重组截短型人角质细胞生长因子1在昆虫细胞中的表达  

Expression of recombinant truncated human keratinocyte growth factor 1 in insect cells

作　　者：薛萍[1,2] 朱小静[3] 刘孝菊[4] 李一兰[3] 南佳[3] 姜潮[2,3] 李校堃[2,3,5] 

机构地区：[1]吉林农业大学生命科学学院,吉林长春130118 [2]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118 [3]温州医学院药学院浙江省生物技术与制药工程重点实验室,浙江温州325035 [4]吉林大学第二医院检验科,吉林长春130041 [5]吉林大学白求恩医学院生物化学教研室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2012年第4期633-639,共7页Journal of Jilin University：Medicine Edition

基　　金：国家863计划项目资助课题(2010AA100069)

摘　　要：目的:探讨重组截短型人角质细胞生长因子1(rhKGF1D23)蛋白在昆虫细胞中表达的可行性,为rhKGF1D23蛋白的生产提供新的途径。方法:PCR法扩增昆虫偏爱密码子优化截短序列kgf1d23亚克隆到pFastbac载体。在辅助质粒的作用下,pFastBac-kgf1d23载体Tn7片段转座到Bacmid中mini-attTn7位点,得到穿梭载体Bacmid-kgf1d23,并通过脂质体法转染sf-9细胞得到杆状病毒P1。继续转染细胞扩增病毒并提高病毒滴度,蛋白表达分析从P3代病毒转染细胞开始,收集细胞超声裂解后进行纯化,MTT法检测纯化后目的蛋白生物活性。结果:昆虫偏好密码子改造的kgf1d23基因构建进转移载体pFastBac-kgf1d23和穿梭载体Bacmid-kgf1d23载体中。SDS-PAGE结果表明,重组蛋白在60h后开始大量表达,优化的收获蛋白时间在84～96h;利用肝素亲和层析和SP阳离子交换层析可以去除90%以上的杂蛋白。纯化的rhKGF1D23蛋白在0.3～25.0μg.L-1时,随浓度的增加HaCat细胞的促有丝分裂能力增强,在25.0μg.L-1时达到平台期。结论:重组截短型角质细胞生长因子rhKGF1D23蛋白在昆虫细胞中得到表达,保持其特有的肝素亲和特性,并具有免疫原性及细胞生物学活性。Objective To explore the feasible of the expression of recombinant truncated human keratinocyte growth factor 1（rhKGF1D23） in insect cells and to provide a new method to product rhKGF1D23.Methods The truncated gene of kgf1d23 optimized by insect prefer codon was amplified and subcloned into pFastBac vector.With the help of helper plasmid,the Tn7 section of pFastBac-kgf1d23 was transposed to the mini-attTn7 site of Bacmid DNA to obtain the recombinant Bacmid-kgf1d23.Bacmid-kgf1d23 shuttle vector was used to transfect sf-9 cells by lipofection kit to produce recombinant baculovirus P1.The baculoviral stock was amplified and the virus titer was increased until P3 stock.The cells were broken by sonic and the lysate liquid and purified by heparin affinity chromatography and cation exchange;MTT method was used to detect the rhKGF1D23 mitogen activity.Results kgf1d23 gene was optimized by insect bias codon and the recombinant pFastBac-kgf1d23 and Bacmid-kgf1d23 had been successfully constructed.The SDS-PAGE results showed that the rhKGF1D23 expressed mainly in cells and until 60 h post-infection,and the optimal harvest time was in 84-96 h.Heparin affinity and SP cation exchange could remove 90% noisy proteins.The MTT results demonstrated that the purified rhKGF1D23 protein with 0.3-25.0 μg·L-1 could significantly stimulate HaCat cells to proliferate in a dose-dependent manner.Conclusion The rhKGF1D23 expression is obtained in sf-9 cells with heparin binding characteristics,as well as immunogenic and cell biology activity.

关 键 词：角质细胞生长因子1 截短突变 sf-9细胞 杆状病毒系统 

分 类 号：Q78[生物学—分子生物学]