【作者】 徐炳森；

【导师】 邵健忠；

【作者基本信息】 浙江大学 ， 遗传学， 2001， 硕士

【摘要】 白细胞介素-2（Interleukin-2，IL-2）是机体复杂免疫网络中起调节作用的关键淋巴因子之一，在人和其他高等脊椎动物中已有广泛的研究与应用，但对低等脊椎动物的IL-2仍知之甚少，鱼类在进化上处于连结低等无脊椎动物和高等脊椎动物的关键地位，开展鱼类IL-2的研究，对了解IL-2的分子起源与进化具有重要意义。 本研究用ConA刺激体外培养的草鱼头肾白细胞，MTT法检测发现体外诱导的草鱼白细胞培养上清液具有促进草鱼头肾淋巴母细胞增殖的作用，观察到了草鱼IL-2活性。在此基础上，开展了草鱼IL-2cDNA基因克隆、序列分析和原核表达研究。 草鱼头肾白细胞用PHA和ConA协同PMA诱导14小时，提取细胞总RNA，用Oligo dT₁₈引物把mRNA逆转录为第一链cDNA。以此cDNA为模板，根据人和其他生物IL-2 cDNA基因两端的保守序列设计引物，做RT-PCR扩增。通过优化扩增条件发现，当退火温度提高到56℃时，可得到特异性的扩增产物，琼脂糖凝胶电泳发现目的条带大小约为500bp。该RT-PCR产物经纯化后，采用T-A克隆的方法，插入pUCm—T载体，用T7启动子引物在ABI377测序仪上测定了产物的全部核苷酸序列。用DNAMAN-和OMIGA软件分析表明，该序列全长492bp，编码164个氨基酸残基，其推算的分子量约为17.5kD，包含编码信号肽的序列和编码草鱼IL-2成熟肽的开放读码框架（ORF）。分析该核苷酸序列推导的氨基酸序列发现，草鱼IL-2多肽中存在潜在糖基化位点，推测为糖蛋白。没有发现Cys残基，无潜在的S—S键结构。通过核苷酸序列同源性比较发现，草鱼IL-2 cDNA基因序列与人和其他高等脊椎动物相比有34-40％的同源性。草鱼IL-2在核苷酸序列和氨基酸序列水平与牙鲆相比分别有40％和23％的同源性。 浙 江大 学 硕 士 学 位 论 文 将克隆到 pUCm—T质粒上的草鱼 IL2 cDNA基因用 Ncol和 BaxnHI切下，连接到表达质粒pET28b上，筛选得到的重组质粒转化 E。。h．BLZI／DE3，用WTG诱导表达，做SDS—PAGE分析，发现在ZlkD 处有特异性的表达产物条带，分子量与预期的重组IL－2（包含部分HIS 等载体序列）分子量大小一致，表明草鱼IL－2蛋白实现了在原核细胞 中的表达，为进一步研究草鱼IL－2的免疫生物学功能奠定了基础。更多还原

【Abstract】 Interleukin-2(IL-2) is a lymphokine which plays pivotal role in the regulation of intricate immunology network. It has been thoroughly studied and wildly aplicated in human and other vertebrates, but the studies on lower vertebrates such as fish are still very poor. In the progress of evalution, fish connects the vertebrates with invertebrates, so it is of interest to investigate the IL-2 in fish which will be helpful to understand the origin and phylogency of IL-2.In this research, a grass carp (ctenopharyngodon idellus) IL-2 was illustrated. First of all, concanavalinA (ConA) was used to stimulate the grass carp pronephric leucocyte in vitro. In the supernatants of cell cultures, activaties of promoting the proliferation of mitogen stimulated grass carp lymphoblasts have been found. Evidence for the existence of grass carp IL-2 was primarily observed.To go deep into the grass carp IL-2, a cDNA coding grass carp IL-2 was cloned and sequenced. After 14 hours’ inductions of phytohaemagg-lutinin (PHA) and ConA cooperated with PMA, the total RNA was extracted from grass carp proniphric leucocyte. The mRNA was trasncripted into first strand cDNA by oligodT(ig) primers, which was served as a template for reverse transcription-polymerase chain-reaction (RT-PCR). The RT-PCR primers were designed based on the conservative region of the 5’and 3’-terminal of human and other vertebrates’ IL-2 cDNA gene. The amplification conditions were optimized and than specific product was obtained by elevating the annealing temperature to 56癈. After purification, this RT-PCR product which was about 500bp shown in 1.5% agrose gel electrophoresis was inserted into the pUCm-T plasmid by T-A cloning and sequenced for necleotide sequences using T7 promoter primerby ABI377 DNA sequencer. The full length of grass carp IL-2 cDNA gene was 492bp coding for 164 ammo acid protein whose molecular weight was calculated to be about 17.5kD. No cysteine residue but two potential N-glycosylation sites were observed in the deduced polypeptide sequence which suggested that grass carp IL-2 was a glycoprotein without potential S-S structure. Analysed with DNAMAN and OMIGA software, we found that Grass carp IL-2 has an nucleotide sequence homology of 34-40% with human and other vertebrates and an amino acid sequence homology of 23% with ftatfish(Paralichthys sp\Grass carp IL-2 cDNA gene fragment was cut out from pUCm-T plasmid by Ncol and BamHI enzyme and then ligated to pET28b express plasmid. The recombinant plasmid pET28b_IL-2 was selected and transferred to E.coli. BL21/DE3. After the induction of IPTG, a specific protein band with expected molecular weight of 21kD were detected by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) analysis. This result indicates that grass carp IL-2 can be expressed in prokaryocyte which might be a basis for further studies on its immuology function .更多还原