人血小板CD36基因220C>T和429+4insg变异真核表达载体的构建及表达  

Construction of eukaryotic expression vector for human platelet CD36 gene 220C>T and 429+ 4insg variants and analysis of their expressions in HEK293T cells

作　　者：徐秀章[1] 丁浩强[1] 刘静[1] 夏文杰[1] 邓晶[1] 陈扬凯[1] 王嘉励[1] 邵媛[1] 陈大伟[1] 叶欣[1] Xu Xiuzhang;Ding Haoqiang;Liu Jing;Xia Wenjie;Deng Jing;Chen Yangkai;Wang Jiali;Shao Yuan;Chen Dawei;Ye Xin(Institute of Blood Transfusion,Guangzhou Blood Center,Guangzhou Municipal Key Laboratory for Medical Research,Guangzhou,Guangdong 510095,China)

机构地区：[1]广州血液中心临床输血研究所,广州市医学重点实验室,510095

出　　处：《中华医学遗传学杂志》2019年第2期124-127,共4页Chinese Journal of Medical Genetics

基　　金：国家自然科学基金(81601451);广东省自然科学基金(2016A030313124,2016A030313123);广州市科技计划项目(201607010007,201707010021).

摘　　要：目的 构建人血小板CD36基因220C>T和429+4insg变异的真核表达载体,并转染HEK293T细胞进行CD36蛋白的表达分析。 方法 提取人血小板总RNA并逆转录为cDNA,经PCR扩增获得分别携带220C>T和429+4insg基因变异的CD36基因片段;经TA克隆将目的基因片段连接到pcDNA3.1/V5-His-TOPO载体并转化TOP10E.coli,蓝白斑筛选获得阳性质粒,提取质粒DNA进行测序分析;将分别含有220C>T和429+4insg基因变异体的质粒DNA在effectene作用下转染HEK293T细胞;应用流式细胞术及Western印迹分别对220C>T和429+4insg基因变异体的CD36蛋白表达进行鉴定分析。 结果 通过TA克隆成功构建了pcDNA3.1/V5-His-CD36 (220C>T/429+4insg)真核表达载体,转染HEK293T细胞后,经流式细胞术和Western印迹分析,证实220C>T和429+4insg基因变异可导致CD36蛋白在HEK293T细胞的缺失表达。 结论 CD36基因220C>T和429+4insg变异均可引起人血小板CD36的缺失表达,明确了220C>T和429+4insg变异对CD36表达的影响,为其他CD36基因变异的研究奠定了基础。Objectives To construct eukaryotic expression vectors for human platelet CD36 gene 220C>T and 429+ 4insg variants and analyze their expressions in HEK293T cells. Methods RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+ 4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+ 4insg variant was used to transfect HEK 293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting.Results An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+ 4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+ 4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting.Conclusion The 220C>T and 429+ 4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+ 4insg, and other variants on the expression of CD36.

关 键 词：血小板 CD36基因 变异 真核表达载体 

分 类 号：R346[医药卫生—基础医学]