was 93.3% according to the mortality by S.agalactiae challenge.All the results suggested that the chimeric DNA vaccine is an effective vaccine candidate against S.agalactiae infection.