转基因哺乳动物细胞自合成多不饱和脂肪酸

Transgenic Production of Polyunsaturated Fatty Acids in Mammalian Cells

亚油酸、亚麻酸是哺乳动物体内的必需脂肪酸,但哺乳动物由于缺乏△12和ω-3脂肪酸脱氢酶而自身不能合成.△12和ω-3脂肪酸脱氢酶存在于真菌、植物和一些低等动物中.为了实现哺乳动物细胞亚油酸的自身合成,克隆了线虫编码△12脂肪酸脱氢酶的FAT-2基因cDNA序列,通过优化密码子,构建真核表达载体,稳定转染细胞,经抗生素筛选获得稳定整合FAT-2基因的CHO细胞.PCR和RNA印迹(Northern blot)验证了基因的整合和表达.气相色谱分析细胞的脂肪酸含量表明,FAT-2基因的表达显著提高了转基因细胞中亚油酸的含量,亚油酸含量为阴性对照细胞的2.4倍.研究结果表明,低等动物△12脂肪酸脱氢酶可以重建哺乳动物多不饱和脂肪酸合成途径,并利用细胞中的油酸合成亚油酸.上述研究为进一步利用转基因技术促进农业动物合成多不饱和脂肪酸从而提高食品营养价值奠定基础.

Linoleic acid （C18 ： 2n-6） and α-linolenic acid （C18 ： 3n-3） are found widely in fungi, plants and some lower animals. However, they can not be synthesized in mammals due to lack of △12 and ω-3 fatty acid desaturases. To enable endogenous production of essential fatty acids in mammalian cells, here the stable expression ofa Caenorhabditis elegans gene FA T-2 encoding △12 fatty acid desaturase in CHO cells was reported. First, the FA T-2 coding sequence was cloned by RT-PCR. To facilitate high level synthesis of heterogeneous protein, the codon usage of the fatty acid desaturase genes was optimized according to the codon preference of mouse by site-directed mutagenesis, 2 synonymous mutations were introduced into FA T-2 gene by overlapping PCR. The codon-modified gene was finally fused to pBudCE4.1 vector （Invitrogen） under the control of CMV promoter. The expression vector pBudCE-FA T2 was linearized with Nhe I, and then transfected CHO cells, the cells were under Zeocin selection for nine days and then propagated, then the transfected cells were harvested. The genome and total RNA were isolated for PCR and Norhern blot ananlysis. The results revealed that FA T-2 gene has been integrated into the genome of CHO cells and expressed properly. Fatty acids of total cellular lipids were analyzed by gas chromatography. The results indicate that the expression and function of △-12 fatty acid desaturase resulted in accumulation of linoleic acid. The levels of linoleic acid in transgenic cells were 2.4-fold higher than those in wild-type cells. The moderate linoleic acid in CHO cells was derived from cell culture media uptaken by cell membrane. The results demonstrate that a heterogenous desaturase gene can function well in mammalian ceils and prove that transgenic approach is an efficient strategy for changing fatty acid composition of mammals.