李学优 曹丁 肖宏艳 黄秀敏 甘祥武

摘要  对重组人溶菌酶真核表达载体的构建筛选、表达与活性鉴定，经过双酶切和测序鉴定表明，重组载体正确。电转化毕赤酵母GWDL菌株，通过G418抗性平板筛选得到高表达高效价菌株，经诱导表达，检测蛋白含量，体外抑菌检测，表达产物15 ku和预期结果一致，筛选得到效价为1 907 U/mL，编号为 SMD1168-pPIC9k-HLZ的高效价菌株;采用重组人溶菌酶制剂，检测样品GWDL对5种菌的最小抑菌浓度（MIC） ，藤黄微球菌的最小抑菌浓度较好，表明重组人溶菌酶在毕赤酵母中成功表达，活性较为稳定。

关键词  毕赤酵母;菌株筛选;高效表达;抑菌活性

中图分类号  S  188文献标识码  A

文章编号  0517-6611（2020）04-0100-03

doi：10.3969/j.issn.0517-6611.2020.04.029

開放科学（资源服务）标识码（OSID）：

Expression of Human Lysozyme in Pichia Pastoris and Its Antibarterial Activity in vitro

LI Xue-you，CAO Ding，XIAO Hong-yan et al  （Guangzhou lnstitute of Microbiology，Guangzhou，Guangdong 510663）

Abstract  The screening， expression and activity identification of constructed recombinant human lysozyme eukartic expression vectors were studied.The recombinant vectors were shown to be correct through double enzyme digestion and sequencing identification.To electro-transform the pichia pastoris GWDL strain， through G418 resistance flat screening to obtain high expression of high-efficiency strains，the expression product（15 ku） was consistent with the expected results after induced expression， detection of protein content and in vitro antiseptic detection，

we obtained No. SMD1168-pPIC9k-HLZ high titer strain with a titer of 1 907 U/mL;To detect the sample GWDL minimum bacterial concentration （MIC） for five bacteria by using Recombinant human lysozyme preparation，the minimum concentration of micrococcus luteus was better， indicating that recombinant human lysozyme in pichia pastoris successfully expressed，the activity was more stable.

Key words  Pichia pastoris;Strain screening;Efficient expression;Antibacterial activity

溶菌酶是小分子碱性蛋白，因其能有效地降解微生物的细胞壁，溶解作用较好，不易产生耐药性，无副作用等优点，被广泛应用于医药制剂、食品防腐等多个行业。溶菌酶可从胎盘中少量提取得到，因提取量较少，有着巨大的应用前景[1-3]。

毕赤酵母是一种真核表达菌株，此系统具有稳定性、高效表达、分泌性等特点，可通过甲醇诱导，提高溶氧量，使得菌株效价更高[4-5]。该研究以毕赤酵母为表达系统，重组质粒，经电击转化SMD1168，菌株经G418抗性筛选，对筛选菌株进行诱导表达，加大溶氧量提高表达量，检测活性，得到毕赤酵母的高效表达，以期为后期扩大培养打下基础。

1  材料与方法

1.1  材料与试剂

大肠杆菌DH5α、pPIC9K质粒、藤黄微球菌、毕赤酵母SMD1168由广东省微生物种质资源库保存。

PFU Mix、DNA 胶回……