CAJ | 학술논문

山羊脂肪酸合酶 (Fatty acid synthase,FAS)是脂肪酸合成的关键酶,对乳腺短、中链脂肪酸合成起重要调控作用.设计了shRNA-5544、shRNA-5936、shRNA-6132 3条针对FAS基因不同区域的小发夹RNA(Short hairpin RNA,shRNA)及一条阴性对照序列shRNA-NC,并构建表达这4条shRNA序列的入门载体及其靶基因与红色荧光蛋白基因的融合表达载体,二者共转染HEK 293细胞进行有效序列筛选,结果显示shRNA-5544和shRNA-5936序列具有明显的干扰效果.在LR Clonase-Ⅱ重组酶作用下,分别将pENTR/CMV-GFP/U6-shRNA-5544、pENTR/CMV-GFP/U6-shRNA-5936及pENTR/CMV-GFP/U6-shRNA-NC入门载体与腺病毒骨架载体pAd/PL-DEST进行LR重组,经氨苄青霉素及氯霉素抗性筛选后成功获得3个重组腺病毒载体,ScaⅠ酶切鉴定及测序分析证实所构建的重组腺病毒载体中插入序列与设计序列一致.重组腺病毒载体经PacⅠ酶切线性化后,利用Lipofectamine 2000转染HEK 293细胞,10×12 d后收集病毒,在HEK 293细胞中反复扩增3次后,获得高滴度的重组腺病毒,利用TCID_(50)法测定重组腺病毒滴度分别为6×10~8 PFU/mL(表达shRNA-5544序列)、5×10~8 PFU/mL(表达shRNA-5936序列)及6×10~8 PFU/mL(表达shRNA-NC序列),为进一步在原代培养的山羊乳腺上皮细胞中进行FAS基因的RNA干扰研究奠定基础.
산양지방산합매 (Fatty acid synthase,FAS)시지방산합성적관건매,대유선단、중련지방산합성기중요조공작용.설계료shRNA-5544、shRNA-5936、shRNA-6132 3조침대FAS기인불동구역적소발협RNA(Short hairpin RNA,shRNA)급일조음성대조서렬shRNA-NC,병구건표체저4조shRNA서렬적입문재체급기파기인여홍색형광단백기인적융합표체재체,이자공전염HEK 293세포진행유효서렬사선,결과현시shRNA-5544화shRNA-5936서렬구유명현적간우효과.재LR Clonase-Ⅱ중조매작용하,분별장pENTR/CMV-GFP/U6-shRNA-5544、pENTR/CMV-GFP/U6-shRNA-5936급pENTR/CMV-GFP/U6-shRNA-NC입문재체여선병독골가재체pAd/PL-DEST진행LR중조,경안변청매소급록매소항성사선후성공획득3개중조선병독재체,ScaⅠ매절감정급측서분석증실소구건적중조선병독재체중삽입서렬여설계서렬일치.중조선병독재체경PacⅠ매절선성화후,이용Lipofectamine 2000전염HEK 293세포,10×12 d후수집병독,재HEK 293세포중반복확증3차후,획득고적도적중조선병독,이용TCID_(50)법측정중조선병독적도분별위6×10~8 PFU/mL(표체shRNA-5544서렬)、5×10~8 PFU/mL(표체shRNA-5936서렬)급6×10~8 PFU/mL(표체shRNA-NC서렬),위진일보재원대배양적산양유선상피세포중진행FAS기인적RNA간우연구전정기출.
Goat fatty acid synthase (FAS),a central multifunctional enzyme in charge of fatty acid synthesis in mammary gland,plays a significant role in regulation of short-and medium-chain fatty acid. This study designed three short hairpin RNA (shRNA) sequences shRNA-5544,shRNA-5936,and shRNA-6132 targeting different area of FAS gene and one negative control sequence,then constructed four entry vectors containing shRNA expression cassette as well as pDsRed1-C1 vector expressing target gene by cotransfecting HEK 293 cell. The result showed that entry vector expressing shRNA-5544 and shRNA-5936 sequences caused an obvious interference effect. Then ampicillin and chloramphenicol were successfully applied to identify three recombinant adenovirus vectors generated by LR recombination between entry vector (pENTR/CMV-GFP/U6-shRNA-5544,pENTR/CMV-GFP/U6-shRNA-5936,pENTR/CMV-GFP/U6-shRNA-NC) and backbone vector (pAd/PL-DEST). ScaⅠdigestion identification and sequencing analysis indicated that the sequence inserted into pAd/PL-DEST vector was the same with designed shRNA template. Recombinant adenovirus vector was transfected into HEK 293 Cell by Lipofectamine 2000 reagent after linearization by PacⅠdigestion. The cell lysates harvested during 10 to 12 days post transfection were employed to perform virus amplification in HEK 293 cells for three times to generate high-titer virus. The titer of the adenoviral stock was determined by TCID_(50) method and respectively reaches 6×10~8 PFU/mL(Expressing shRNA-5544 sequence),5×10~8 PFU/mL(Expressing shRNA-5936 sequence),and 6×10~8 PFU/mL(Expressing shRNA-NC sequence),respectively. The attaining of recombinant virus lays a foundation for RNA interference research of FAS gene in primary goat mammary epithelial cells.