中国对虾蛋白磷酸酶1催化亚基β基因的克隆表达及特性分析

Cloning,Expression and Characterization of Protein Phosphatase 1 Catalytic Subunit Beta Isoform in Shrimp Fenneropenaeus chinensis

本论文利用RACE（Rapid-amplification of cDNA ends）技术克隆获得中国对虾（Fenneropenaeus chinensis）蛋白磷酸酶1催化亚基β（Protein phosphatase 1catalytic subunit beta isoform,PP1β）基因cDNA序列全长。该基因全长1 214bp,包含一个987bp的开放阅读框,编码328个氨基酸。同源性分析显示,中国对虾PP1β氨基酸序列与不同物种PP1β的相似性高达90%~91%,表现出高度保守性。多序列比对结果显示,不同物种PP1β均含有丝氨酸/苏氨酸特异性蛋白磷酸酶家族的特征基序GDxHG、GDxVDRG和GNHE。系统进化树分析显示,甲壳动物PP1β聚为一大支,中国对虾PP1β和凡纳滨对虾（Litopenaeus vannamei）聚为一小支。实时荧光定量PCR分析显示,PP1β在健康的中国对虾各组织中均有不同程度的表达,其中在性腺中表达最高,血细胞次之。白斑症病毒（White spot syndrome virus,WSSV）注射感染健康中国对虾后,血细胞和性腺中PP1β基因均呈上调表达,并在12h达到峰值,且在血细胞中上调表达更显著。构建了中国对虾PP1β基因原核重组表达载体pET28a-PP1β,转化大肠杆菌后成功诱导表达重组PP1β蛋白（rPP1β）,分子量为41kDa。将亲和层析纯化的rPP1β免疫BALB/c小鼠制备抗血清,通过制备中国对虾血细胞滴片,应用间接免疫荧光法检测PP1β在血细胞中的分布情况,结果显示,中国对虾PP1β在血细胞的核区及细胞质内均有分布。本研究结果为进一步解析中国对虾PP1β与WSSV感染的相互关系提供了数据。

Protein phosphatase 1（PP1）of host cells was documented to play a crucial role in virus infection in mammals,participating in the transcription,replication and life cycle regulation of virus.PP1 in hemocytes of Fenneropenaeus chinensis was up-regulated significantly after WSSV infection as was demonstrated in our previous work,which indicated that PP1 of F.chinensis was involved in WSSV infection.To further illustrate the role of PP1 in WSSV infection,in present work,protein phosphatase 1catalytic subunit beta isoform（PP1β）gene of F.chinensis was cloned and sequenced by rapid amplification of cDNA ends approaches（RACE）.The full-length cDNA sequence of PP1βgene was 1,214 bp,and contained an open reading frame（ORF）of 987 bp that encoded for a polypeptide of 328 amino acids.Homology comparison showed that PP1βof F.chinensis shared 90%~91% amino acids with thst of other species,indicating the high conservation of PP1βgene.Multiple sequence alignment was performed using the ClustalW Multiple Alignment program.It was demonstrated that all amino acid sequences of PP1βfrom various species contained three conserved catalytic domains which were specific for Ser/Thr phosphatases,GDxHG,GDxVDRG and GNHE.A neighbor-joining（NJ）tree was constructed based on the protein sequences of PP1βfrom 17 species by the NJ algorithm using MEGA 4.0software package and Clustal X usingα-lactalbumin as outgroup.The result showed that F.chinensis was clustered with L.vannamei,and PP1βof crustaceans gathered in one branch.By quantitative real-time RT-PCR,PP1βgene mRNA was observed in all the eight tissues of healthy F.chinensis,with the high transcription level in gonad and hemocytes.The high transcription level of PP1βgene in gonad suggested that PP1βpossibly involved in germ cell differentiation and spermatogenesis as was reported in mammals.Moreover,the gene in the above two tissues was up-regulated after WSSV infection,with the peak value found at 12 h,while the fold change of PP1βgene transcript abundance in hemocytes was higher than that in gonad.The higher transcription level of PP1βgene observed in hemocytes suggested that PP1βmight play an important role in resistance to WSSV infection.The PP1βgene ORF was cloned into pET-28 aexpression plasmid and the recombinant plasmid was transformed into E.coli BL21（DE3）.SDS-PAGE analysis showed that the molecular weight of recombinant protein was 41 kDa.Subsequently,rPP1βwas purified by using affinity chromatography,and the polyclonal antibody against rPP1βwas produced by immunizing mouse.The localization of PP1βin F.chinensis haemocytes was determined by indirect immunofluorescence assay（IIFA）.The results showed that PP1βwas synthesized in cell nucleus and cytoplasm,which implied that PP1βmight mediate kinds of biological processes such as the signal transduction in F.chinensis.Overall,this study provided important data for illustrating the relationship between F.chinensis PP1βand WSSV infection.