CPT-Ⅱ基因4号外显子质粒构建与变异分析  

Plasmid construction and variation analysis of exon-4 of carnitine palmitoyltransferase-Ⅱ gene

作　　者：姚登兵[1] 姚敏[2] 王理[2] 董志珍[3] 姚登福[4] 

机构地区：[1]南通大学神经再生重点实验室,江苏省南通市226001 [2]南通大学附属医院医学检验中心,江苏省南通市226001 [3]南通大学医学院诊断学教研室,江苏省南通市226001 [4]南通大学附属医院临床医学研究中心,江苏省南通市226001

出　　处：《中国组织工程研究与临床康复》2011年第50期9437-9440,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：南通大学自然科学基金项目资助(09ZY014;09B16;09ZY007);江苏省高校自然科学基金项目资助(09KJB310012);江苏省自然科学基金研究项目(BK2010282)部分资助~~

摘　　要：背景:肉毒碱棕榈酰转移酶Ⅱ(Carnitine palmitoyltransferase-Ⅱ,CPT-Ⅱ)位于细胞线粒体内膜上,是脂肪酸氧化过程中起关键作用的酶之一。目前关于流感病毒感染与CPT-Ⅱ基因变异尚未见报道。目的:构建CPT-Ⅱ第4号外显子(E4)表达质粒及其核苷酸变异分析。方法:从流感病毒感染2例患者外周血中提取DNA,以PCR法扩增CPT-Ⅱ基因的第4号外显子,将其片段插入pGEM-T载体,以T4DNA连接酶,构建T-CPT-ⅡE4,转化大肠杆菌DH5α细胞,增菌,制备质粒,EcoRI酶切、测序和变异分析。结果与结论:成功构建重组表达质粒pGEM-T-CPT-ⅡE4,经DNA测序,证实质粒中插入CPT-Ⅱ第4号外显子完整序列,全长1305个核苷酸,编码435个氨基酸;与Genebank源序列对照比较,发现含有两个变异位点:1618(GTC→ATC)和1858(TTT→TCT),对应氨基酸为V368I和F448L。BACKGROUND： Carnitine palmitoyltransferase- 11 （CPT- Ⅱ ） is located in the inner mitochondrial membrane and is one of the pivotal enzymes during the fatty acid oxidation. Currently, there are no studies about influenza virus infection and variation of CPT- Ⅱ gene. OBJECTIVE： To construct the plasmid expressing exon-4 of CPT- Ⅱgene （CPT- 11 -E4） and analyze its mutation. METHODS： Human CPT- ⅡDNA from the blood of two patients infected with influenza virus was involved. After the PCR amplification, the product of CPT- Ⅱ -E4 was cloned into pGEMD-T vector. The T-CPT- Ⅱ E4 was transferred to E. coli DH5a cells after prepared with T4 DNA higase. Then propagation of bacteria, preparation of plasmid, EcoRI digestion, sequencing, and variation analysis were performed. RESULTS AND CONCLUSION： The recombinant pGEM-T-CPT- Ⅱ E4 was proved to contain whole sequence of CPT- Ⅱ -E4 with 1305 nucleotides which could code 435 amino acids. According to the original sequence from Genebank, two variable sites were found at 1618 （GTC→ATC） and 1858 （TTT→TCT） which code amino acids of V3681 and F448L respectively.

关 键 词：肉毒碱棕榈酰转移酶Ⅱ(CPT-Ⅱ) 外显子4 基因扩增 变异 流感病毒A 

分 类 号：R318[医药卫生—生物医学工程]