奶牛乳腺上皮细胞HSP70 mRNA 荧光实时定量 - 河北农业大学学报.PDF

33 5 Vol.33 No.5 20 10 9 JOURNA L OF AGRICULT URAL UNIVERSITY OF HEBEI 　 　 Sep .2 0 10 :1000-1573(2010)05-0089-05 HSP70 RNA P CR 赵丽红, 　高艳霞, 　李秋凤, 　李建国 ① ( , 071001) : HSP70 RNA , RNA , cDNA, cDNA PCR, , pGEM-T-HSP70 , , PCR 。 HSP70 PCR , 259 bp 10 3 , , 2.5×10 copies/ L, 2.5×10 copies/ L, (R=0.99), HSP70 。 　　:HSP70;PCR; :S 811.4 :A The HSP70 mRNA of mammary epithelial cells by real time FQ-PCR standard plasmid preparation and standard curve establishment ZHAO Li-hong, GAO Yan-x ia, LI Qiu-feng, LI Jian-guo (College of Ani al Science and Technology,Agricultural University of Hebei, Baoding 071000, China) Abstract:To clone dairy cow HSP70 cDNA and use it as the standard for real-ti e quantifying HSP70 RNA.Total RNA was extractedfro v it ro raised cow a ary gland epithelial cells and reverse transcribed to cDNA.A pified HSP70 and identifiedwith poly eraseclain reaction (PCR),and then were subjected to sequencing.Total RNA extractedfro dairy caw a ary gland epithelial cells were perfected.The PCR of HSP70 plas id was a259 bp clear stripe, and the DNA sequencing of HSP70 was according with ai ed frag ent(100%).The original con- 10 3 centration was 2.5×10 copies/ L,and then diluted to 2.5×10 copies/ L with ultiple pro- portions,perfected standard cure could be all received(R =0.99).The reco bined plas ids con- taining dairy cow HSP70 RNA have been constructed successfully.