虎纹捕鸟蛛Kunitz型毒素基因的分子改造和表达  

Molecular modification and expression of genes encoding Kunitz toxins from the spider Ornithoctonus huwena

作　　者：罗玉娇[1,2] 舒衡平[1] 李滨[1] 蒋立平[1,3] 

机构地区：[1]中南大学基础医学院医学寄生虫学系,湖南长沙410078 [2]中南大学湘雅二医院血液内科分子血液病研究室,湖南长沙410011 [3]中南大学药学院药理学系,湖南长沙410013

出　　处：《中国病原生物学杂志》2016年第8期677-684,共8页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30901874);湖南省自然科学基金项目(No.14JJ2032;No.2015JJ2192);中南大学教师研究基金项目(No.2014JSJJ027)

摘　　要：目的构建HW11c40突变体,寻找合适的表达系统表达HW11c40突变体和HWTX-XI(虎纹捕鸟蛛毒素-XI),解决HWTX-XI来源问题和HW11c40改造后毒素难于获取的难题,为合适HW11c40突变体的筛选和治疗急性胰腺炎高效新药的研制奠定基础。方法用PCR定点突变的方法突变HW11c40相关氨基酸残基,构建HW11c40突变体。HW11c40突变体和HWTX-XI基因经PCR扩增后插入到pET-40b(+)载体,基因5'端插入肠激酶切割位点,再重组到大肠埃希菌BL21(DE3)进行原核周质表达,获得相应的融合蛋白并进行SDS-PAGE鉴定,用镍柱纯化融合蛋白。融合蛋白用肠激酶酶切,释放出的目的蛋白用Tricine-SDS-PAGE进行鉴定,用镍柱分离目的蛋白,进行高效液相色谱进行进一步分离纯化后作质谱鉴定。对HW11c40突变体和HWTX-XI的表达时间、温度和IPTG浓度进行优化。结果成功获得HW11c40的4个突变体,并正确构建HW11c40突变体和HWTX-XI的原核表达载体。经SDS-PAGE和Tricine-SDS-PAGE鉴定,重组质粒转化菌在无IPTG诱导的情况下成功表达所有HW11c40突变体和HWTX-XI的融合蛋白,经肠激酶酶切均获得对应的目的蛋白,其中HWTX-XI表达量为3.4mg/L。结论成功构建出HW11c40突变体和HWTX-XI的重组质粒并在原核细胞内高效表达出相应的目的蛋白,开辟了基因工程表达HWTX-XI的新途径,同时解决了HW11c40改造后毒素获取难题,突破了整个系列研究的瓶颈,为突变体的筛选及治疗急性胰腺炎高效新药的研制奠定了基础。Objectives To construct HW11c40 mutants and determine a suitable system for expression of HW11c40 mutants and HWTX-XI（Huwentoxin XI）in order to resolve the difficulty of obtaining sources of HWTX-XI and HW11c40 modified toxins and to lay the foundation for screening suitable mutants to develop s novel and highly efficient treatment for acute pancreatitis. Methods HW11c40 mutants were designed by mutating HW11c40 amino acid residues using PCR site-directed mutagenes.The genes of HW11c40 mutants and HWTX-XI amplified with PCR were inserted into the prokaryotic vector pET-40b（＋）to construct recombinant plasmids.An enterokinase cleavage site was inserted at the 5’-terminus of the genes.The recombinant plasmids were then transformed into the E.coli strain BL21（DE3）.Screening for positive clones,PCR identification,and DNA sequencing yielded the target genes.SDS-PAGE was used to analyze expression of the fusion proteins.After purification with an Ni-NTA column,the fusion proteins were digested with enterokinase to release target proteins,and then the target proteins verified with Tricine-SDS-PAGE and separated with an NiNTA column and further purified with RP-HPLC.Finally,the target proteins were identified using MALDI-TOF mass spectrometry.Moreover,the expression of HW11c40M1 and HWTX-XI was optimized in terms of time,temperature,and the concentration of IPTG. Results The recombinant vectors were identified with PCR and DNA sequencing verified that prokaryotic expression vectors（pET40b-HW11c40M1,pET40b-HW11c40M2,EHW11c40M3,EHW11c40M4,and pET40b-HWTX-XI）for the four HW11c40 mutants and HWTX-XI were successfully constructed.SDS-PAGE and Tricine-SDS-PAGE confirmed that the fusion proteins of HW11c40 mutants and HWTX-XI were expressed in the periplasm of E.coli in the absence of IPTG,and the fusion proteins were digested with enterokinase to successfully obtain the corresponding target proteins.The target proteins HWTX-XI and HW11c40M1 were further purified with an NiNTA column and RP-HPLC.Target protei

关 键 词：虎纹捕鸟蛛毒素Ⅺ HW11c40突变体 胰蛋白酶抑制活性 KUNITZ 原核周质表达 

分 类 号：R384[医药卫生—医学寄生虫学]