大乳头水螅增殖细胞核抗原基因的克隆及再生进程中的表达分析

CLONING AND EXPRESSION ANALYSIS OF PROLIFERATING CELL NUCLEAR ANTIGEN GENE DURING REGENERATION IN HYDRA MAGNIPAPILLATA

研究以增殖细胞标记物-增殖细胞核抗原(Proliferating cell nuclear antigen,PCNA)蛋白为切入点,采用RACE(Rapid amplification of cDNA ends)方法克隆了大乳头水螅(Hydra magnipapillata)PCNA基因的cDNA序列。该cDNA序列包含1240 bp,其中837 bp为编码区,编码的蛋白质预测分子量为30.93 kD。把PCNA基因ORF中的部分序列克隆到原核表达质粒pET-28b(+)中,重组质粒转化E.coli BL21(DE3)菌株,IPTG诱导后成功表达重组PCNA蛋白,再用该重组蛋白免疫新西兰兔制备多克隆抗体用于PCNA蛋白的免疫印迹分析(Western blotting assay,WB)。采用实时定量PCR(quantitative Real-time PCR,qPCR)及WB方法检测水螅头部再生进程中其PCNA表达水平的动态变化,结果表明在再生进程的中后期阶段水螅PCNA表达量呈现一定程度的上调。结果暗示水螅头部再生进程的中后期阶段其伤口及附近区域可能存在细胞分裂活动。

Freshwater polyp Hydra is an excellent model organism for studying tissue regeneration, and its strong regeneration capacity originates from a large number of stem cell-like cells in the body. However, whether these cells undergo dedifferentiation or mitosis to produce new cells during regeneration is still unclear. Here, we focus on proliferating cell nuclear antigen(PCNA) protein, a marker of proliferating cells. The cDNA sequence of PCNA in Hydra magnipapillata was cloned by rapid amplification of cDNA ends(RACE) method. The total length of the obtained cDNA is1240 bp, including a 101 bp 5′ non-coding region, a 302 bp 3′ non-coding region and a 837 bp open reading frame(ORF). The PCNA of H. magnipapillata has 278 amino acids with a putative molecular weight of 30.93 kD. H. magnipapillata PCNA ORF fragment was subcloned into the prokaryotic expression plasmid pET-28 b(+), and then transformed into E. coli BL21(DE3) strain, and finally the recombinant PCNA protein was successfully expressed after induction by IPTG(isopropyl-β-d-thiogalactopyranoside). The recombinant protein was used to prepare the rabbit polyclonal antibodies for western blotting assay(WB). The quantitative real-time PCR(qPCR) and the WB results showed that PCNA expression increased at the middle and late regeneration stage in H. magnipapillata. In conclusion, our results suggest that there might be cell proliferation at wound site and nearby areas of hydra at the middle and late regeneration stage.