重组人肝细胞生长因子改造后在酵母中二级结构活性的研究  

Study on Two-structure with Biological Activities of Recombinant Human Hepatocyte Growth Factor in Transformation.

作　　者：吕云福[1] 董永红[2] 宫晓光[1] 刘宁[1] 常顺伍[1] 邱庆安[1] 黄海[1] 王保春[1] 

机构地区：[1]海南省人民医院普外科,570311 [2]山西省人民医院普外科,030001

出　　处：《世界最新医学信息文摘》2012年第3期26-30,共5页World Latest Medicine Information Electronic Version

基　　金：海南省自然科学基金资助项目（琼科函[2006]291号）

摘　　要：目的探讨人肝细胞生长因子（hHGF）改造后重组hHGF（rhHGF）蛋白在P．pastoris酵母中二级结构的活性。方法根据酵母偏爱密码子和Kex2酶切识别位点，通过套叠PCR技术对hHGFcDNA进行改造并与pGEM—TEasy载体连接，蓝白癍筛选，Xba I／Sal I双酶切鉴定后测序。构建穿梭载体pPICZdA．rhHGF，XbaI／Xbo1酶切鉴定。将穿梭载体质粒用SacI单酶切线性化，转化Rpastoris GS115细胞，表型鉴定，拷贝数确定。采用甲醇诱导表达，选择表达量最高的l株进行温度控制性优化表达。对表达产物进行Westernblot分析和生物学活性测定。结果Xba1／SalI双酶切获得2．1kb大小的片段，测序结果显示AAG序列插入到目的位置，其余序列正确。穿梭载体构建后，XbaI／XhoI双酶切鉴定显示为两条带，分别为580bp和1074bp，与预期一致。转化Rpastoris GS115细胞，PCR鉴定表型均呈mut＋，Zeocin浓度筛选拷贝数均为l。选取其中6个转化茵甲醇诱导，表达rhHGF的浓度分别为14．5mg／L，14．6mg／L，16．1mg／L，18．7mg／L，19．3mg／L，20．5mg／L。还原状态下，SDS．PAGE凝胶电泳显示为两条带。Western blot检测显示在约80kDa处有一条浓聚带，证实rhtlGF表达产物免疫原性存在。结论经过基因改造的rhHGF在P．pastoris中获得表达，其表达产物的二级结构具备生物学活性，有增强HGF的作用，但表达量尚待进一步提高。Objective To probe into human hepatocyte growth factor（hHGF） cDNA by transformation, biological activities of two-structure. Methods The initial part of hHGF cDNA was modified according to optimal expression codons and the connection part between et and β-chain was done according to protease expressed in Pichia pastoris itself. The rhHGF was ligated to pGEM-T Easy vector, which was sequenced after blue/white color screening and identification by Xba I /Sal I cleavage. The interest gene cleavaged by Xba I /Sal I was inserted into pP1CZ a A vector. The linearized pPICZ a A-rhHGF plasmid DNA with Sac I was transformed into Pichia pastoris GS115 by electroporation. Phenotypes were identified by PCR, and copy number was screened on YPD plate containing different concentration of Zeocin. After induce expression with methanol, a productionimproved method by lower temperature, was filrther applied to the most expression strain. Immunity and biologi- cal activity ofrhHGF was detected, respectively. Results An about 2.1 kb DNA fragment was seen after Xba I / Sal I cleavage, and the sequenced result showed that AAG was inserted into the aimed site. The recombined pPICZα A-rhHGF was cleavaged into two bands,580 bp and 1074 bp as expected. Phenotypes identified by PCR were all mut＋, and copy number was 1 screened on YPD plate containing different concentrations of Zeocin. Pick- ing 6 colonies and inducing expression with methanol, the concentration of rbHGF was 14.Smg/L, 14.6mg/L, 16.1 rag/L, 18.7rag/L, 19.3mg/L,20.5mg/L,respectively. The result of SDS-PAGE gel showed to be two bonds un- der reduction state. A strong bond was seen at 80 kDa by Western blot under non-reduced state, which confirmed that rhHGF protein had a certain immunity. Conclusion hHGF cDNA was successfully modified by overlap PCR,and rhHGF was expressed in Pichia pastoris. Expressed rhHGF have biological activities of two-structure. The level ofrhHGF expression in Pichia pastoris is too low and to be improved.

关 键 词：肝细胞生长因子 酵母/巴斯德毕赤 改造 活性 二级结构 

分 类 号：R730[医药卫生—肿瘤]