摘要
【目的】旨在进一步研究草鱼(Ctenopharygodon idella)莫洛尼鼠白血病病毒前病毒整合基因1(Proviral integration of moloney murineleukemia virus,Pim-1)的功能。【方法】采用RACE法克隆草鱼Pim-1(CiPim-1)的全长cDNA序列,并对其全长序列进行生物信息学分析。采用实时荧光定量PCR(qRT-PCR)检测CiPim-1在健康草鱼肝脏、肾脏、头肾、脾脏、肠、心脏、鳃、皮肤、肌肉及血液中的相对表达量,同时将CiPim-1基因(246~318 aa)片段克隆到原核表达质粒pGEX-4T-1中,转入E.coli Rosetta菌株,经IPTG诱导后成功表达重组蛋白pGEX-4T-1-Pim1,随后采用纯化后的融合蛋白免疫新西兰大白兔获得抗血清,并通过ELISA测定抗血清效价,最后采用免疫印迹(Western Blotting)分析抗体的特异性。【结果】CiPim-1基因序列全长1082 bp,编码318个氨基酸,分子量为36.14 ku,具有1个蛋白激酶结构域(Protein kinase domain,35~287 aa)。同源性及系统进化结果显示,CiPim-1氨基酸序列与其它鱼类及哺乳类Pim-1均具有较高的相似性和一致性,与斑马鱼(Danio rerio)Pim-1亲缘关系最近。qRT-PCR结果表明,CiPim-1在草鱼血液和组织中均广泛表达,在心脏组织中的相对表达量最丰富,显著高于相对表达量最低的肝脏组织(P>0.05)。ELISA与Western blotting结果表明,经抗原亲和纯化后获得的CiPim-1多克隆抗体,效价高,能够特异性识别CiPim-1重组蛋白。【结论】研究克隆了CiPim-1基因,发现该基因在草鱼组织中广泛表达,且成功制备了CiPim-1多克隆抗体,为深入研究CiPim-1在草鱼细胞周期调控、增殖、分化及凋亡等生理过程中的作用奠定了基础。
[Objective]This study aims to further investigate the function of Pim-1 in grass carp(Ctenopharygodon idella).[Methods]The full-length cDNA sequence of Pim-1 in grass carp,named as CiPim-1,was cloned by using RACE method.The sequence of CiPim-1 was analyzed by bioinformatics software.The expression of CiPim-1 in liver,kidney,head kidney,spleen,intestine,heart,gills,skin,muscle and blood of healthy grass carp was detected by qRT-PCR.In addition,the fragment of CiPim-1 gene(246-318 aa)was cloned into the prokaryotic expression plasmid pGEX-4T-1,the recombinant plasmid was transformed into Escherichia coli(E.coli)Rosetta strain,and the recombinant protein pGEX-4T-1-Pim1 was successfully expressed after IPTG induction.The purified recombinant protein was used to immunize New Zealand white rabbits to obtain antiserum,and titer of the antiserum was determined by ELISA.The polyclonal antibody was purified by antigen affinity and the specificity of the antibody was analyzed by Western Blotting.[Results]The results showed that the full length of CiPim-1 was 1082 bp,encoding 318 amino acids with a molecular weight of36.14 ku,and a protein kinase domain(35-287 aa).Homology and phylogenetic analysis showed the amino acid sequence of CiPim-1 had high similarity and consistency with other fish and mammalian Pim-1,and it had the closest genetic relationship with Pim-1 of Danio rerio.Besides,CiPim-1 was widely expressed in grass carp blood and tissues,with the highest relative expression in heart tissues,which was significantly higher than that in liver tissues with the lowest relative expression(P>0.05).The results of ELISA and Western Blotting showed that CiPim-1 polyclonal antibody had high titer and could specifically recognize CiPim-1 recombinant protein.[Conclusion]CiPim-1gene was successfully cloned,which is widely expressed in grass carp tissues.The polyclonal antibody of CiPim-1 was prepared,which lays a solid foundation for studying the role of CiPim-1 in cell cycle regulation,proliferation,differentiation,apoptosis and other physiological processes of grass carp.
作者
王辉
刘林
阮记明
梁惜梅
隗黎丽
WANG Hui;LIU Lin;RUAN Jiming;LIANG Ximei;WEI Lili(College of Animal Science and Technology,Jiangxi Agricultural University,Nanchang 330045,China)
出处
《江西农业大学学报》
CAS
CSCD
北大核心
2022年第3期670-678,共9页
Acta Agriculturae Universitatis Jiangxiensis
基金
国家自然科学基金项目(31760764,31460146)。
关键词
草鱼
PIM-1
克隆
组织表达
原核表达
多克隆抗体
grass carp
Pim-1
cloning
tissue expression
prokaryotic expression
polyclonal antibody
