摘要
利用构建的文蛤cDNA文库,克隆得到了文蛤C1q基因全长cDNA序列,该序列全长1213bp,5′UTR为316bp,3′UTR为372bp,开放阅读框长度为525bp,编码174个氨基酸。在文蛤C1q(MmC1q)蛋白中,C1q结构域与软体动物、两栖类动物和脊椎动物中的C1q结构域均具有较高的同源性。通过荧光实时定量PCR,MmC1q mRNA在所检测的各个组织中均有表达,在肝胰脏中表达量最高。在细菌感染试验中,文蛤受鳗弧菌感染后,C1q相对表达水平有明显的上升调节,注射2h,MmC1qmRNA表达量最高,为对照组的2.19倍。包含成熟肽的MmC1q cDNA片段被重组,并在大肠杆菌BL21(DE3)中表达,采用牛津杯法对MmC1q重组蛋白进行体外抑菌试验,但并未检测到明显的抑菌活性。
C1q as the target recognition protein of the classical complement pathway is crucial for the clearance of pathogens and apoptotic cells.In the present study,the C1q gene in hard clam（Meretrix meretrix）,one of commercial bivalves in China,was cloned through SMART cDNA libraries.The full-length cDNA of MmC1q was found to be 1213 base pairs（bp） consisting of a 5′-terminal untranslated region（UTR） of 316 bp and a 3′UTR of 372 bp with a poly（A） tail.The MmC1q cDNA was shown to contain an open read frame（ORF） of 525 bp,to encod a polypeptide of 174 amino acids.The C1q-domain in MmC1q was found to exhibit homology with those in C1qDC proteins from mollusks,amphibians and higher vertebrates.By fluorescent quantitative real-time PCR,mRNA transcripts of MmC1q were detected in hepatopancreas,hemocytes,gill,mantle,and adductor,the maximum mRNA expression in hepatopancreas.In the bacterial challenge experiment,there was a significant up-regulation in the relative expression level of MmC1q 2 h post-injection in the hard clam challenged by Vibrio anguillarum,the maximum mRNA expression,2.19-fold higher than that in control group.The cDNA fragment encoding the mature peptide of MmC1q was recombined and expressed in Escherichia coli BL21（DE3）,indicating that MmC1q recombinant protein has no obvious antibacterial activity through bacteriostasis in vitro.
出处
《水产科学》
CAS
北大核心
2012年第6期321-328,共8页
Fisheries Science
基金
国家自然科学基金资助项目(31101899)
上海市教委第五期重点学科海洋生物学建设项目(J50701)
大连市科技基金资助项目(2010J21DW033)
