摘要
腺苷一磷酸脱氨酶(Adenosine monophosphate deaminase,AMPD)是嘌呤代谢过程中一种关键的酶。只在真核生物中发现的一种酶,由多基因家族编码。该基因的活化可保护肌细胞免受能量缺乏,其功能缺失会导致一种代谢性肌肉疾病。本研究通过对猪AMPD1基因改变其表达和功能,达到改进猪肌肉质量和风味的可能目的。本实验以Genbank里猪的AMPD1基因序列为基础,采用PCR方法克隆了民猪AMPD1基因的cDNA序列;采用双酶切与过夜连接的方法将AMPD1基因的完整编码区片断连入pcDNA3.1(+)真核表达载体;采用常规方法对猪的胎儿成纤维细胞和PK15细胞进行培养,设计了4组不同的转染体系,即质粒:脂质体=1:2/1:3/2:5/2:7,转染结束后对其转染效率进行比对分析;采用脂质体法将构建好的质粒分别转入成纤维细胞和PK15细胞,转染后选择在6天致死细胞的G418浓度作为转染细胞所用最适的药物筛选浓度,14天致死细胞的G418浓度作为筛选后的维持浓度,最后用PCR法鉴定所得阳性转基因细胞株。本实验主要研究结果如下:
1)成功克隆了民猪AMPD1基因的完整编码区2207个bp序列。
2)经双酶切鉴定后成功构建了猪的AMPD1基因真核表达载体pcDNA3.1-AMPD1。
3)针对pcDNA3.1-AMPD1质粒成纤维细胞中的最佳转染条件为200μg/mlG418筛选浓度,100μg/ml维持浓度;在PK15细胞中的最佳转染条件为1000μg/ml G418筛选浓度,500μg/ml维持浓度。在两种细胞内质粒:脂质体均为2:5,稳定转染6h后转染效果最佳。
4)在稳定表达猪AMPD1基因的成纤维细胞和PK15细胞中均扩增出目的基因,表明转染成功。
5)获得了稳定整合AMPD1基因的PK15细胞单克隆。
Adenosine monophosphate deaminase(AMPD)is one of the key enzymes in the purine nucleotide cycle.It has only been found in all eukaryote . AMPD is a complex allosteric enzyme encoded by a multigene family in mammals .The genic activate can protect muscle cells from lack of energy. Its function is lost will lead to a metabolic muscle disease .,The study changed expression and function research of AMPD1gene ,in order to produce livestock which have more muscle and more flavor. This study was designed to clone the cDNA of pig boar AMPD1 gene based on the sequences of AMPD1 gene of Genbank. The method by polymerase chain reaction (PCR) to cloned cDNA sequence of AMPD1 gene of pigs .The method by double enzyme cut and overnight connection to complete coding region of AMPD1 gene connected pcDNA3.1 (+) eukaryotic expression vector.fibroblast and pk15 cell were successfully established by conventional method. Design for four different of transfection system, namely, DNA: Lipofectamine = 1:2/1:3/2:5/2:7, Comparison analysis of transfection efficiency afer over the transfection.Construct plasmid was introduced into fetal fibroblast and pk15 cell of pig by lipofectamin-mediated DNA transfection methods, Cells selecting with G418 for 6 days, then go on selecting with G418 for 14 days of cells. At last, the positive cells were identified by PCR. The main results are as follows:
1)The complete two thousand two hundred and seven tcDNA sequences of minpig AMPD1 gene were cloned .
2)Constructs pcDNA3.1 that AMPD1 gene of pig eukaryotic expression vector after double enzyme.
3)Fibroblast of pig selecting with G418(200μg/ml) for pcDNA3.1(+)-AMPD1 plasmid, and PK15 cell of pig selecting with G418(1000μg/ml) for pcDNA3.1(+)-AMPD1 plasmid .After transfection, ,then fibroblast of pig and pk15 cell of pig go on select with 100μg/ml G418 and 500μg/mlG418. DNA and Lipofectamin than for 2:5, stable transfection 6 hours are optimized condition for transfection in two kinds of cells.
4)Two cells that stable expression AMPD1gene all have purpose gene amplification,it shows that transfection success.
5)Get stable integration AMPD1 gene of pk15 monoclonal cell.
引文
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