【作者】 王颖；

【导师】 罗云波；

【作者基本信息】 中国农业大学 ， 食品科学， 2003， 博士

【摘要】 人溶菌酶是存在于人体正常体液和组织中的一种非特异性免疫因子。它对人体完全无毒副作用，同时具有抗菌、消炎、抗病毒、抗肿瘤等作用，可被开发成口含片、胶囊、口香糖及口腔喷雾剂等产品形式。 由于人溶菌酶来源有限，因此本研究提出通过遗传工程方法，利用毕赤酵母细胞生产人溶菌酶，一方面解决了人溶菌酶资源稀缺的问题，另一方面则能创造更大的经济效益。由于人胎盘组织中富含hLZM基因的mRNA，因此从人胎盘组织中提取细胞总RNA作为模板，根据GENEBANK中已发表的hLZM基因的cDNA序列，设计合成特异引物，经逆转录合成hLZM基因的cDNA序列，并以此cDNA序列为模板，经PCR扩增hLZM编码基因。经DNA序列分析证明与已发表的hLZM cDNA序列完全一致。 通过EcoRⅠ和NotⅠ双酶切作用，将hLZM的编码基因按照质粒阅读框的转译方向插入到表达质粒pPIC9K的MCS中，构建重组表达质粒pPIC9K／hLZM。利用电转化方法转化表型为His~-的宿主菌KM71，利用MD培养基筛选到近1 200个His~+转化子。再经G418抗性的二次筛选，获得15株含高拷贝人溶菌酶基因的重组毕赤酵母转化子。当以甲醇进行诱导表达时，成功获得了1株高效分泌表达的重组菌株Pp135，其表达量为317mg／L，酶活性为32035u／mL。 菌株Pp135经甲醇诱导，可将人溶菌酶分泌到培养基中，因此可在发酵上清液中直接检测到人溶菌酶的活性。该检测采用以溶壁微球菌(Micrococcus lysodeikticus)为试验菌的琼脂平板扩散法。研究发现培养基BGMM、BMM、MMC等均能诱导菌株Pp135表达人溶菌酶，由于培养基BGMM和MMC中存在干扰溶菌酶纯化的杂蛋白，并使溶菌酶产品带有异味和色素，因而确定利用BMM培养基进行重组菌株Pp135的诱导表达。实验最终获得381mg／L的表达量，若继续优化发酵条件，还有可能进一步提高菌株Pp135的表达水平。 本研究为人溶菌酶的工业化规模生产奠定理论基础，这对带动人溶菌酶的规模化生产具有特殊意义，其推广应用将给社会带来相当的经济效益。更多还原

【Abstract】 Human lysozyme (hLZM ) is a kind of unspecific immunal factor consisting in normal human body liquids and tissues. It was completely no harmful and side-effect to human body, and has the function of antibacterial, anti-inflammatory, anti-viral and anti-tumor. Therefore, lysozyme had already been made into multiform products such as troche keeping in mouth, capsule, chewing gum and stomatitis aerosols.Due to lacking human lysozyme resources in nature, the research which produce this lysozyme by Pichiapastoris was carried out by a genetic engeering method. It will be settle for unenough amount of human lysozyme resource in application, and will be made more ecnomic profits to society. Because of the mRNA of hLZM abounding in human placenta tissue, the total RNA was extracted from placenta to be used as template for inverse transcriptional synthesis of hLZM cDNA sequence. According to published hLZM cDNA sequence in GENEBANK, the special primers were designed and synthesized for leading amplification of hLZM encoding sequence by PCR. The obtained sequence was proved that completely identical with the published hLZM cDNA sequece.Cutting by EcoR I and Not I the hLZM encoding sequence was inserted in MCS of pPIC9K according to the right reading frame translational direction. Transforming His" phenotype KM71 with recombinant plasmid pPIC9K/hLZM by electroporation, 1200 His+ transformants were acquired by MD plate. Sreening by G418 secondly, 15 recombinant transformants with multiple copies of hLZM gene were obtained. After inducing by methanol, one high-level expressing strain Ppl35 was successfully obtained, and its expression level was amount to 317mg/L which enzyme activity was 32035 u/mL.Strain Ppl35 can secrete human lysozyme in media when inducing by methanol, so it can be determinated for human lysozyme bacteriolytic activity straightly in fermentative supernatants by agar plate diffusion method which used Microccous lysodeikticus as the test bacterium. It was found that the media such as BGMM, BMM and MMC all could induce strain Ppl35 to expression human lysozyme. Because of media BGMM and MMC being of some proteins which can interfere the purification of lysozyme and enduing the product with odour and pigment, finally the media BMM was confirmed to induce expression hLZM of the strain Ppl35. The result showed that the expressing level was 381mg/L, and the Ppl35 has the potential to continually increase the expressing level which will be achieved by optimizing the fermentation factors.This research will make an academic base for industrial scale production of human lysozyme. It will be significiant to drive the development of scale producing human lysozyme. The spresding and applicating of this technics will be considerablely benefit to the world.更多还原