Cloning Of Human Lysozyme Coden Gene And Expressing In Pichia Pastoris For Natural Food Antiseptic

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.