pET3c-FGF2^(K18S/S69C)突变体的构建及表达条件优化  

Construction of pET3c-FGF2^(K18S/S69C) Mutant and Optimization of Its Expression Condition

作　　者：董媛 张家琦 唐一鑫 董雪 马巍 侯寒进 辛本凯 刘彦 王会岩[2] Dong Yuan;Zhang Jiaqi;Tang Yixin;Dong Xue;Ma Wei;Hou Hanjin;Xin Benkai;Liu Yan;Wang Huiyan(Academy of Laboratory,Jilin Medical University,Jilin,132013;Jilin Collaborative Innovation Center for Antibody Engineering,Jilin Medical University,Jilin,132013)

机构地区：[1]吉林医药学院检验学院,吉林132013 [2]吉林医药学院吉林省抗体工程创新中心,吉林132013

出　　处：《基因组学与应用生物学》2021年第2期489-493,共5页Genomics and Applied Biology

基　　金：吉林省教育厅项目(JJKH20180822KJ);吉林省科技厅项目(20180623045TC);吉林省大学生创新创业训练项目(201813743007)共同资助。

摘　　要：构建和表达人FGF2^(K18S/S69C)突变体,并筛选最佳的表达条件。对GenBank数据库FGF2序列进行分析后,发现了位于第18位和第69位的氨基酸定向突变之后,能够增加FGF2稳定性。基因合成FGF2^(K18S/S69C)突变体,与载体pET3c连接后转化至大肠杆菌BL21中,用乳糖诱导,以蛋白质表达量为指标,考察诱导剂浓度、装液体积、诱导时机、时间和温度对表达量的影响。成功构建pET3c-FGF2^(K18S/S69C)突变体重组质粒,经菌落PCR、双酶切法和测序法证实质粒构建正确,目的片段长度约447 bp。在BL21中成功表达,FGF2^(K18S/S69C)最佳发酵工艺为:装液量30 mL/250 mL锥形瓶,A600为0.8,乳糖浓度0.5 g/L,37℃、180 r/min诱导4 h。Western blot分析该表达产物表明,该蛋白质可以和人FGF2抗体特异性结合。通过构建pET3c-FGF2突变体基因工程菌,并优化其表达条件,为后续研究FGF2^(K18S/S69C)突变体提供了基础。Human FGF2^(K18S/S69C) mutants were constructed and expressed,and the expression conditions were optimized.Bioinformatics analysis in GenBank database showed that the site-directed mutagenesis at 18 th and 69 th amino acids can increase the stability of FGF2.The FGF2^(K18S/S69C)mutant was synthesized,connected with the carrier pET3 c,and transformed into the strain BL21(DE3)for expression.Afterwards,the effects of inducer concentration,induction time,temperature,loading volume and OD value on protein expression were investigated.The positive recombinant plasmid was identified by PCR,double enzyme digestion and sequencing.The results indicated that the target fragment was about 447 bp,which was consistent with the expected fragment length.The recombinant plasmid of pET3 c-FGF2 mutant was constructed and expressed successfully.The optimal expression conditions were as follows:growth density of the strain(A600)0.8,lactose concentration 0.5 g/L,loading volume 30 mL/250 mL and induction at 37℃and 180 r/min for 4 h.Western blot analysis indicated that the expression product could specifically be bound to human FGF2 antibody.The pET3 c-FGF2 mutant was constructed and its expression conditions were optimized,which provides a basis for the subsequent study of FGF2^(K18S/S69C)mutant.

关 键 词：成纤维细胞生长因子2 突变体 乳糖诱导 表达 优化 

分 类 号：Q78[生物学—分子生物学]