青蛤Ⅰ型溶菌酶在毕赤酵母中的重组DNA表达  

Recombinant DNA Expression of Ⅰ-Type Lysozyme of Clam Cyclina sinensis in Yeast Pichia pastoris

作　　者：冯付霭 赵震 陶妍[1,2] 谢晶 钱韻芳[1,2] FENG Fu′ai;ZHAO Zhen;TAO Yan;XIE Jing;QIAN Yunfang(College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China;Shanghai Engineering Research Center of Aquatic-Product Processing&Preservation,Shanghai 201306,China)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海水产品加工及贮藏工程技术研究中心,上海201306

出　　处：《水产科学》2022年第5期836-843,共8页Fisheries Science

基　　金：国家“十三五”重点研发计划项目(2016YFD0400106)。

摘　　要：Ⅰ型溶菌酶是无脊椎动物免疫系统中一种富含半胱氨酸的重要蛋白质免疫因子,具有良好的抗病原微生物功能。通过反转录PCR自青蛤闭壳肌中克隆到Ⅰ型溶菌酶(CslyⅠ)基因,以pPⅠCZαA为表达载体、毕赤酵母X-33为工程菌,构建重组毕赤酵母菌株X-33/pPⅠCZαA-CslyⅠ,经高质量浓度博莱霉素培养基筛选获得高拷贝重组菌株。该菌株在28℃、250r/min条件下,经1%甲醇诱导表达72h后获得蛋白质类表达产物。Western blot和MALDⅠ-TOF/TOF质谱分析表明,该表达产物为预期的重组青蛤Ⅰ型溶菌酶;进一步的抑菌试验证明,该重组青蛤Ⅰ型溶菌酶具有抗金黄色葡萄球菌、大肠杆菌和副溶血性弧菌的活性,并且显示良好的热稳定性。本研究可为无脊椎动物来源Ⅰ型溶菌酶的制备提供基于重组毕赤酵母的生物合成技术。Ⅰ-type lysozyme as a cysteine-rich protein immune factor mainly expressed in invertebrate has strong activity against pathogenic microorganisms. Ⅰn the present study, the cDNA encoding Ⅰ-type lysozyme of clam Cyclina sinensis was cloned by RT-PCR, known as CslyⅠ gene. Using pPⅠCZαA as expression vector and yeast Pichia pastoris X-33 as engineering bacteria to construct recombinant strain X-33/pPⅠCZαA-CslyⅠ. A high-copy recombinant strain was obtained by using the culture medium containing high concentrations of zeocin. The strain created protein-like expression products after 72 h induced by 1% methanol at 28 ℃, and 250 r/min. The expressed product was identified by Western-blot and MALDⅠ-TOF/TOF mass spectrometry, indicating that it was the expected recombinant CslyⅠ. The bacteriostatic tests demonstrated that the recombinant CslyⅠ had obvious activities against pachogens Staphylococcus aureus, Escherichia coli and Vibrio parahaemolyticus. Ⅰn addition, it also showed a good thermal stability. The findings provide a biosynthetic technology approach based on recombinant yeast P. pastoris for the preparation of Ⅰ-type lysozyme derived from invertebrate.

关 键 词：青蛤 Ⅰ型溶菌酶 重组毕赤酵母菌株 蛋白质表达 抑菌活性 

分 类 号：S917.4[农业科学—水产科学]