摘要
为了从蛋白水平和mRNA水平上比较和分析MHC Ⅱα和MHC Ⅱβ基因在卵形鲳鲹不同组织中的表达情况,本研究构建了重组载体p ET32a(+)/M-Ⅱα和p ET32a(+)/M-Ⅱβ来诱导其表达,并制备了多克隆抗体,而且通过qRT-PCR技术分析了MHC Ⅱ类基因在卵形鲳鲹正常组织中的表达.结果显示,本研究成功表达了卵形鲳鲹MHC Ⅱα和MHC Ⅱβ基因的融合蛋白,并制备出了效价高和特异性强的多克隆抗体;在利用Western Blot技术分析MHC Ⅱα或MHC Ⅱβ基因在卵形鲳鲹不同组织中对蛋白的表达情况时,均检测到一条大小约70k Da的疑似MHC Ⅱαβ二聚体的单一条带,且该MHC Ⅱαβ二聚体在脾脏、头肾、肠道、鳃中的表达量较高,而在胃、心、脑、肝中的表达量稍低.qRT-PCR的分析结果一致表明,MHC Ⅱ类基因广泛分布在不同组织中,尤其在与免疫相关的组织(脾脏、头肾、肠道、鳃)中,其具有较高表达量.
To analyze the expression of MHC Ⅱα and MHC Ⅱβ in the different tissues of golden pompano(Trachinotus ovatus),in the study,the recombinant vectors,p ET32 a(+)/M-Ⅱα and p ET32 a(+)/M-Ⅱβ were constructed,the protein expression were induced,the polyclonal antibodies were prepared,and qRT-PCR were used to determine the expression of MHC Ⅱ class in the normal tissue of golden pompano(Trachinotus ovatus).The results indicated that the fusion proteins of r Trov-MHC Ⅱα and r Trov-MHC Ⅱβ were successfully expressed;the high titer and specific polyclonal antibodies were prepared; one specific band with a molecular mass of 70 KDa,which might be the MHC Ⅱαβ dimmer,were observed with western blotting; the expression of MHC Ⅱαβdimer in spleen,head kidney,intestine and gill were higher than that in other tissues,however,its expression in stomach,heart,brain and liver were lower than that in other tissues. qRT-PCR analysis results suggested that MHC Ⅱ class genes were ubiquitously expressed in eight tested tissues and the expression in immune-related tissues(spleen,head kidney,intestine and gill) were higher than that in other tissues.
作者
张小其
孙云
周永灿
曹贞洁
Zhang Xiaoqi, Sun Yun, Zhou Yongcan, Cao Zhenjie(State Key Laboratory of Marine Resource Utilization in South China Sea, The Ocean College, Hainan University, Haikou 570228, China)
出处
《海南大学学报(自然科学版)》
CAS
2018年第2期101-109,共9页
Natural Science Journal of Hainan University
基金
国家自然科学基金(31702379,3156072)
海南省重大项目(ZDKJ2016011)
海南省自然科学基金(2016CXTD005,20163054)
海南大学校内团队培育项目专项(hdkytg201404)
关键词
卵形鲳鲹
MHC
Ⅱ类分子
原核表达
多克隆抗体
组织表达
Trachinotus ovatus
major histocompatibility complex class Ⅱ( MHC Ⅱ) molecular
prokaryotic ex-pression
polyclonal antibody
tissues expression