中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2013年
2期
315-319
,共5页
李立%石小云%张登文%张传汉%姚文龙
李立%石小雲%張登文%張傳漢%姚文龍
리립%석소운%장등문%장전한%요문룡
组织构建%组织构建细胞学实验%细胞周期末期促进复合物%Cdh1%定点突变%聚合酶联反应%真核表达质粒%神经系统%发育%神经损伤修复%基因治疗%国家自然科学基金
組織構建%組織構建細胞學實驗%細胞週期末期促進複閤物%Cdh1%定點突變%聚閤酶聯反應%真覈錶達質粒%神經繫統%髮育%神經損傷脩複%基因治療%國傢自然科學基金
조직구건%조직구건세포학실험%세포주기말기촉진복합물%Cdh1%정점돌변%취합매련반응%진핵표체질립%신경계통%발육%신경손상수복%기인치료%국가자연과학기금
背景:细胞周期末期促进复合物调节亚基Cdh1的活性受到磷酸化调节,磷酸化的Cdh1不能与细胞周期末期促进复合物结合,从而抑制细胞周期末期促进复合物的活性.目的:构建突变型Cdh1基因真核表达质粒及鉴定.方法:采用RT-PCR方法,从大鼠海马组织扩增出Cdh1基因编码序列.通过限制性内切酶EcoRⅠ和XbaⅠ双酶切PCR回收产物和pBluescript质粒将Cdh1基因克隆到pBluescript质粒上.根据定点突变技术原理,以含Cdh1编码序列的pBluescript-Cdh1质粒为模版,针对Cdh1第40、151、163位丝氨酸(S)和第121位苏氨酸(T)设计4对突变引物,将4个氨基酸位点全部突变为丙氨酸(A).最后通过测序鉴定.结果与结论:经电泳鉴定PCR扩增产物大小约为1500 bp,包括Cdh1基因完整的编码序列、编码序列两端引入的酶切位点以及KOZAK序列,与预期相符.重组质粒pBluescript-Cdh1经EcoRⅠ和XbaⅠ双酶切鉴定与预期结果符合.DNA测序比对发现Cdh1(BC162059.1)编码序列第930位碱基A在重组质粒pBluescript-Cdh1上突变为G,但氨基酸无变化,为同义突变,其他DNA序列无突变.经测序鉴定pBluescript-Cdh1-4A40、121、151、163示实验成功构建磷酸化位点突变型Cdh1基因真核表达质粒.突变质粒第40、121、151、163位氨基酸全部突变为丙氨酸.提
揹景:細胞週期末期促進複閤物調節亞基Cdh1的活性受到燐痠化調節,燐痠化的Cdh1不能與細胞週期末期促進複閤物結閤,從而抑製細胞週期末期促進複閤物的活性.目的:構建突變型Cdh1基因真覈錶達質粒及鑒定.方法:採用RT-PCR方法,從大鼠海馬組織擴增齣Cdh1基因編碼序列.通過限製性內切酶EcoRⅠ和XbaⅠ雙酶切PCR迴收產物和pBluescript質粒將Cdh1基因剋隆到pBluescript質粒上.根據定點突變技術原理,以含Cdh1編碼序列的pBluescript-Cdh1質粒為模版,針對Cdh1第40、151、163位絲氨痠(S)和第121位囌氨痠(T)設計4對突變引物,將4箇氨基痠位點全部突變為丙氨痠(A).最後通過測序鑒定.結果與結論:經電泳鑒定PCR擴增產物大小約為1500 bp,包括Cdh1基因完整的編碼序列、編碼序列兩耑引入的酶切位點以及KOZAK序列,與預期相符.重組質粒pBluescript-Cdh1經EcoRⅠ和XbaⅠ雙酶切鑒定與預期結果符閤.DNA測序比對髮現Cdh1(BC162059.1)編碼序列第930位堿基A在重組質粒pBluescript-Cdh1上突變為G,但氨基痠無變化,為同義突變,其他DNA序列無突變.經測序鑒定pBluescript-Cdh1-4A40、121、151、163示實驗成功構建燐痠化位點突變型Cdh1基因真覈錶達質粒.突變質粒第40、121、151、163位氨基痠全部突變為丙氨痠.提
배경:세포주기말기촉진복합물조절아기Cdh1적활성수도린산화조절,린산화적Cdh1불능여세포주기말기촉진복합물결합,종이억제세포주기말기촉진복합물적활성.목적:구건돌변형Cdh1기인진핵표체질립급감정.방법:채용RT-PCR방법,종대서해마조직확증출Cdh1기인편마서렬.통과한제성내절매EcoRⅠ화XbaⅠ쌍매절PCR회수산물화pBluescript질립장Cdh1기인극륭도pBluescript질립상.근거정점돌변기술원리,이함Cdh1편마서렬적pBluescript-Cdh1질립위모판,침대Cdh1제40、151、163위사안산(S)화제121위소안산(T)설계4대돌변인물,장4개안기산위점전부돌변위병안산(A).최후통과측서감정.결과여결론:경전영감정PCR확증산물대소약위1500 bp,포괄Cdh1기인완정적편마서렬、편마서렬량단인입적매절위점이급KOZAK서렬,여예기상부.중조질립pBluescript-Cdh1경EcoRⅠ화XbaⅠ쌍매절감정여예기결과부합.DNA측서비대발현Cdh1(BC162059.1)편마서렬제930위감기A재중조질립pBluescript-Cdh1상돌변위G,단안기산무변화,위동의돌변,기타DNA서렬무돌변.경측서감정pBluescript-Cdh1-4A40、121、151、163시실험성공구건린산화위점돌변형Cdh1기인진핵표체질립.돌변질립제40、121、151、163위안기산전부돌변위병안산.제
@@@@ BACKGROUND:The activity of anaphase promoting complex-Cdh1 is regulated by phosphorylation. Phosphorylated Cdh1 cannot be combined with anaphase promoting complex, thereby inhibiting the activity of the anaphase promoting complex. OBJECTIVE:To construct and identify a mutated-Cdh1 eukaryotic expressing vector. METHODS:The entire coding sequence of the Cdh1 gene was amplified from rat hippocampal mRNA by reverse transcription-PCR. Then the PCR product of Cdh1 was cloned into pBluescript plasmid by double digestion with restriction endonucleases EcoR1 and Xba1, and ligation. Based on site-directed mutagenesis, the pBluescript-Cdh1 plasmid containing Cdh1 coding sequence was used as a template. The 40, 151, 163 serine (S) and 121 threonine (T) in Cdh1 gene was mutated to alanine (A) by multiple PCR with four pairs of mutated primers. The mutated Cdh1 vector was identified by DNA sequencing. RESULTS AND CONCLUSION:The PCR product was about 1 500 bp by electrophoresis, including the entire coding sequence of Cdh1, restriction sites at both ends of Cdh1 and KOZAK sequence. The recombinant pBluescript-Cdh1 plasmid was identified by digestion with restriction endonuclease EcoR1 and Xba1, which was consistent with the expected results. DNA sequencing showed that A at the 930th base of Cdh1 (BC162059.1) coding sequence was mutated to G in the recombinant plasmid of pBluescript-Cdh1. But the sequence of amino acids was not affected. The 40, 121, 151, 163 amino acids in pBluescript-Cdh1-4A40, 121, 151, 163 mutant plasmids were al mutated to alanine. The mutated Cdh1 gene expressing plasmid at phosphorylation site was successful y constructed, which provides a good foundation for further studies of Cdh1 gene function.
