天花粉蛋白的定点突变及聚乙二醇修饰

目的对天花粉蛋白(Trichosanthin,TCS)进行定点突变及聚乙二醇(PEG)修饰,并对修饰产物进行DNA酶活性分析。方法选择TCS分子上可能的抗原决定簇位点(YFF81-83和KR173-174)进行定点突变,将所构建的两个突变体(TCSYFF81-83ACS和TCSKR173-174CG)在大肠杆菌中表达,并纯化表达产物。通过第82位和173位引入的半胱氨酸残基,分别对突变体蛋白进行PEG定点修饰。通过与突变型TCS比较,分析两个PEG修饰型TCS的DNA酶活性。结果突变的TCS经酶切鉴定及测序分析,证明质粒构建正确,表达的目的蛋白相对分子质量约为30000,经PEG修饰后,相对分子质量约为40000。初步分析显示,与突变型TCS相比,所构建的两个PEG修饰型TCS也显示出类似DNA酶活性。结论成功进行了TCS的定点突变及PEG修饰,为基因工程及化学修饰方法改造TCS提供了一条可行的途径。

Site-directed Mutation of Trichosanthin and Chemical Modification of Mutant by Polyethylene Glycol

Objective To induce site-directed mutation of trichosanthin (TCS),modify the mutant by polyethylene glycol (PEG) and analyze its DNase-like activity.Methods Two potential antigenic determinants of TCS,YFF81-83 and KR173-174,were selected by computer modeling,based on which the genes encoding two site-directed mutants of TCS,TCSYFF81-83ACS and TCSKR173-174CG,were amplified by PCR,inserted into pRSET-A vector and transformed to E.coli DH5α.The recombinant plasmid was extracted,identified by restriction analysis and sequencing and transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot and modified by PEG.The DNase-like activities of the two PEG-modified mutants were analyzed and compared with those of unmodified mutants.Results Both restriction analysis and sequencing result showed that recombinant plasmids were constructed correctly.The relative molecular masses of the expressed products before and after modification were about 30 000 and about 40 000 respectively.Like unmodified TCS mutants,the two mutants modified by PEG showed DNase-like activity.Conclusion The site-directed mutation of TCS was successfully induced,and the mutants were modified by PEG.It provided a feasible approach for reconstructing TCS by gene engineering and chemical modification.