摘要
CARD6蛋白广泛存在于除脑组织以外的所有组织中,在心肌和骨骼肌组织中表达最多,它存在于细胞核和核DNA中。CARD6通过调节RIP激酶家族的功能来调节NF-κB激活作用,由于受到CARD4,Rip2的抑制作用的影响,CARD6基因作为NF-κB激活作用的选择性调节因子,与CARD家族的所有成员一道,共同参与机体的炎症应答和遗传性免疫调节。
CARD7(又称NALP1/DEFCAP/NAC)是NOD家族中唯一含有N末端pyrin区域,同时连接着NBD-LRR和C末端CARD区域的蛋白,由"apoptosome"和"inflammasome"组成的大蛋白合成物,CARD7的mRNA在各种组织中广泛表达,在血粒细胞、胸腺、脾和心脏中表达最高,在脱噬作用、炎症性胱门蛋白酶激活和胱门蛋白酶原-1β进程中均扮演着重要作用。
RIP2是核苷酸结合寡聚化结构域胞浆蛋白家族成员之一,它是一种在C终点含有氨基末端激酶域的丝氨酸/苏氨酸激酶,介于激酶域和CARD域之间的媒介领域。其生理和病理作用是调解参与多个NF-κB的活化,对先天的和适宜性免疫反应具有重要意义的信号转导途径。
本研究应用比较基因组学的相关原理和方法,利用猪CARD6基因、CARD7基因和RIP2基因的mRNA序列,采用RT-PCR法,首次得到了猪相应基因的CDS部分序列。同时利用GENBANK数据库中不同物种三种基因的CDS区域序列及氨基酸序列,运用Mega4.0软件构建三种基因DNA序列的NJ关系树。结果显示:
(1)从猪骨骼肌组织中克隆得到了2222bp的猪CARD6基因cDNA序列,与人、猩猩、牛、马及鼠的核苷酸同源性分别为:82.5%、82.6%、85.3%、84.0%及71.7%,氨基酸同源性分别为:72.4%、72.3%、78.1%、73.8%及61.6%。(2)从猪肾脏组织中克隆得到了一个1836bp的猪CARD7基因cDNA序列,与人、牛、猩猩及鼠的核苷酸同源性分别为:74.3%、78.4%、74.6%及62.0%,氨基酸同源性分别为:52.3%、58.9%、52.5%及46.7%。(3)从猪脑组织中克隆得到了一个1511bp的猪RIP2基因cDNA序列,与人、牛、狗及鼠的核苷酸同源性分别为:89.1%、89.7%、88.8%及82.5%,氨基酸同源性分别为:87.4%、87.4%、85.6%及82.4%。(4)猪的CARD6基因、CARD7基因和RIP2基因核酸序列和氨基酸序列聚类图中都是牛与猪的有关基因聚为一类。
The CARD6 protein is found in almost all tissues except for brain, the larger transcript was present only in heart and skeletal muscle. It is found in the nucleus and nuclear DNA.CARD6 is a regulator of NF-κB activation that modulates the functions of RIP kinase family members.CAR-D6 represents a selective modulator of NF-κB activation by Cardiak and Nod1, adding to the repertoire of CARD-family proteins implicated in inflammatory responses and innate immunity.
The CARD7 (NALP1/DEFCAP/NAC) protein is unique in having a Nterminal pyrin domain, followed by NBD-LRR and a C-terminal CARD domain,it's a component of two important and large protein complexes: the "apoptosome" and the "inflammasome" complexes. CARD7 mRNAs were widely expressed in human tissues, with highest levels found in blood leukocytes, thymus, spleen, and heart,it has been shown to play a central role in the apoptosis, activation of inflammatory caspases and processing of pro-IL1β(pro-interleukin-1β).
RIP2 is a member of the nucleotide binding oligomerization structural domain plasmosin family,it is a intermediary domain between amino terminal kinase domain and card,is a serin /threonine kinase which have C-terminal amino terminal kinase domain. Its physio-function and patho-function are modulate participation of several NF-κB activation,It's a important signal transduction pathway of innate and suitable immunity reaction.
This study apply the mRNA sequences of pig CARD6 gene, CARD7 gene and RIP2 gene by comparative genomics theory, we obtained the corresponding mero- CDS sequences in yak by RT-PCR method first time. By using the CDS domain sequence and amino acid sequence of the upper three genes from the GENBAN,we also construct the NJ-relationtree of the DNA sequence. The results show that:
(1)We obtain a cDNA sequence about 2222bp of CARD6 gene from skeletal muscle tissue, compare to the human being, chimpanzee,cattle,horse and mouse,the nucleotide homology are 82.5%、82.6%、85.3%、84.0% and 71.7%, the amino acids homology are 72.4%、72.3%、78.1%、73.8% and 61.6%.(2)We obtain a cDNA sequence about 1836bp of CARD7 gene from kidney tissue,compare to the human being, cattle,chimpanzee and mouse,the nucleotide homology are 74.3%、78.4%、74.6%and 62.0%, the amino acids homology are 52.3%、58.9%、52.5%and 46.7%.(3)We obtain a cDNA sequence about 1511bp of RIP2 gene from brain tissue, compare to the human being, cattle,dog and mouse,the nucleotide homology are 89.1%, 89.7%, 88.8%and 82.5%, the amino acids homology are 87.4%, 87.4%, 85.6%and 82.4%.(4)The pig and cattle gather to a group Not only in the nucleotide sequence dendrogram but also in the amino acid sequence dendrogram of CARD6 gene, CARD7 gene and RIP2 gene.
引文
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