α-2b干扰素重组质粒的建立及在毕赤酵母中的表达-生物化学与分子生物学专业论文.docxVIP

华 华 中 科 技 大 学 硕 士 学 位 论 文 I I 摘 要 自从1957年，英国科学家Alick Isaacs和Jean Lindenmann发现干扰素(Interferon, IFN)以来，干扰素是目前研究最多的细胞因子。α-干扰素不仅具有抗病毒和调节免 疫作用，还可以抑制肿瘤细胞增殖，一直是病毒学、细胞学、分子生物学、临床医 学、免疫学、肿瘤学等相关领域的研究热点。 本研究根据基因编码区中同义密码子的非均匀使用，编码区中碱基的构成及密 码子内和密码子之间的碱基关联与基因表达水平之间的关系，利用密码子的简并性 将人干扰素 α-2b 基因编码区进行重新设计，使之成为预期可在毕赤酵母中高效表达 的外源基因。应用化学合成法，合成寡核苷酸小片段，然后组装成完整的人干扰素 α-2b 基因片段，并克隆在 pPIC9K 质粒中。经序列分析证实，合成并克隆的人干扰 素 α-2b 基因序列与设计完全相符。同时构建了表达质粒 pPIC9K-IFNα-2b，经 PCR 分析，证实构建正确。用电转化法转化 Pichia pastoris GS115，筛选出整合型 His+Mut+ 菌株，进一步用氨基糖甙类新霉素衍生物 Geneticin（G418）筛选获得高拷贝转化子， 经 5d 诱导表达后 SDS检测，SDS结果显示目的蛋白已成功表达。 关键词：α-2b 干扰素；毕赤酵母；pPIC9K；SDS II II Abstract Since interferon was discovered in 1957, it has been hailed as a significant antiviral agent. In the 1970s, interferon made a big splash as a symbol of recombinant gene technology and the medical breakthroughs it would bring. The natural antiviral and antitumor activity of interferon alpha and beta promoted investigation for therapeutic use. Interferon therapy has been successfully implemented for skin malignancies, viral infections, kaposi sarcoma, multiple sclerosis, leukima, et al. According to the relation between gene expression levels and synonymcodon usage, base composition in coding region，base pair correlation in a codon and base pair correlation between codons. Human Interferon α-2b gene was designed by using degenracy of codon. The designed gene was expected to be expressed highly in Pichia pastroris. Oligo DNA was synthesized to form complete human interferon α-2b gene fragment that was inserted into the vector pPIC9K. DNA sequencing show that Human interferon α-2b gene sequence of synthesis and insertion is completely consistent with that of the designed gene. Meantime, the expression plamid pPIC9K-IFNα-2b was constructed. By PCR analysis, construction was proved to be right. After electroporation of Pichia pastoris GS115, some high-copy transformants (His+Mut+) were picked up by Geneticin(G418). After 5 days induction, the result was t