摘要
[目的]构建CMV-3flag-h PROKR2(343YFK/AAA345,351HWR/AAA353)真核表达质粒并观察其蛋白表达。[方法]将NotⅠ的酶切位点(GCGGCCGCC)引入第一次PCR的后引物,扩增并突变h PROKR2的1-1035 bp(343YFK/AAA345),再将NotⅠ的酶切位点和3个连续甘氨酸突变碱基GCCGCCGCA引入第二次PCR的前引物扩增并突变h PROKR2的1036-1152bp(351HWR/AAA353),通过NotⅠ酶切位点连接前后两个片段,即可构建pc DNA3.1-h PROKR2-myc-his(343YFK/AAA345,351HWR/AAA353),最后通过测序鉴定突变是否成功。扩增h PROKR2(343YFK/AAA345,351HWR/AAA353),连接入CMV-3flag。Western Blot检测相应蛋白的表达。[结果]成功构建CMV-3flag-h PROKR2(343YFK/AAA345,351HWR/AAA353)质粒,并验证到相应蛋白表达。[结论]利用NotⅠ酶的碱基序列特性(GCGGCCGCC)进行3个连续氨基酸突变的方法简单有效。质粒CMV-3flag-h PROKR2(343YFK/AAA345,351HWR/AAA353)的成功构建为后期进一步实验创造条件。
[Objective] To construct the plasmid CMV-3 flag-h PROKR2（343 YFK/AAA345,343 HWR/AAA353） and observe the expression of corresponding protein.[Methods] We designed the NotⅠ（GCGGCCGCC） in the reverse primer of the first PCR reaction,the target gene was amplified and the mutational h PROKR2 1-1035 bp（343 YFK/AAA345） was got.Then we designed the NotⅠ（GCGGCCGCC） and GCCGCCGCA in the forward primer of the second PCR reaction,the target gene was amplified and the mutational hPROKR2 1036-1152 bp（351 HWR/AAA353） was got.The enzyme NotⅠ was used to connect the two PCR amplified fragments,the plasmid pcDNA3.1-h PROKR2-myc-his（343 YFK/AAA345,343 HWR/AAA353） was constructed,the mutation was finally identified by sequencing.The h PROKR2（343 YFK/AAA345,351 HWR/AAA353） was amplified and connected into the plasmid CMV-3 flag.The expression of corresponding proteinwas identified by Western Blot.[Results] The plasmid CMV-3 flag-h PROKR2（343 YFK/AAA345,343 HWR/AAA353） was successfully constructed and the expression of corresponding protein was verified.[Conclusion] The method was simple and effective to mutate 3 consecutive amino acid by using the base sequence of Not Ⅰ（GCGGCCGCC）.The construction of plasmid CMV-3 flag-h PROKR2（343 YFK/AAA345,351 HWR/AAA353） provide the condition for the further experiment.
作者
周晓涛
周文涛
彭震
刘化蝶
陈丹娜
夏开德
迪丽娜尔.波拉提
赵云娟
李家大
Xiaotao Zhou;Wentao Zhou;Zhen Peng;Huadie Liu;Danna Chen;Kaide Xia;Dilinaer · Polati;Yunjuan Zhao;Jiada Li(Department of Immunology of Basic Medical College, Xinjiang Medical University, Urumqi 830011, China;Department of Traditional Chinese Medicine in the Fifth Affiliated Hospital, Xinjiang Medical University, Urumqi 830000, China;Key Laboratory of Medical Genetics, Centralsouth University, Changsha 410000, China;Guizhou Maternity and Child Care Hospital, Guiyang 550000, China)
出处
《生物技术》
CAS
2018年第2期113-118,150,共7页
Biotechnology
基金
国家自然科学基金项目("PROKR2互作蛋白SNAPIN对其功能的调控机制研究"
No.31401218)
新疆维吾尔自治区自然科学基金("SNAPIN对PROKR2功能调控的机制研究"
No.2015211C034)
